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Sample records for genetically-encoded ratiometric indicator

  1. MagIC, a genetically encoded fluorescent indicator for monitoring cellular Mg2+ using a non-Förster resonance energy transfer ratiometric imaging approach

    NASA Astrophysics Data System (ADS)

    Koldenkova, Vadim Pérez; Matsuda, Tomoki; Nagai, Takeharu

    2015-10-01

    Intracellular Mg roles are commensurate with its abundance in the cell cytoplasm. However, little is known about Mg subcellular dynamics, primarily due to the lack of suitable Mg-selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a Mg-sensitive indicator-MagIC (indicator for Magnesium Imaging in Cell) -composed of a functionalized yellow fluorescent protein (FP) variant fused to a red-emitting FP serving as a reference, thus allowing ratiometric imaging of Mg. MagIC expressed in mammalian cells is homogeneously distributed between the cytosol and nucleus but its fusion with appropriate targeting sequences redirects it to mitochondria or the endoplasmic reticulum. MagIC shows little interference by intracellular Ca [Kd(Mg2+)=5.1 mM Kd(Ca2+)=4.8 mM] and its kinetic properties (k=84 s-1) approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic Mg increase provoked by application of 50 mM MgCl2 in the medium. This effect is, however, mimicked by 75 mM KCl or 150 mM D-sorbitol addition, indicating that it is a response to the associated hyperosmotic shock and not to Mg itself. Our results confirm the functionality of MagIC as a useful tool for the long-awaited possibility of prolonged and organelle-specific monitoring of cellular Mg.

  2. Genetically Encoded Voltage Indicators in Circulation Research

    PubMed Central

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-01-01

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided. PMID:26370981

  3. Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Wang, Da; Hurst, Tamiika K.; Thompson, Richard B.; Fierke, Carol A.

    2011-08-01

    Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. One type of biosensor uses carbonic anhydrase (CA) as the recognition element based on its tunable affinity, superior metal selectivity, and fluorescence signal from aryl sulfonamide ligands coupled to zinc binding. Here, we fuse carbonic anhydrase with a red fluorescent protein to create a series of genetically-encoded Förster resonance energy transfer-based excitation ratiometric zinc sensors that exhibit large signal increases in response to alterations in physiological-free zinc concentrations. These sensors were applied to the prokaryotic model organism Escherichia coli to quantify the readily exchangeable zinc concentration. In minimal media, E. coli BL21(DE3) cells expressing the CA sensor, exhibit a median intracellular readily exchangeable zinc concentration of 20 pM, much less than the total cellular zinc concentration of ~0.2 mM. Furthermore, the intracellular readily exchangeable zinc concentration varies with the concentration of environmental zinc.

  4. Properties of genetically-encoded indicators used for cytosolic and organellar Ca2+ measurements

    PubMed Central

    Park, J. Genevieve; Palmer, Amy E.

    2016-01-01

    For the last 15 years, there has been an explosion in the development of genetically-encoded biosensors that report enzyme activity, chemical transformation, or concentration of ions and molecules in living cells. Now, there are well over 120 biosensors of different cellular targets. As a general design principle, these sensors convert a molecular event, such as the binding of a molecule to a sensing domain or a signal-induced change in protein conformation, into a change in the sensors’ fluorescence properties. In contrast to small-molecule sensors, genetically-encoded sensors are generated as cells, tissues, or organisms translate sensor-encoding nucleic acid sequences, which have been introduced by transgenic technologies. One of the best developed classes of biosensors is that of genetically-encoded Ca2+ indicators (GECIs). Here, we briefly summarize the properties of ratiometric GECIs that permit them to be used to quantify Ca2+ in specific cellular locations, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria. We also provide two protocols that describe in detail how to carry out such quantitative measurements in cultured mammalian cells. PMID:25561625

  5. Monitoring activity in neural circuits with genetically encoded indicators

    PubMed Central

    Broussard, Gerard J.; Liang, Ruqiang; Tian, Lin

    2014-01-01

    Recent developments in genetically encoded indicators of neural activity (GINAs) have greatly advanced the field of systems neuroscience. As they are encoded by DNA, GINAs can be targeted to genetically defined cellular populations. Combined with fluorescence microscopy, most notably multi-photon imaging, GINAs allow chronic simultaneous optical recordings from large populations of neurons or glial cells in awake, behaving mammals, particularly rodents. This large-scale recording of neural activity at multiple temporal and spatial scales has greatly advanced our understanding of the dynamics of neural circuitry underlying behavior—a critical first step toward understanding the complexities of brain function, such as sensorimotor integration and learning. Here, we summarize the recent development and applications of the major classes of GINAs. In particular, we take an in-depth look at the design of available GINA families with a particular focus on genetically encoded calcium indicators (GCaMPs), sensors probing synaptic activity, and genetically encoded voltage indicators. Using the family of the GCaMP as an example, we review established sensor optimization pipelines. We also discuss practical considerations for end users of GINAs about experimental methods including approaches for gene delivery, imaging system requirements, and data analysis techniques. With the growing toolbox of GINAs and with new microscopy techniques pushing beyond their current limits, the age of light can finally achieve the goal of broad and dense sampling of neuronal activity across time and brain structures to obtain a dynamic picture of brain function. PMID:25538558

  6. Photoactivatable Genetically-Encoded Calcium Indicators for targeted neuronal imaging

    PubMed Central

    Berlin, Shai; Carroll, Elizabeth C.; Newman, Zachary L.; Okada, Hitomi O.; Quinn, Carson M.; Kallman, Benjamin; Rockwell, Nathan C.; Martin, Shelley S.; Lagarias, J. Clark; Isacoff, Ehud Y.

    2015-01-01

    Circuit mapping requires knowledge of both structural and functional connectivity between cells. While optical tools have been made to assess either the morphology and projections of neurons or their activity and functional connections, few probes integrate this information. We have generated a family of photoactivatable Genetically Encoded Ca2+ Indicators (pa-GECIs) that combines attributes of high-contrast photo-labeling with high-sensitivity Ca2+ detection in a single-color, protein-sensor. We demonstrate the utility of pa-GECIs in cultured neurons and in vivo in Drosophila and zebrafish larvae. We show how single cells can be selected out of dense populations for Golgi-like visualization of morphology and high signal-to-noise measurements of activity, synaptic transmission and connectivity. Our design strategy is readily transferrable to other sensors based on circularly permutated GFP (cpGFP). PMID:26167640

  7. Calcium imaging with genetically encoded indicators in behaving primates.

    PubMed

    Seidemann, Eyal; Chen, Yuzhi; Bai, Yoon; Chen, Spencer C; Mehta, Preeti; Kajs, Bridget L; Geisler, Wilson S; Zemelman, Boris V

    2016-01-01

    Understanding the neural basis of behaviour requires studying brain activity in behaving subjects using complementary techniques that measure neural responses at multiple spatial scales, and developing computational tools for understanding the mapping between these measurements. Here we report the first results of widefield imaging of genetically encoded calcium indicator (GCaMP6f) signals from V1 of behaving macaques. This technique provides a robust readout of visual population responses at the columnar scale over multiple mm(2) and over several months. To determine the quantitative relation between the widefield GCaMP signals and the locally pooled spiking activity, we developed a computational model that sums the responses of V1 neurons characterized by prior single unit measurements. The measured tuning properties of the GCaMP signals to stimulus contrast, orientation and spatial position closely match the predictions of the model, suggesting that widefield GCaMP signals are linearly related to the summed local spiking activity. PMID:27441501

  8. Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage Indicators

    PubMed Central

    Nakajima, Ryuichi; Jung, Arong; Yoon, Bong-June; Baker, Bradley J.

    2016-01-01

    The age of genetically encoded voltage indicators (GEVIs) has matured to the point that changes in membrane potential can now be observed optically in vivo. Improving the signal size and speed of these voltage sensors has been the primary driving forces during this maturation process. As a result, there is a wide range of probes using different voltage detecting mechanisms and fluorescent reporters. As the use of these probes transitions from optically reporting membrane potential in single, cultured cells to imaging populations of cells in slice and/or in vivo, a new challenge emerges—optically resolving the different types of neuronal activity. While improvements in speed and signal size are still needed, optimizing the voltage range and the subcellular expression (i.e., soma only) of the probe are becoming more important. In this review, we will examine the ability of recently developed probes to report synaptic activity in slice and in vivo. The voltage-sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the rhodopsin family of probes are all good at reporting changes in membrane potential, but all have difficulty distinguishing subthreshold depolarizations from action potentials and detecting neuronal inhibition when imaging populations of cells. Finally, we will offer a few possible ways to improve the optical resolution of the various types of neuronal activities. PMID:27547183

  9. Calcium imaging with genetically encoded indicators in behaving primates

    PubMed Central

    Seidemann, Eyal; Chen, Yuzhi; Bai, Yoon; Chen, Spencer C; Mehta, Preeti; Kajs, Bridget L; Geisler, Wilson S; Zemelman, Boris V

    2016-01-01

    Understanding the neural basis of behaviour requires studying brain activity in behaving subjects using complementary techniques that measure neural responses at multiple spatial scales, and developing computational tools for understanding the mapping between these measurements. Here we report the first results of widefield imaging of genetically encoded calcium indicator (GCaMP6f) signals from V1 of behaving macaques. This technique provides a robust readout of visual population responses at the columnar scale over multiple mm2 and over several months. To determine the quantitative relation between the widefield GCaMP signals and the locally pooled spiking activity, we developed a computational model that sums the responses of V1 neurons characterized by prior single unit measurements. The measured tuning properties of the GCaMP signals to stimulus contrast, orientation and spatial position closely match the predictions of the model, suggesting that widefield GCaMP signals are linearly related to the summed local spiking activity. DOI: http://dx.doi.org/10.7554/eLife.16178.001 PMID:27441501

  10. pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH

    PubMed Central

    Robertson, J. Brian; Johnson, Carl Hirschie

    2012-01-01

    We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H+ specific; neither Ca++, Mg++, Na+, nor K+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate. PMID:22905204

  11. Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

    NASA Astrophysics Data System (ADS)

    Ermakova, Yulia G.; Bilan, Dmitry S.; Matlashov, Mikhail E.; Mishina, Natalia M.; Markvicheva, Ksenia N.; Subach, Oksana M.; Subach, Fedor V.; Bogeski, Ivan; Hoth, Markus; Enikolopov, Grigori; Belousov, Vsevolod V.

    2014-10-01

    Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca2+ uptake.

  12. Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

    PubMed Central

    Ermakova, Yulia G.; Bilan, Dmitry S.; Matlashov, Mikhail E.; Mishina, Natalia M.; Markvicheva, Ksenia N.; Subach, Oksana M.; Subach, Fedor V.; Bogeski, Ivan; Hoth, Markus; Enikolopov, Grigori; Belousov, Vsevolod V.

    2015-01-01

    Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca2+ uptake. PMID:25330925

  13. Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

    PubMed Central

    Wagener, Kerstin C.; Kolbrink, Benedikt; Dietrich, Katharina; Kizina, Kathrin M.; Terwitte, Lukas S.; Kempkes, Belinda; Bao, Guobin

    2016-01-01

    Abstract Aims: Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. Results: Our redox indicator mice widely express Thy1-driven roGFP1 (reduction–oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. Innovation and Conclusion: Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell- and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding

  14. A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.

    PubMed

    Kim, Sung Bae; Ozawa, Takeaki; Umezawa, Yoshio

    2016-01-01

    We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147-149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: •Cytokine activities are determined within 2 h after stimulation.•Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.•Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity. PMID:27489781

  15. Genetically encoded voltage indicators for large scale cortical imaging come of age.

    PubMed

    Knöpfel, Thomas; Gallero-Salas, Yasir; Song, Chenchen

    2015-08-01

    Electrical signals are fundamental to cellular sensing, communication and motility. In the nervous system, information is represented as receptor, synaptic and action potentials. Understanding how brain functions emerge from these electrical signals is one of the ultimate challenges in neuroscience and requires a methodology to monitor membrane voltage transients from large numbers of cells at high spatio-temporal resolution. Optical voltage imaging holds longstanding promises to achieve this, and has gained a fresh powerful momentum with the development of genetically encoded voltage indicators (GEVIs). With a focus on neuroimaging studies on intact mouse brains, we highlight recent advances in this field. PMID:26115448

  16. Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology

    PubMed Central

    Kotlikoff, Michael I

    2007-01-01

    This article reviews genetically encoded Ca2+ indicators (GECIs), with a focus on the use of these novel molecules in the context of understanding complex cell signalling in mammals, in vivo. The review focuses on the advantages and limitations of specific GECI design strategies and the results of experiments in which these molecules have been expressed in transgenic mice, concentrating particularly on recent experiments from our laboratory in which physiological signalling could be monitored in vivo. Finally, newer strategies for effective genetic specification of GECIs are briefly reviewed. PMID:17038427

  17. An expanded palette of genetically encoded Ca²⁺ indicators.

    PubMed

    Zhao, Yongxin; Araki, Satoko; Wu, Jiahui; Teramoto, Takayuki; Chang, Yu-Fen; Nakano, Masahiro; Abdelfattah, Ahmed S; Fujiwara, Manabi; Ishihara, Takeshi; Nagai, Takeharu; Campbell, Robert E

    2011-09-30

    Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging. PMID:21903779

  18. Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments

    PubMed Central

    Bilan, Dmitry S.; Matlashov, Mikhail E.; Gorokhovatsky, Andrey Yu.; Schultz, Carsten; Enikolopov, Grigori; Belousov, Vsevolod V.

    2014-01-01

    Background The ratio of NAD+/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD+/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD+/NADH are fundamentally new approach for studying the NAD+/NADH dynamics. Methods We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy. Results The sensor, named RexYFP, reports changes in the NAD+/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD+/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD+/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore. Conclusion RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments. General significance RexYFP has several advantages over existing NAD+/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging. PMID:24286672

  19. Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

    PubMed

    Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang; Johnson, Carl Hirschie

    2016-01-01

    "pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator. PMID:27424899

  20. Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS.

    PubMed

    Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A; Moore, Christina A; Vella, Stephen A; Hortua Triana, Miryam A; Liu, Jing; Garcia, Celia R S; Pace, Douglas A; Moreno, Silvia N J

    2015-11-01

    Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait. PMID:26374900

  1. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain

    PubMed Central

    Mishina, Yukiko; Mutoh, Hiroki; Song, Chenchen; Knöpfel, Thomas

    2014-01-01

    Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP) prototypical design or on the voltage-dependent state transitions of microbial opsins. We recently introduced a new VSFP design in which the voltage-sensing domain (VSD) is sandwiched between a fluorescence resonance energy transfer pair of fluorescent proteins (termed VSFP-Butterflies) and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians. PMID:25324718

  2. Imaging the Awake Visual Cortex with a Genetically Encoded Voltage Indicator

    PubMed Central

    Rossi, L. Federico; Sato, Tatsuo K.; Benucci, Andrea; Knöpfel, Thomas

    2015-01-01

    Genetically encoded voltage indicators (GEVIs) promise to reveal the membrane potential of genetically targeted neuronal populations through noninvasive, chronic imaging of large portions of cortical space. Here we test a promising GEVI in mouse cortex during wakefulness, a challenging condition due to large hemodynamic activity, and we introduce a straightforward projection method to separate a signal dominated by membrane voltage from a signal dominated by hemodynamic activity. We expressed VSFP-Butterfly 1.2 plasmid in layer 2/3 pyramidal cells of visual cortex through electroporation in utero. We then used wide-field imaging with two cameras to measure both fluorophores of the indicator in response to visual stimuli. By taking weighted sums and differences of the two measurements, we obtained clear separation of hemodynamic and voltage signals. The hemodynamic signal showed strong heartbeat oscillations, superimposed on slow dynamics similar to blood oxygen level-dependent (BOLD) or “intrinsic” signals. The voltage signal had fast dynamics similar to neural responses measured electrically, and showed an orderly retinotopic mapping. We compared this voltage signal with calcium signals imaged in transgenic mice that express a calcium indicator (GCaMP3) throughout cortex. The voltage signal from VSFP had similar signal-to-noise ratios as the calcium signal, it was more immune to vascular artifacts, and it integrated over larger regions of visual space, which was consistent with its reporting mostly subthreshold activity rather than the spiking activity revealed by calcium signals. These results demonstrate that GEVIs provide a powerful tool to study the dynamics of neural populations at mesoscopic spatial scales in the awake cortex. PMID:25568102

  3. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

    PubMed Central

    Akerboom, Jasper; Carreras Calderón, Nicole; Tian, Lin; Wabnig, Sebastian; Prigge, Matthias; Tolö, Johan; Gordus, Andrew; Orger, Michael B.; Severi, Kristen E.; Macklin, John J.; Patel, Ronak; Pulver, Stefan R.; Wardill, Trevor J.; Fischer, Elisabeth; Schüler, Christina; Chen, Tsai-Wen; Sarkisyan, Karen S.; Marvin, Jonathan S.; Bargmann, Cornelia I.; Kim, Douglas S.; Kügler, Sebastian; Lagnado, Leon; Hegemann, Peter; Gottschalk, Alexander; Schreiter, Eric R.; Looger, Loren L.

    2013-01-01

    Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics. PMID:23459413

  4. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.

    PubMed

    Gee, J Michael; Gibbons, Meredith B; Taheri, Marsa; Palumbos, Sierra; Morris, S Craig; Smeal, Roy M; Flynn, Katherine F; Economo, Michael N; Cizek, Christian G; Capecchi, Mario R; Tvrdik, Petr; Wilcox, Karen S; White, John A

    2015-01-01

    Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity

  5. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation

    PubMed Central

    Gee, J. Michael; Gibbons, Meredith B.; Taheri, Marsa; Palumbos, Sierra; Morris, S. Craig; Smeal, Roy M.; Flynn, Katherine F.; Economo, Michael N.; Cizek, Christian G.; Capecchi, Mario R.; Tvrdik, Petr; Wilcox, Karen S.; White, John A.

    2015-01-01

    Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5–14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal

  6. Imaging voltage in zebrafish as a route to characterizing a vertebrate functional connectome: promises and pitfalls of genetically encoded indicators.

    PubMed

    Kibat, Caroline; Krishnan, Seetha; Ramaswamy, Mahathi; Baker, Bradley J; Jesuthasan, Suresh

    2016-06-01

    Neural circuits are non-linear dynamical systems that transform information based on the pattern of input, current state and functional connectivity. To understand how a given stimulus is processed, one would ideally record neural activity across the entire brain of a behaving animal, at cellular or even subcellular resolution, in addition to characterizing anatomical connectivity. Given their transparency and relatively small size, larval zebrafish provide a powerful system for brain-wide monitoring of neural activity. Genetically encoded calcium indicators have been used for this purpose, but cannot directly report hyperpolarization or sub-threshold activity. Voltage indicators, in contrast, have this capability. Here, we test whether two different genetically encoded voltage reporters, ASAP1 and Bongwoori, can be expressed and report activity in the zebrafish brain, using widefield, two-photon and light sheet microscopy. We were unable to express ASAP1 in neurons. Bongwoori, in contrast expressed well, and because of its membrane localization, allowed visualization of axon trajectories in 3D. Bongwoori displayed stimulus-evoked changes in fluorescence, which could be detected in single trials. However, under high laser illumination, puncta on neural membranes underwent spontaneous fluctuations in intensity, suggesting that the probe is susceptible to blinking artefacts. These data indicate that larval zebrafish can be used to image electrical activity in the brain of an intact vertebrate at high resolution, although care is needed in imaging and analysis. Recording activity across the whole brain will benefit from further developments in imaging hardware and indicators. PMID:27328843

  7. Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

    SciTech Connect

    Rodríguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Akerboom, Jasper; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

    2008-07-01

    The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.

  8. Comparison of genetically encoded calcium indicators for monitoring action potentials in mammalian brain by two-photon excitation fluorescence microscopy

    PubMed Central

    Podor, Borbala; Hu, Yi-ling; Ohkura, Masamichi; Nakai, Junichi; Croll, Roger; Fine, Alan

    2015-01-01

    Abstract. Imaging calcium transients associated with neuronal activity has yielded important insights into neural physiology. Genetically encoded calcium indicators (GECIs) offer conspicuous potential advantages for this purpose, including exquisite targeting. While the catalogue of available GECIs is steadily growing, many newly developed sensors that appear promising in vitro or in model cells appear to be less useful when expressed in mammalian neurons. We have, therefore, evaluated the performance of GECIs from two of the most promising families of sensors, G-CaMPs [Nat. Biotechnol. 19(2), 137–141 (2001)11175727] and GECOs [Science 333(6051), 1888–1891 (2011)21903779], for monitoring action potentials in rat brain. Specifically, we used two-photon excitation fluorescence microscopy to compare calcium transients detected by G-CaMP3; GCaMP6f; G-CaMP7; Green-GECO1.0, 1.1 and 1.2; Blue-GECO; Red-GECO; Rex-GECO0.9; Rex-GECO1; Carmine-GECO; Orange-GECO; and Yellow-GECO1s. After optimizing excitation wavelengths, we monitored fluorescence signals associated with increasing numbers of action potentials evoked by current injection in CA1 pyramidal neurons in rat organotypic hippocampal slices. Some GECIs, particularly Green-GECO1.2, GCaMP6f, and G-CaMP7, were able to detect single action potentials with high reliability. By virtue of greatest sensitivity and fast kinetics, G-CaMP7 may be the best currently available GECI for monitoring calcium transients in mammalian neurons. PMID:26158004

  9. Enhanced ratiometric fluorescent indicators for magnesium based on azoles of the heavier chalcogens.

    PubMed

    Afzal, Mohammad S; Pitteloud, Jean-Philippe; Buccella, Daniela

    2014-10-01

    Red-shifted fluorescent indicators for magnesium were developed by incorporation of sulfur or selenium in the azole moiety of 'fura' fluorophores. Single atom replacement in the acceptor of these ITC probes affords longer excitation and emission wavelengths as well as greater separation between excitation bands, valuable for ratiometric intracellular Mg(2+) imaging. PMID:25164869

  10. Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

    PubMed

    Huynh Nhat, Kim Phuong; Watanabe, Takayoshi; Yoshikoshi, Kensuke; Hohsaka, Takahiro

    2016-08-01

    Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules. PMID:26896314

  11. Validation of optical voltage reporting by the genetically encoded voltage indicator VSFP-Butterfly from cortical layer 2/3 pyramidal neurons in mouse brain slices

    PubMed Central

    Empson, Ruth M; Goulton, Chelsea; Scholtz, David; Gallero-Salas, Yasir; Zeng, Hongkui; Knöpfel, Thomas

    2015-01-01

    Understanding how behavior emerges from brain electrical activity is one of the ultimate goals of neuroscience. To achieve this goal we require methods for large-scale recording of the electrical activity of specific neuronal circuits. A very promising approach is to use optical reporting of membrane voltage transients, particularly if the voltage reporter is genetically targeted to specific neuronal populations. Targeting in this way allows population signals to be recorded and interpreted without blindness to neuronal diversity. Here, we evaluated the voltage-sensitive fluorescent protein, VSFP Butterfly 2.1, a genetically encoded voltage indicator (GEVI), for monitoring electrical activity of layer 2/3 cortical pyramidal neurons in mouse brain slices. Standard widefield fluorescence and two-photon imaging revealed robust, high signal-to-noise ratio read-outs of membrane voltage transients that are predominantly synaptic in nature and can be resolved as discrete areas of synaptically connected layer 2/3 neurons. We find that targeted expression of this GEVI in the cortex provides a flexible and promising tool for the analysis of L2/3 cortical network function. PMID:26229003

  12. Dual optical recordings for action potentials and calcium handling in induced pluripotent stem cell models of cardiac arrhythmias using genetically encoded fluorescent indicators.

    PubMed

    Song, LouJin; Awari, Daniel W; Han, Elizabeth Y; Uche-Anya, Eugenia; Park, Seon-Hye E; Yabe, Yoko A; Chung, Wendy K; Yazawa, Masayuki

    2015-05-01

    Reprogramming of human somatic cells to pluripotency has been used to investigate disease mechanisms and to identify potential therapeutics. However, the methods used for reprogramming, in vitro differentiation, and phenotyping are still complicated, expensive, and time-consuming. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free induced pluripotent stem cells (iPSCs) from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from patients with Timothy syndrome into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared with the controls. The results are consistent with previous reports using a retroviral method for reprogramming and an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording the action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and that these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to investigate mechanisms underlying cardiac arrhythmias and to test potential therapeutics. PMID:25769651

  13. ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions.

    PubMed

    Roussakis, Emmanuel; Voutsadaki, Styliani; Pinakoulaki, Eftychia; Sideris, Dionisia P; Tokatlidis, Kostas; Katerinopoulos, Haralambos E

    2008-09-01

    A new fluorescent Zn2+ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn2+ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3',2':3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn2+ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn2+ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn2+ probe. PMID:18243303

  14. Fluorescent Ratiometric Indicators Based on Cu(II)-Induced Changes in Poly(NIPAM) Microparticle Volume

    PubMed Central

    Osambo, John; Seitz, W. Rudolf; Kennedy, Daniel P.; Planalp, Roy P.; Jones, Aaron M.; Jackson, Randy K.; Burdette, Shawn

    2013-01-01

    Microparticles consisting of the thermal responsive polymer N-isopropyl acrylamide (polyNIPAM), a metal ion-binding ligand and a fluorophore pair that undergoes fluorescence resonance energy transfer (FRET) have been prepared and characterized. Upon the addition of Cu(II), the microparticles swell or contract depending on whether charge is introduced or neutralized on the polymer backbone. The variation in microparticle morphology is translated into changes in emission of each fluorophore in the FRET pair. By measuring the emission intensity ratio between the FRET pair upon Cu(II) addition, the concentration of metal ion in solution can be quantified. This ratiometric fluorescent indicator is the newest technique in an ongoing effort to use emission spectroscopy to monitor Cu(II) thermodynamic activity in environmental water samples. PMID:23337337

  15. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

    PubMed Central

    Benčina, Mojca

    2013-01-01

    Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments. PMID:24316570

  16. Engineering Genetically Encoded FRET Sensors

    PubMed Central

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  17. A near-infrared ratiometric fluorescent probe for cysteine detection over glutathione indicating mitochondrial oxidative stress in vivo.

    PubMed

    Yin, Kun; Yu, Fabiao; Zhang, Weiwei; Chen, Lingxin

    2015-12-15

    We establish a near-infrared (NIR) ratiometric fluorescent probe Cy-NB for the selective detection of cysteine (Cys) over glutathione (GSH) and homocysteine (Hcy) in mitochondria to indicate oxidative stress. Heptamethine cyanine dye is chosen as the fluorophore of Cy-NB whose emission locates in NIR region. And p-nitrobenzoyl is employed as the fluorescent modulator due to its capability of selective-Cys response. Once triggered by Cys, the uncaged p-nitrobenzoyl rearranges the polymethine π-electron system of the fluorophore, which leads to a remarkable spectrum shifts in absorption and emission profiles. Taking advantage of these spectroscopic properties, we construct a ratiometric fluorescent signal for the detection of Cys with a detection limit of 0.2 µM within 5 min. Our probe Cy-NB can sensitively detect the mitochondrial Cys pool changes under different oxidative stress status in HepG2 cells. We also successfully employ Cy-NB to imaging Cys level changes in living mice. It suggests that mitochondrial Cys can be used as an oxidative stress biomarker with simple potential clinical applications. And our probe Cy-NB is of great potential for further utilizing in exploring the physiological function of Cys in biological systems. PMID:26141101

  18. Toward Better Genetically Encoded Sensors of Membrane Potential.

    PubMed

    Storace, Douglas; Sepehri Rad, Masoud; Kang, BokEum; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J

    2016-05-01

    Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions. Optical signals of membrane potential are useful because membrane potential changes are a direct sign of both synaptic and action potentials. Here we describe recent improvements in the in vitro and in vivo signal size and kinetics of genetically encoded voltage indicators (GEVIs) and discuss their relationship to alternative sensors of neural activity. PMID:27130905

  19. Imaging Cellular Inorganic Phosphate in Caenorhabditis elegans Using a Genetically Encoded FRET-Based Biosensor

    PubMed Central

    Banerjee, Swayoma; Versaw, Wayne K.; Garcia, L. Rene

    2015-01-01

    Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal. PMID:26484766

  20. A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging

    PubMed Central

    Wu, Jiahui; Abdelfattah, Ahmed S.; Miraucourt, Loïs S.; Kutsarova, Elena; Ruangkittisakul, Araya; Zhou, Hang; Ballanyi, Klaus; Wicks, Geoffrey; Drobizhev, Mikhail; Rebane, Aleksander; Ruthazer, Edward S.; Campbell, Robert E.

    2016-01-01

    The introduction of calcium ion (Ca2+) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca2+ dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650–1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca2+ indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca2+ indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca2+ and glutamate imaging in organotypic cultures. PMID:25358432

  1. Glutamine Flux Imaging Using Genetically Encoded Sensors

    PubMed Central

    Besnard, Julien; Okumoto, Sakiko

    2014-01-01

    Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories. One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed. PMID:25146898

  2. Design and application of genetically encoded biosensors

    PubMed Central

    Palmer, Amy E.; Qin, Yan; Park, Jungwon Genevieve; McCombs, Janet E.

    2012-01-01

    In the past 5–10 years, the power of the green fluorescent protein (GFP) and its numerous derivatives has been harnessed toward the development of genetically encoded fluorescent biosensors. These sensors are incorporated into cells or organisms as plasmid DNA, which leads the transcriptional and translational machinery of the cell to express a functional sensor. To date, over 100 different genetically encoded biosensors have been developed for targets as diverse as ions, molecules and enzymes. Such sensors are instrumental in providing a window into the real-time biochemistry of living cells and whole organisms, and are providing unprecedented insight into the inner workings of a cell. PMID:21251723

  3. Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon

    PubMed Central

    Hennig, Grant W.; Gould, Thomas W.; Koh, Sang Don; Corrigan, Robert D.; Heredia, Dante J.; Shonnard, Matthew C.; Smith, Terence K.

    2015-01-01

    Genetically encoded Ca2+ indicators (GECIs) have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100–1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity

  4. Genetically encoded fluorescent sensors of membrane potential

    PubMed Central

    Baker, B. J.; Mutoh, H.; Dimitrov, D.; Akemann, W.; Perron, A.; Iwamoto, Y.; Jin, L.; Cohen, L. B.; Isacoff, E. Y.; Pieribone, V. A.; Hughes, T.; Knöpfel, T.

    2009-01-01

    Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments. PMID:18679801

  5. Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments.

    PubMed

    Waseem, Tatyana; Mukhtarov, Marat; Buldakova, Svetlana; Medina, Igor; Bregestovski, Piotr

    2010-10-30

    Chloride (Cl) participates in a variety of physiological functions. To study processes connected with Cl homeostasis we need effective and quantitative probes allowing measurements of intracellular Cl concentration ([Cl(-)](i)) in different cell types, particularly in specialized small cellular compartments such as dendrites and dendritic spines. Of the different tools proposed for monitoring [Cl(-)](i), the genetically encoded Cl-sensitive indicators are the most promising. Recently, a ratiometric CFP-YFP based construct, termed "Cl-Sensor", with a relatively high sensitivity to Cl has been proposed (Markova et al., 2008). In the present study, we have developed conditions for the efficient expression of Cl-Sensor in tiny neuronal compartments including distal dendrites and spines. We also propose a new approach for the calibration of intracellularly expressed probes using a natural triterpenoid saponin, β-escin. We have mapped [Cl(-)](i) distribution in different neuronal compartments of cultured hippocampal and spinal cord neurons. The maximum Cl concentration was observed in the soma and it had a tendency to decrease gradually along dendritic branches, reaching minimum values in thin distal dendrites. We have also monitored transient increases in intracellular Cl in dendritic spines caused by glutamate application. These results demonstrate that Cl-Sensor enables non-invasive monitoring of the [Cl(-)](i) distribution in different types of neurons with variable morphology. This probe represents an effective tool for the quantitative estimation of [Cl(-)](i) in various cellular compartments including dendritic spines. PMID:20705097

  6. A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system

    PubMed Central

    Raimondo, Joseph V.; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E.; Srinivas, Shankar; Akerman, Colin J.

    2013-01-01

    Within the nervous system, intracellular Cl− and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl− and pH are often co-regulated, and network activity results in the movement of both Cl− and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl− and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN—a new genetically-encoded ratiometric Cl− and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl− and H+ concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons. PMID:24312004

  7. Probing the endocytic pathways of the filamentous bacteriophage in live cells using ratiometric pH fluorescent indicator.

    PubMed

    Tian, Ye; Wu, Man; Liu, Xiangxiang; Liu, Zhi; Zhou, Quan; Niu, Zhongwei; Huang, Yong

    2015-02-18

    Viral nanoparticles have attracted extensive research interests in diverse applications of diagnosis and therapy. In particular, filamentous M13 bacteriophages have shown great potential in biomedical applications. However, its pathways entering into cells still remain unclear, and this greatly hinders its further use as a drug or gene carrier. Here, a ratiometric M13 pH probe is designed by conjugating two fluorescent dyes onto the surface of M13. Since the intensity ratio is not influenced by probe concentration, ion strength, temperature, photobleaching, and optical path length, this ratiometric probe can be used to investigate the intracellular pH map of M13. More importantly, the internalization mechanism of M13 can be elucidated. It is found that this filamentous phage shows great cell-type dependence in interaction with cells and internalization mechanism. The phage tends to be bounded on the cell membrane of only epithelial cells, not endothelial cells. Furthermore, the M13 phage enters into cells through endocytosis with specific mechanism: clathrin-mediated endocytosis and macropinocytosis for HeLa; vesicular transport, clathrin-mediated endocytosis, and macropinocytosis for MCF-7; caveolae-mediated endocytosis for human dermal microvascular endothelial cell (HDMEC). This work provides key notes for cancer diagnosis and therapy based on filamentous bacteriophage, especially for design of pH-sensitive drug delivery systems. PMID:25308797

  8. Measuring the in situs Kd of a genetically-encoded Ca2+ sensor

    PubMed Central

    Park, J. Genevieve; Palmer, Amy E.

    2016-01-01

    The use of genetically-encoded Ca2+ sensors (GECIs) for long-term monitoring of intracellular Ca2+ has become increasingly common in the last decade. Emission-ratiometric GECIs, such as those in the yellow cameleon family, are capable of making quantitative measurements, meaning that their fluorescence signals can be converted to free Ca2+ concentrations ([Ca2+]free). This conversion is only as accurate as the sensor’s apparent dissociation constant for Ca2+ (Kd’), which depends on temperature, pH, and salt concentration. This protocol describes a method for performing a titration, in living cells (in situ), of cytosolic, nuclear, or mitochondrial sensors. An excellent example of calibration of an ER-targeted sensor is presented elsewhere (Rudolf et al. 2006). PMID:25561615

  9. A genetically encoded, high-signal-to-noise maltose sensor

    PubMed Central

    Marvin, Jonathan S; Schreiter, Eric R; Echevarría, Ileabett M; Looger, Loren L

    2011-01-01

    We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes. PMID:21989929

  10. A genetically encoded, high-signal-to-noise maltose sensor

    SciTech Connect

    Marvin, Jonathan S.; Schreiter, Eric R.; Echevarría, Ileabett M.; Looger, Loren L.

    2012-10-23

    We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.

  11. MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor

    PubMed Central

    Oortwijn, Jorn; Aper, Stijn J. A.; Merkx, Maarten

    2013-01-01

    Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg2+, MagFRET-1. This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (Kd = 148 µM) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (Kd of 10 µM) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling. PMID:24312622

  12. Protocol 1: Verifying the function and localization of genetically-encoded Ca2+ sensors and converting FRET ratios to Ca2+ concentrations

    PubMed Central

    Park, J. Genevieve; Palmer, Amy E.

    2016-01-01

    Genetically-encoded, ratiometric, fluorescent, Ca2+ biosensors can be used in living cells to quantitatively measure free Ca2+ concentrations in the cytosol or in organelles. This protocol describes how to perform a calibration of a Ca2+ sensor expressed in cultured mammalian cells as images are acquired using a widefield fluorescence microscope. This protocol also explains how to calculate Förster Resonance Energy Transfer (FRET) ratios from acquired images and how to convert FRET ratios to Ca2+ concentrations. PMID:25561614

  13. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

    PubMed

    Hanna, David A; Harvey, Raven M; Martinez-Guzman, Osiris; Yuan, Xiaojing; Chandrasekharan, Bindu; Raju, Gheevarghese; Outten, F Wayne; Hamza, Iqbal; Reddi, Amit R

    2016-07-01

    Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines. PMID:27247412

  14. Genetically encoded optical activation of DNA recombination in human cells.

    PubMed

    Luo, J; Arbely, E; Zhang, J; Chou, C; Uprety, R; Chin, J W; Deiters, A

    2016-06-30

    We developed two tightly regulated, light-activated Cre recombinase enzymes through site-specific incorporation of two genetically-encoded photocaged amino acids in human cells. Excellent optical off to on switching of DNA recombination was achieved. Furthermore, we demonstrated precise spatial control of Cre recombinase through patterned illumination. PMID:27277957

  15. A genetically encoded metabolite sensor for malonyl-CoA

    PubMed Central

    Ellis, Jessica M.; Wolfgang, Michael J.

    2012-01-01

    Summary Malonyl-CoA is the rate determining metabolite for long chain de novo fatty acid synthesis and allosterically inhibits the rate-setting step in long chain fatty acid β-oxidation. We have developed a cell-based genetically encoded biosensor based on the malonyl-CoA responsive Bacillus subtilis transcriptional repressor, FapR, for living mammalian cells. Here we show that fluctuations in malonyl-CoA, in mammalian cells, can regulate the transcription of a FapR-based malonyl-CoA biosensor. The biosensor reflects changes in malonyl-CoA flux regulated by malonyl-CoA decarboxylase and AMP-Activated Protein Kinase in a concentration-dependent manner. To gain further insight into the regulatory mechanisms that effect fatty acid metabolism, we utilized the malonyl-CoA sensor to screen and identify several novel kinases. LIMK1 was identified and its expression was shown to alter both fatty acid synthesis and oxidation rates. This simple genetically encoded biosensor can be used to study the metabolic properties of live mammalian cells and enable screens for novel metabolic regulators. PMID:23102226

  16. Extraordinarily Adaptive Properties of the Genetically Encoded Amino Acids

    NASA Astrophysics Data System (ADS)

    Ilardo, Melissa; Meringer, Markus; Freeland, Stephen; Rasulev, Bakhtiyor; Cleaves, H. James, II

    2015-03-01

    Using novel advances in computational chemistry, we demonstrate that the set of 20 genetically encoded amino acids, used nearly universally to construct all coded terrestrial proteins, has been highly influenced by natural selection. We defined an adaptive set of amino acids as one whose members thoroughly cover relevant physico-chemical properties, or ``chemistry space.'' Using this metric, we compared the encoded amino acid alphabet to random sets of amino acids. These random sets were drawn from a computationally generated compound library containing 1913 alternative amino acids that lie within the molecular weight range of the encoded amino acids. Sets that cover chemistry space better than the genetically encoded alphabet are extremely rare and energetically costly. Further analysis of more adaptive sets reveals common features and anomalies, and we explore their implications for synthetic biology. We present these computations as evidence that the set of 20 amino acids found within the standard genetic code is the result of considerable natural selection. The amino acids used for constructing coded proteins may represent a largely global optimum, such that any aqueous biochemistry would use a very similar set.

  17. Extraordinarily Adaptive Properties of the Genetically Encoded Amino Acids

    PubMed Central

    Ilardo, Melissa; Meringer, Markus; Freeland, Stephen; Rasulev, Bakhtiyor; Cleaves II, H. James

    2015-01-01

    Using novel advances in computational chemistry, we demonstrate that the set of 20 genetically encoded amino acids, used nearly universally to construct all coded terrestrial proteins, has been highly influenced by natural selection. We defined an adaptive set of amino acids as one whose members thoroughly cover relevant physico-chemical properties, or “chemistry space.” Using this metric, we compared the encoded amino acid alphabet to random sets of amino acids. These random sets were drawn from a computationally generated compound library containing 1913 alternative amino acids that lie within the molecular weight range of the encoded amino acids. Sets that cover chemistry space better than the genetically encoded alphabet are extremely rare and energetically costly. Further analysis of more adaptive sets reveals common features and anomalies, and we explore their implications for synthetic biology. We present these computations as evidence that the set of 20 amino acids found within the standard genetic code is the result of considerable natural selection. The amino acids used for constructing coded proteins may represent a largely global optimum, such that any aqueous biochemistry would use a very similar set. PMID:25802223

  18. Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids.

    PubMed

    Hu, C; Wang, J

    2016-01-01

    We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First, we considered the unnatural amino acids and the protein scaffold separately. The scaffold is designed by traditional protein design methods. The unnatural amino acids are inspired by natural structure and organic chemistry methods, and synthesized by either organic chemistry methods or enzymatic conversion. With the increasing number of published unnatural amino acids with various functions, we described an unnatural amino acids toolkit containing metal chelators, redox mediators, and click chemistry reagents. These efforts enable a researcher to search the toolkit for appropriate unnatural amino acids for the study, rather than design and synthesize the unnatural amino acids from the beginning. After the first step, the model enzyme was optimized by computational methods and directed evolution. Lastly, we describe a general method for evolving aminoacyl-tRNA synthetase and expressing unnatural amino acids incorporated into a protein. PMID:27586330

  19. Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses

    PubMed Central

    Jackson, Rachel E.; Burrone, Juan

    2016-01-01

    Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses. PMID:27507942

  20. Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses.

    PubMed

    Jackson, Rachel E; Burrone, Juan

    2016-01-01

    Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses. PMID:27507942

  1. Cyclic Peptides Made by Linking Synthetic and Genetically Encoded Fragments.

    PubMed

    Palei, Shubhendu; Mootz, Henning D

    2016-03-01

    Cyclic peptides can be highly valuable as bioactive molecules, both for biomedical applications and in basic research. We introduce a new fragment-based approach to access cyclic peptide structures in which one fragment is of synthetic origin and the other is genetically encoded. The synthetic peptide, which can contain one or more non-proteinogenic building blocks, is coupled to the recombinantly expressed peptide through two bonds, one formed by protein trans-splicing with a split intein and the other by oxime ligation. Semisynthetic macrocycles were obtained with high efficiency for various sequences and ring sizes; they can be prepared in quantities sufficient for initial bioactivity tests. We also prepared lipidated and d-amino-acid-containing peptides that were inspired by the peptide antibiotic daptomycin. Such structures are not accessible by other methods that harness the power of simple genetic diversification in the DNA-encoded part of the peptide. PMID:26691013

  2. Genetically encoded force sensors for measuring mechanical forces in proteins

    PubMed Central

    Wang, Yuexiu; Sachs, Frederick

    2011-01-01

    There are three sources of free energy for cells: chemical potential, electrical potential and mechanical potential. There is little known about the last one since there have not been simple ways to measure stress in proteins in cells. we have now developed genetically encoded force sensors to assess the stress in fibrous proteins in living cells. These FReT based fluorescence sensors can be read out at video rates and provide real time maps of the stress distribution in cells, tissues and animals. The sensors can be inserted into specific proteins and in general do not disturb the normal function or anatomy. The original sensors used mutant GFPs linked by elastic linkers. These sensors provide a linear output with applied stress but the response is linear in strain. To improve contrast and dynamic range we have now developed a new class of sensors that are smaller making them less invasive, and have much higher intrinsic sensitivity since force modulates the angle between the donor and acceptor much more than the distance between them. Known as cpstFRET, the probe shows improved biocompatibility, wider dynamic range and higher sensitivity. PMID:21966553

  3. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    PubMed Central

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.

    2015-01-01

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  4. Genetically encoded sensors of protein hydrodynamics and molecular proximity

    PubMed Central

    Hoepker, Alexander C.; Wang, Ariel; Le Marois, Alix; Suhling, Klaus; Yan, Yuling; Marriott, Gerard

    2015-01-01

    The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1′-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin. PMID:25931526

  5. KillerRed and miniSOG as genetically encoded photosensitizers for photodynamic therapy of cancer

    NASA Astrophysics Data System (ADS)

    Shirmanova, Marina V.; Serebrovskaya, Ekaterina O.; Snopova, Ludmila B.; Kuznetsova, Maria M.; Ryumina, Alina P.; Turchin, Ilya V.; Sergeeva, Ekaterina A.; Ignatova, Nadezhda I.; Klementieva, Natalia V.; Lukyanov, Konstantin A.; Lukyanov, Sergey A.; Zagaynova, Elena V.

    2013-06-01

    Despite of the success of photodynamic therapy (PDT) in cancer treatment, the problems of low selective accumulation of a photosensitizer in a tumor and skin phototoxicity have not resolved yet. The idea of encoding of a photosensitizer in genome of cancer cells is attractive, particularly because it can provide highly selective light induced cell killing. This work is aimed at the development of new approach to PDT of cancer, namely to using genetically encoded photosensitizers. A phototoxicity of red fluorescent GFP-like protein KillerRed and FMN-binding protein miniSOG was investigated on HeLa tumor xenografts in nude mice. The tumors were generated by subcutaneous injection of HeLa cells stably expressing the phototoxic proteins. The tumors were irradiated with 594 nm or 473 nm laser at 150 mW/cm2 for 20 or 30 min, repeatedly. Fluorescence intensity of the tumors was measured in vivo before and after each treatment procedure. Detailed pathomorphological analysis was performed 24 h after the therapy. On the epi-fluorescence images in vivo photobleaching of both proteins was observed indicating photodynamic reaction. Substantial pathomorphological abnormalities were found in the treated KillerRed-expressing tumor tissue, such as vacuolization of cytoplasm, cellular and nuclear membrane destruction, activation of apoptosis. In contrast, miniSOG-expressing tumors displayed no reaction to PDT, presumably due to the lack of FMN cofactor needed for fluorescence recovery of the flavoprotein. The results are of interest for photodynamic therapy as a proof of possibility to induce photodamages in cancer cells in vivo using genetically encoded photosensitizers.

  6. Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

    PubMed Central

    Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A.

    2013-01-01

    Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. PMID:23734096

  7. Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

    PubMed Central

    Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

    2012-01-01

    Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots. PMID:22407646

  8. Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling.

    PubMed

    Sidik, Saima M; Hortua Triana, Miryam A; Paul, Aditya S; El Bakkouri, Majida; Hackett, Caroline G; Tran, Fanny; Westwood, Nicholas J; Hui, Raymond; Zuercher, William J; Duraisingh, Manoj T; Moreno, Silvia N J; Lourido, Sebastian

    2016-04-29

    The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition. PMID:26933036

  9. Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

    PubMed

    Lee, Sungmoo; Piao, Hong Hua; Sepheri-Rad, Masoud; Jung, Arong; Sung, Uhna; Song, Yoon-Kyu; Baker, Bradley J

    2016-01-01

    Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera. PMID:26890551

  10. Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling*

    PubMed Central

    Sidik, Saima M.; Hortua Triana, Miryam A.; Paul, Aditya S.; El Bakkouri, Majida; Hackett, Caroline G.; Tran, Fanny; Westwood, Nicholas J.; Hui, Raymond; Zuercher, William J.; Duraisingh, Manoj T.; Moreno, Silvia N. J.; Lourido, Sebastian

    2016-01-01

    The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition. PMID:26933036

  11. Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

    PubMed Central

    Shinnawi, Rami; Huber, Irit; Maizels, Leonid; Shaheen, Naim; Gepstein, Amira; Arbel, Gil; Tijsen, Anke J.; Gepstein, Lior

    2015-01-01

    Summary The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing. PMID:26372632

  12. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    PubMed Central

    Leyton-Mange, Jordan S.; Mills, Robert W.; Macri, Vincenzo S.; Jang, Min Young; Butte, Faraz N.; Ellinor, Patrick T.; Milan, David J.

    2014-01-01

    Summary In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  13. Rapid cellular phenotyping of human pluripotent stem cell-derived cardiomyocytes using a genetically encoded fluorescent voltage sensor.

    PubMed

    Leyton-Mange, Jordan S; Mills, Robert W; Macri, Vincenzo S; Jang, Min Young; Butte, Faraz N; Ellinor, Patrick T; Milan, David J

    2014-02-11

    In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  14. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

    NASA Astrophysics Data System (ADS)

    Okumoto, Sakiko; Looger, Loren L.; Micheva, Kristina D.; Reimer, Richard J.; Smith, Stephen J.; Frommer, Wolf B.

    2005-06-01

    Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery. aspartate | hippocampal neuron | neurotransmitter | secretion | transport

  15. Genetically encoded sensors enable real-time observation of metabolite production

    PubMed Central

    Rogers, Jameson K.; Church, George M.

    2016-01-01

    Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules. PMID:26858408

  16. Genetically encoded sensors enable real-time observation of metabolite production.

    PubMed

    Rogers, Jameson K; Church, George M

    2016-03-01

    Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules. PMID:26858408

  17. Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors.

    PubMed

    Hessels, Anne M; Taylor, Kathryn M; Merkx, Maarten

    2016-02-01

    The Zn(2+)-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn(2+) from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn(2+) release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn(2+) concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn(2+), eZinCh-2 (Kd = 1 nM at pH 7.1) and eCALWY-4 (Kd = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn(2+) and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn(2+) in both cytosol and ER, suggesting that Zn(2+) was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn(2+) levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn(2+), nor in experiments in which cytosolic and ER Zn(2+) were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF-ionomycin treatment does not result in significant changes in cytosolic Zn(2+) levels as a result from Zn(2+) release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn(2+) dyes. PMID:26739447

  18. A genetically-encoded photoactivatable Rac controls the motility of living cells

    PubMed Central

    Wu, Yi I.; Frey, Daniel; Lungu, Oana I.; Jaehrig, Angelika; Schlichting, Ilme; Kuhlman, Brian; Hahn, Klaus M.

    2009-01-01

    The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties1 or using photoreactive small molecule ligands2. However, this requires use of toxic UV wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (i.e. through microinjection). We have developed a new approach to produce genetically-encoded photo-activatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics3,4. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin5,6, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458 or 473 nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, while PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicron precision7,8. Their mutual regulation remains controversial9, with data indicating that Rac inhibits and/or activates Rho10,11. Rac was shown to inhibit RhoA in living cells, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins. PMID:19693014

  19. Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells.

    PubMed

    Hoppmann, Christian; Lacey, Vanessa K; Louie, Gordon V; Wei, Jing; Noel, Joseph P; Wang, Lei

    2014-04-01

    The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation. PMID:24615769

  20. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  1. Long Wavelength Fluorescence Ratiometric Zinc Biosensor

    PubMed Central

    Zeng, Hui Hui; Matveeva, Evgenia; Stoddard, Andrea K.; Fierke, Carol A.; Thompson, Richard B.

    2013-01-01

    A protein-based emission ratiometric fluorescence biosensor is described that exhibits sensitivity to free zinc ion solutions down to picomolar concentrations. Ratiometric measurements are widely used to assure accurate quantitation, and emission ratios are preferred for laser scanning microscopes such as confocal fluorescence microscopes. The relatively long emission wavelengths used are well suited to studies in tissues and other matrices which exhibit significant fluorescence background, and the apo-carbonic anhydrase moiety recognizes zinc ion with high and controllable specificity. PMID:23345045

  2. Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging

    PubMed Central

    Bhargava, Yogesh; Hampden-Smith, Kathryn; Chachlaki, Konstantina; Wood, Katherine C.; Vernon, Jeffrey; Allerston, Charles K.; Batchelor, Andrew M.; Garthwaite, John

    2013-01-01

    Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations. PMID:24068983

  3. Dual-emitting nanocomposites derived from rare-earth compound nanotubes for ratiometric fluorescence sensing applications.

    PubMed

    Sun, Tian-Ying; Zhang, Da-Quan; Yu, Xue-Feng; Xiang, Yang; Luo, Min; Wang, Jia-Hong; Tan, Guo-Long; Wang, Qu-Quan; Chu, Paul K

    2013-02-21

    A new class of ratiometric fluorescence sensors composed of rare-earth (RE) compound nanotubes is described. Polyethylenimine-coated yttrium hydroxide fluoride nanotubes (YHF NTs) that were synthesized hydrothermally exhibit highly efficient fluorescence when doped with RE ions. The polyethylenimine on the NTs facilitates the incorporation of phosphors such as quantum dots or organic dyes onto the NT surface to produce dual-emitting nanocomposites which are excellent ratiometric fluorescence sensors. The phosphor layer and underlying tubes in the nanocomposites act as the indicator and reference probes, respectively. This ratiometric fluorescence method which can be applied to the detection of heavy metals in solutions, temperature sensing, and pH sensing boasts high sensitivity and selectivity as well as better accuracy than traditional intensity-based fluorescence methods. PMID:23334204

  4. Dual-emitting nanocomposites derived from rare-earth compound nanotubes for ratiometric fluorescence sensing applications

    NASA Astrophysics Data System (ADS)

    Sun, Tian-Ying; Zhang, Da-Quan; Yu, Xue-Feng; Xiang, Yang; Luo, Min; Wang, Jia-Hong; Tan, Guo-Long; Wang, Qu-Quan; Chu, Paul K.

    2013-01-01

    A new class of ratiometric fluorescence sensors composed of rare-earth (RE) compound nanotubes is described. Polyethylenimine-coated yttrium hydroxide fluoride nanotubes (YHF NTs) that were synthesized hydrothermally exhibit highly efficient fluorescence when doped with RE ions. The polyethylenimine on the NTs facilitates the incorporation of phosphors such as quantum dots or organic dyes onto the NT surface to produce dual-emitting nanocomposites which are excellent ratiometric fluorescence sensors. The phosphor layer and underlying tubes in the nanocomposites act as the indicator and reference probes, respectively. This ratiometric fluorescence method which can be applied to the detection of heavy metals in solutions, temperature sensing, and pH sensing boasts high sensitivity and selectivity as well as better accuracy than traditional intensity-based fluorescence methods.A new class of ratiometric fluorescence sensors composed of rare-earth (RE) compound nanotubes is described. Polyethylenimine-coated yttrium hydroxide fluoride nanotubes (YHF NTs) that were synthesized hydrothermally exhibit highly efficient fluorescence when doped with RE ions. The polyethylenimine on the NTs facilitates the incorporation of phosphors such as quantum dots or organic dyes onto the NT surface to produce dual-emitting nanocomposites which are excellent ratiometric fluorescence sensors. The phosphor layer and underlying tubes in the nanocomposites act as the indicator and reference probes, respectively. This ratiometric fluorescence method which can be applied to the detection of heavy metals in solutions, temperature sensing, and pH sensing boasts high sensitivity and selectivity as well as better accuracy than traditional intensity-based fluorescence methods. Electronic supplementary information (ESI) available: EA and TGA profiles, emission spectra of YHF:Eu NTs. See DOI: 10.1039/c2nr33217e

  5. Synthesis of Non-linear Protein Dimers through a Genetically Encoded Thiol-ene Reaction

    PubMed Central

    Torres-Kolbus, Jessica; Chou, Chungjung; Liu, Jihe; Deiters, Alexander

    2014-01-01

    Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylTCUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins. PMID:25181502

  6. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light

    PubMed Central

    Bozhanova, Nina G.; Sharonov, George V.; Staroverov, Dmitriy B.; Egorov, Evgeny S.; Ryabova, Anastasia V.; Solntsev, Kyril M.; Mishin, Alexander S.; Lukyanov, Konstantin A.

    2015-01-01

    Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity. PMID:26679300

  7. A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors

    PubMed Central

    2011-01-01

    Background Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility. Results To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library. Conclusion Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%). PMID:22074568

  8. Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane

    PubMed Central

    Jinno, Yuka; Shoda, Keiko; Rial-Verde, Emiliano; Yuste, Rafael; Miyawaki, Atsushi; Tsutsui, Hidekazu

    2014-01-01

    Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its β-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential. PMID:25505870

  9. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles.

    PubMed

    Stanley, Sarah A; Sauer, Jeremy; Kane, Ravi S; Dordick, Jonathan S; Friedman, Jeffrey M

    2015-01-01

    Means for temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and would have therapeutic implications. Here we report the development of a genetically encoded system for remote regulation of gene expression by low-frequency radio waves (RFs) or a magnetic field. Iron oxide nanoparticles are synthesized intracellularly as a GFP-tagged ferritin heavy and light chain fusion. The ferritin nanoparticles associate with a camelid anti-GFP-transient receptor potential vanilloid 1 fusion protein, αGFP-TRPV1, and can transduce noninvasive RF or magnetic fields into channel activation, also showing that TRPV1 can transduce a mechanical stimulus. This, in turn, initiates calcium-dependent transgene expression. In mice with stem cell or viral expression of these genetically encoded components, remote stimulation of insulin transgene expression with RF or a magnet lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology and therapeutics. PMID:25501906

  10. Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane.

    PubMed

    Jinno, Yuka; Shoda, Keiko; Rial-Verde, Emiliano; Yuste, Rafael; Miyawaki, Atsushi; Tsutsui, Hidekazu

    2014-01-01

    Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its β-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential. PMID:25505870

  11. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles

    PubMed Central

    Kane, Ravi S; Dordick, Jonathan S; Friedman, Jeffrey M

    2016-01-01

    Means for temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and would have therapeutic implications. Here we report the development of a genetically encoded system for remote regulation of gene expression by low-frequency radio waves (RFs) or a magnetic field. Iron oxide nanoparticles are synthesized intracellularly as a GFP-tagged ferritin heavy and light chain fusion. The ferritin nanoparticles associate with a camelid anti-GFP–transient receptor potential vanilloid 1 fusion protein, αGFP-TRPV1, and can transduce noninvasive RF or magnetic fields into channel activation, also showing that TRPV1 can transduce a mechanical stimulus. This, in turn, initiates calcium-dependent transgene expression. In mice with stem cell or viral expression of these genetically encoded components, remote stimulation of insulin transgene expression with RF or a magnet lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology and therapeutics. PMID:25501906

  12. Genetically encoded proton sensors reveal activity-dependent pH changes in neurons

    PubMed Central

    Raimondo, Joseph V.; Irkle, Agnese; Wefelmeyer, Winnie; Newey, Sarah E.; Akerman, Colin J.

    2011-01-01

    The regulation of hydrogen ion concentration (pH) is fundamental to cell viability, metabolism, and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission, and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilized to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E2GFP, and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons. PMID:22666186

  13. Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

    PubMed

    Singh, Vijay; Wang, Shenliang; Kool, Eric T

    2013-04-24

    Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy. PMID:23590213

  14. Genetically encoded fluorescent biosensors for live-cell visualization of protein phosphorylation

    PubMed Central

    Oldach, Laurel; Zhang, Jin

    2014-01-01

    Fluorescence-based, genetically encodable biosensors are widely used tools for real-time analysis of different biological process. Over the last few decades the number of available genetically encodable biosensors and the types of processes they can monitor has increased rapidly. In this review we aim to introduce the reader to general principles and best practices in biosensor development and highlight some of the ways in which biosensors can be used to illuminate outstanding questions of biological function. Specifically, we will focus on sensors developed for monitoring kinase activity and use them to illustrate some common considerations for biosensor design. We will describe several uses to which kinase and second-messenger biosensors have been put, and conclude with considerations for the use of biosensors once they are developed. Overall, as fluorescence-based biosensors continue to diversify and improve we expect them to continue to be widely used as reliable and fruitful tools for gaining deeper insights into cellular and organismal function. PMID:24485761

  15. A Genetically Encoded β-Lactamase Reporter for Ultrasensitive (129) Xe NMR in Mammalian Cells.

    PubMed

    Wang, Yanfei; Roose, Benjamin W; Palovcak, Eugene J; Carnevale, Vincenzo; Dmochowski, Ivan J

    2016-07-25

    Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub-micromolar concentrations. Particularly useful are genetically encoded, single-protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Herein, we report TEM-1 β-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) (129) Xe NMR, with significant saturation contrast at 0.1 μm. Xenon chemical exchange saturation transfer (CEST) interactions with the primary allosteric site in bla give rise to a unique saturation peak at 255 ppm, well removed (≈60 ppm downfield) from the (129) Xe-H2 O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells. PMID:27305488

  16. The study of hydrogen peroxide level under cisplatin action using genetically encoded sensor hyper

    NASA Astrophysics Data System (ADS)

    Belova, A. S.; Orlova, A. G.; Maslennikova, A. V.; Brilkina, A. A.; Balalaeva, I. V.; Antonova, N. O.; Mishina, N. M.; Shakhova, N. M.; Belousov, V. V.

    2014-03-01

    The aim of the work was to study the participation of hydrogen peroxide in reaction of cervical cancer cell line HeLa Kyoto on cisplatin action. Determination of hydrogen peroxide level was performed using genetically encoded fluorescent sensor HyPer2. The dependence of cell viability on cisplatin concentration was determined using MTT assay. Mechanisms of cell death as well as HyPer2 reaction was revealed by flow cytometry after 6-hours of incubation with cisplatin in different concentrations. Cisplatin used in low concentrations had no effect on hydrogen peroxide level in HeLa Kyoto cells. Increase of HyPer2 fluorescence was detected only after exposure with cisplatin in high concentration. The reaction was not the consequence of cell death.

  17. Genetically encoded dendritic marker sheds light on neuronal connectivity in Drosophila

    PubMed Central

    Nicolaï, Laura J. J.; Ramaekers, Ariane; Raemaekers, Tim; Drozdzecki, Andrzej; Mauss, Alex S.; Yan, Jiekun; Landgraf, Matthias; Annaert, Wim; Hassan, Bassem A.

    2010-01-01

    In recent years, Drosophila melanogaster has emerged as a powerful model for neuronal circuit development, pathology, and function. A major impediment to these studies has been the lack of a genetically encoded, specific, universal, and phenotypically neutral marker of the somatodendritic compartment. We have developed such a marker and show that it is effective and specific in all neuronal populations tested in the peripheral and central nervous system. The marker, which we name DenMark (Dendritic Marker), is a hybrid protein of the mouse protein ICAM5/Telencephalin and the red fluorescent protein mCherry. We show that DenMark is a powerful tool for revealing novel aspects of the neuroanatomy of developing dendrites, identifying previously unknown dendritic arbors, and elucidating neuronal connectivity. PMID:21059961

  18. A Genetically Encodable System for Sequence-Specific Detection of RNAs in Two Colors.

    PubMed

    Kellermann, Stefanie J; Rentmeister, Andrea

    2016-05-17

    Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non-fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths. PMID:26919688

  19. Compartmentalized AMPK Signaling Illuminated by Genetically Encoded Molecular Sensors and Actuators

    PubMed Central

    Miyamoto, Takafumi; Rho, Elmer; Sample, Vedangi; Akano, Hiroki; Magari, Masaki; Ueno, Tasuku; Gorshkov, Kirill; Chen, Melinda; Tokumitsu, Hiroshi; Zhang, Jin; Inoue, Takanari

    2015-01-01

    Summary AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell vitality, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPKα1, not AMPKα2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation. PMID:25892241

  20. Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen activating proteins

    PubMed Central

    Maji, Suvrajit; Huang, Fang; Szent-Gyorgyi, Chris; Lidke, Diane S.; Lidke, Keith A.; Bruchez, Marcel P.

    2014-01-01

    The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen activating protein has been exploited to produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and superresolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved using physiological buffers and cellular media, and does not require additives or switching buffer to obtain superresolution images. We evaluate superresolution properties and images obtained from a selected fluorogen activating protein clone fused to actin, and show that the photon counts per object fall between those typically reported for fluorescent proteins and switching dye-pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures. PMID:24194371

  1. Reporting from the Field: Genetically Encoded Fluorescent Reporters Uncover Signaling Dynamics in Living Biological Systems

    PubMed Central

    Mehta, Sohum; Zhang, Jin

    2015-01-01

    Real-time visualization of a wide range of biochemical processes in living systems is being made possible through the development and application of genetically encoded fluorescent reporters. These versatile biosensors have proven themselves tailor-made to the study of signal transduction, and in this review, we discuss some of the unique insights that they continue to provide regarding the spatial organization and dynamic regulation of intracellular signaling networks. In addition, we explore the more recent push to expand the scope of biological phenomena that can be monitored using these reporters, while also considering the potential to integrate this highly adaptable technology with a number of emerging techniques that may significantly broaden our view of how networks of biochemical processes shape larger biological phenomena. PMID:21495849

  2. A fully genetically-encoded protein architecture for optical control of peptide ligand concentration

    PubMed Central

    Schmidt, Daniel; Tillberg, Paul W.; Chen, Fei; Boyden, Edward S.

    2014-01-01

    Ion channels are amongst the most important proteins in biology - regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible fashion, and without requiring chemical co-factors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin’s local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology. PMID:24407101

  3. Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors

    PubMed Central

    2015-01-01

    Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability. PMID:25010185

  4. Genetically Encoded Sender–Receiver System in 3D Mammalian Cell Culture

    PubMed Central

    2013-01-01

    Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin–Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell–cell communication networks in 3D cell culture. PMID:24313393

  5. A fully genetically encoded protein architecture for optical control of peptide ligand concentration

    NASA Astrophysics Data System (ADS)

    Schmidt, Daniel; Tillberg, Paul W.; Chen, Fei; Boyden, Edward S.

    2014-01-01

    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.

  6. A fully genetically encoded protein architecture for optical control of peptide ligand concentration.

    PubMed

    Schmidt, Daniel; Tillberg, Paul W; Chen, Fei; Boyden, Edward S

    2014-01-01

    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K(+) channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology. PMID:24407101

  7. Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy

    PubMed Central

    Kahle, Kristopher T; Merner, Nancy D; Friedel, Perrine; Silayeva, Liliya; Liang, Bo; Khanna, Arjun; Shang, Yuze; Lachance-Touchette, Pamela; Bourassa, Cynthia; Levert, Annie; Dion, Patrick A; Walcott, Brian; Spiegelman, Dan; Dionne-Laporte, Alexandre; Hodgkinson, Alan; Awadalla, Philip; Nikbakht, Hamid; Majewski, Jacek; Cossette, Patrick; Deeb, Tarek Z; Moss, Stephen J; Medina, Igor; Rouleau, Guy A

    2014-01-01

    The KCC2 cotransporter establishes the low neuronal Cl− levels required for GABAA and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl−-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EGly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE. PMID:24928908

  8. Reversibly switchable photoacoustic tomography using a genetically encoded near-infrared phytochrome

    NASA Astrophysics Data System (ADS)

    Yao, Junjie; Kaberniuk, Andrii A.; Li, Lei; Shcherbakova, Daria M.; Zhang, Ruiying; Wang, Lidai; Li, Guo; Verkhusha, Vladislav V.; Wang, Lihong V.

    2016-03-01

    Optical imaging of genetically encoded probes has revolutionized biomedical studies by providing valuable information about targeted biological processes. Here, we report a novel imaging technique, termed reversibly switchable photoacoustic tomography (RS-PAT), which exhibits large penetration depth, high detection sensitivity, and super-resolution. RS-PAT combines advanced photoacoustic imaging techniques with, for the first time, a nonfluorescent photoswitchable bacterial phytochrome. This bacterial phytochrome is the most near-infrared shifted genetically encoded probe reported so far. Moreover, this bacterial phytochrome is reversibly photoconvertible between its far-red and near-infrared light absorption states. Taking maximum advantage of the powerful imaging capability of PAT and the unique photochemical properties of the phytochrome, RS-PAT has broken through both the optical diffusion limit for deep-tissue imaging and the optical diffraction limit for super-resolution photoacoustic microscopy. Specifically, with RS-PAT we have achieved an unprecedented detection sensitivity of ~2 μM, or as few as ~20 tumor cells, at a centimeter depth. Such high sensitivity is fully demonstrated in our study by monitoring tumor growth and metastasis at whole-body level with ~100 μm resolution. Moreover, our microscopic implementation of RS-PAT is capable of imaging mammalian cells with a sub-diffraction lateral resolution of ~140 nm and axial resolution of ~400 nm, which are respectively ~2-fold and ~75-fold finer than those of our conventional photoacoustic microscopy. Overall, RS-PAT is a new and promising imaging technology for studying biological processes at different length scales.

  9. A ratiometric strategy to detect hydrogen sulfide with a gold nanoclusters based fluorescent probe.

    PubMed

    Yang, Yan; Lei, Yingjie; Zhang, Xinrong; Zhang, Sichun

    2016-07-01

    The emergence of ratiometric fluorescent probes have offered more convincing results to the bioanalytical field of research. In particular, using nanoparticles as scaffolds for the construction of ratiometric systems has received increasing attention. In this work, a novel design strategy was implemented for ratiometric sensing of hydrogen sulfide (H2S), in which bovine serum albumin templated gold nanoclusters (BSA-AuNCs) was served as the internal reference fluorophore and HSip-1, a azamacrocyclic Cu(2+) complex based fluorescent probe toward H2S, acted as both the signal indicator and specific recognition element. Under single wavelength excitation, the nanohybrid probe HSip-1@AuNC emitted dual fluorescence at 519 and 632nm, coming from HSip-1 and AuNCs respectively. The effective fluorescence response of organic dye to H2S and constant fluorescence of AuNCs enabled the proposed HSip-1@AuNC to achieve the ratiometric measurement with a dynamic linear range of 7-100μM and a detection limit of 0.73μM. This probe also possesses high selectivity, stability against pH change and continuously light illumination. In addition, we provided HSip-1@AuNC as a valuable tool to analyze sulfides in serum samples and perfect recoveries verified its potential in biological applications. PMID:27154665

  10. Unconventional ratiometric-enhanced optical sensing of oxygen by mixed-phase TiO2

    NASA Astrophysics Data System (ADS)

    Lettieri, S.; Pallotti, D. K.; Gesuele, F.; Maddalena, P.

    2016-07-01

    We show that mixed-phase titanium dioxide (TiO2) can be effectively employed as an unconventional, inorganic, dual-emitting, and ratiometric optical sensor of O2. Simultaneous availability of rutile and anatase TiO2 photoluminescence (PL) and their peculiar "anti-correlated" PL responses to O2 allow using their ratio as a measurement parameter associated with the O2 concentration, leading to an experimental responsivity being by construction larger than the one obtainable for single-phase PL detection. A proof of this concept is given, showing a two-fold enhancement of the optical responsivity provided by the ratiometric approach. Besides the peculiar ratiometric-enhanced responsivity, other characteristics of mixed phase TiO2 can be envisaged as favorable for O2 optical probing, namely (a) low production costs, (b) absence of heterogeneous components, and (c) self-supporting properties. These characteristics encourage experimenting with its use for applications requiring high indicator quantities at a competitive price, possibly also tackling the need to develop supporting matrixes that carry the luminescent probes and avoiding issues related to the use of different components for ratiometric sensing.

  11. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

    NASA Astrophysics Data System (ADS)

    Lang, Kathrin; Davis, Lloyd; Torres-Kolbus, Jessica; Chou, Chungjung; Deiters, Alexander; Chin, Jason W.

    2012-04-01

    The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit ‘turn-on’ fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

  12. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    DOE PAGESBeta

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; Tjhung, Katrina F.; Gerlits, Oksana O.; Deng, Lu; Kasper, Brian; Sood, Amika; Paschal, Beth M.; Zhang, Ping; et al

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 outmore » of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

  13. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    SciTech Connect

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; Tjhung, Katrina F.; Gerlits, Oksana O.; Deng, Lu; Kasper, Brian; Sood, Amika; Paschal, Beth M.; Zhang, Ping; Ling, Chang-Chun; Klassen, John S.; Noren, Christopher J.; Mahal, Lara K.; Woods, Robert J.; Coates, Leighton; Derda, Ratmir

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

  14. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    PubMed

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. PMID:26908592

  15. A genetically encoded biosensor for in vitro and in vivo detection of NADP(.).

    PubMed

    Zhao, Feng-Lan; Zhang, Chang; Zhang, Chen; Tang, Yun; Ye, Bang-Ce

    2016-03-15

    NADP(+), the oxidized form of nicotinamide adenine dinucleotide phosphate, plays an essential role as a coenzyme in cellular electron transfer reactions. The concentration of NADP(+) in cytoplasm or organelles is dynamic due to its conversion to many important derivatives. To track the NADP(+) concentration in single living cells, we developed a genetically encoded NADP(+) biosensor by inserting a reporter element, ketopantoate reductase (KPR), between the Förster resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). This recombinant sensor showed a NADP(+) concentration-dependent decrease in the fluorescence ratio in vitro assay. In order to optimize this biosensor, we performed peptide-length optimization and site-directed mutagenesis in the binding pocket of KPR guided by predictions from computational protein redesign. This modified biosensor showed a 70% Δratio increase compared to the wild type and was found to be highly specific to NADP(+), with a detection limit of 1 μM. The sensor also reported NADP(+) real-time cellular dynamics in Escherichia coli (E. coli) after the addition of its precursor, nicotinic acid (NA). Altogether, these results demonstrate the feasibility of the biosensor for visualizing NADP(+) both in vitro and in vivo. PMID:26524720

  16. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

    PubMed

    Ewald, Jennifer C; Reich, Sabrina; Baumann, Stephan; Frommer, Wolf B; Zamboni, Nicola

    2011-01-01

    Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation. PMID:22164251

  17. Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors

    PubMed Central

    Depry, Charlene; Mehta, Sohum; Zhang, Jin

    2012-01-01

    Cells rely on a complex, interconnected network of signaling pathways to sense and interpret changes in their extracellular environment. The development of genetically encoded fluorescent protein (FP)-based biosensors has made it possible for researchers to directly observe and characterize the spatiotemporal dynamics of these intracellular signaling pathways in living cells. However, detailed information regarding the precise temporal and spatial relationships between intersecting pathways is often lost when individual signaling events are monitored in isolation. As the development of biosensor technology continues to advance, it is becoming increasingly feasible to image multiple FP-based biosensors concurrently, permitting greater insights into the intricate coordination of intracellular signaling networks by enabling parallel monitoring of distinct signaling events within the same cell. In this review, we discuss several strategies for multiplexed imaging of FP-based biosensors, while also underscoring some of the challenges associated with these techniques and highlighting additional avenues that could lead to further improvements in parallel monitoring of intracellular signaling events. PMID:23138230

  18. Bacterial XylRs and synthetic promoters function as genetically encoded xylose biosensors in Saccharomyces cerevisiae.

    PubMed

    Teo, Wei Suong; Chang, Matthew Wook

    2015-02-01

    Lignocellulosic biomass is a sustainable and abundant starting material for biofuel production. However, lignocellulosic hydrolysates contain not only glucose, but also other sugars including xylose which cannot be metabolized by the industrial workhorse Saccharomyces cerevisiae. Hence, engineering of xylose assimilating S. cerevisiae has been much studied, including strain optimization strategies. In this work, we constructed genetically encoded xylose biosensors that can control protein expression upon detection of xylose sugars. These were constructed with the constitutive expression of heterologous XylR repressors, which function as protein sensors, and cloning of synthetic promoters with XylR operator sites. Three XylR variants and the corresponding synthetic promoters were used: XylR from Tetragenococcus halophile, Clostridium difficile, and Lactobacillus pentosus. To optimize the biosensor, two promoters with different strengths were used to express the XylR proteins. The ability of XylR to repress yEGFP expression from the synthetic promoters was demonstrated. Furthermore, xylose sugars added exogenously to the cells were shown to regulate gene expression. We envision that the xylose biosensors can be used as a tool to engineer and optimize yeast that efficiently utilizes xylose as carbon source for growth and biofuel production. PMID:24975936

  19. Organoids and the genetically encoded self‐assembly of embryonic stem cells

    PubMed Central

    Baillie‐Johnson, Peter

    2015-01-01

    Understanding the mechanisms of early embryonic patterning and the timely allocation of specific cells to embryonic regions and fates as well as their development into tissues and organs, is a fundamental problem in Developmental Biology. The classical explanation for this process had been built around the notion of positional information. Accordingly the programmed appearance of sources of Morphogens at localized positions within a field of cells directs their differentiation. Recently, the development of organs and tissues from unpatterned and initially identical stem cells (adult and embryonic) has challenged the need for positional information and even the integrity of the embryo, for pattern formation. Here we review the emerging area of organoid biology from the perspective of Developmental Biology. We argue that the events underlying the development of these systems are not purely linked to “self‐organization,” as often suggested, but rather to a process of genetically encoded self‐assembly where genetic programs encode and control the emergence of biological structures. PMID:26666846

  20. Apollo-NADP(+): a spectrally tunable family of genetically encoded sensors for NADP(+).

    PubMed

    Cameron, William D; Bui, Cindy V; Hutchinson, Ashley; Loppnau, Peter; Gräslund, Susanne; Rocheleau, Jonathan V

    2016-04-01

    NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP(+) redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP(+) (Apollo-NADP(+)) that exploits NADP(+)-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose-stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP(+) to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean-tagged version of Apollo-NADP(+) with the H2O2 sensor HyPer. PMID:26878383

  1. Inhibitory luminopsins: genetically-encoded bioluminescent opsins for versatile, scalable, and hardware-independent optogenetic inhibition

    PubMed Central

    Tung, Jack K.; Gutekunst, Claire-Anne; Gross, Robert E.

    2015-01-01

    Optogenetic techniques provide an unprecedented ability to precisely manipulate neural activity in the context of complex neural circuitry. Although the toolbox of optogenetic probes continues to expand at a rapid pace with more efficient and responsive reagents, hardware-based light delivery is still a major hurdle that limits its practical use in vivo. We have bypassed the challenges of external light delivery by directly coupling a bioluminescent light source (a genetically encoded luciferase) to an inhibitory opsin, which we term an inhibitory luminopsin (iLMO). iLMO was shown to suppress action potential firing and synchronous bursting activity in vitro in response to both external light and luciferase substrate. iLMO was further shown to suppress single-unit firing rate and local field potentials in the hippocampus of anesthetized rats. Finally, expression of iLMO was scaled up to multiple structures of the basal ganglia to modulate rotational behavior of freely moving animals in a hardware-independent fashion. This novel class of optogenetic probes demonstrates how non-invasive inhibition of neural activity can be achieved, which adds to the versatility, scalability, and practicality of optogenetic applications in freely behaving animals. PMID:26399324

  2. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria

    PubMed Central

    Elbaz, Johann; Yin, Peng; Voigt, Christopher A.

    2016-01-01

    The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32–205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3′-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 μm2) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their production as well as applications in living cells. PMID:27091073

  3. Applications of Genetically-Encoded Biosensors for the Construction and Control of Biosynthetic Pathways

    PubMed Central

    Michener, Josh K.; Thodey, Kate; Liang, Joe C.; Smolke, Christina D.

    2011-01-01

    Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability. PMID:21946159

  4. Inhibitory luminopsins: genetically-encoded bioluminescent opsins for versatile, scalable, and hardware-independent optogenetic inhibition.

    PubMed

    Tung, Jack K; Gutekunst, Claire-Anne; Gross, Robert E

    2015-01-01

    Optogenetic techniques provide an unprecedented ability to precisely manipulate neural activity in the context of complex neural circuitry. Although the toolbox of optogenetic probes continues to expand at a rapid pace with more efficient and responsive reagents, hardware-based light delivery is still a major hurdle that limits its practical use in vivo. We have bypassed the challenges of external light delivery by directly coupling a bioluminescent light source (a genetically encoded luciferase) to an inhibitory opsin, which we term an inhibitory luminopsin (iLMO). iLMO was shown to suppress action potential firing and synchronous bursting activity in vitro in response to both external light and luciferase substrate. iLMO was further shown to suppress single-unit firing rate and local field potentials in the hippocampus of anesthetized rats. Finally, expression of iLMO was scaled up to multiple structures of the basal ganglia to modulate rotational behavior of freely moving animals in a hardware-independent fashion. This novel class of optogenetic probes demonstrates how non-invasive inhibition of neural activity can be achieved, which adds to the versatility, scalability, and practicality of optogenetic applications in freely behaving animals. PMID:26399324

  5. Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

    PubMed Central

    Zhu, Xinyue; Wang, Jianxi; Zhang, Jianjian; Chen, Zhenjie; Zhang, Haixia; Zhang, Xiaoyu

    2015-01-01

    A reaction-based two-photon (TP) ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS) as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM). PMID:25594597

  6. Ratiometric oxygen sensing using lanthanide luminescent emitting interfaces.

    PubMed

    Lehr, Joshua; Tropiano, Manuel; Beer, Paul D; Faulkner, Stephen; Davis, Jason J

    2015-11-14

    Herein we describe the first example of a ratiometric lanthanide luminescent oxygen sensing interface. Immobilisation of terbium and europium cyclen complexes on glass substrates was achieved by a novel aryl nitrene photografting approach. The resulting interfaces demonstrated a ratiometric oxygen response between 0 and 0.2 atm partial oxygen pressure. PMID:26376829

  7. Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer.

    PubMed

    Westberg, Michael; Holmegaard, Lotte; Pimenta, Frederico M; Etzerodt, Michael; Ogilby, Peter R

    2015-02-01

    Singlet oxygen, O(2)(a(1)Δ(g)), plays a key role in many processes of cell signaling. Limitations in mechanistic studies of such processes are generally associated with the difficulty of controlling the amount and location of O(2)(a(1)Δ(g)) production in or on a cell. As such, there is great need for a system that (a) selectively produces O(2)(a(1)Δ(g)) in appreciable and accurately quantifiable yields and (b) can be localized in a specific place at the suborganelle level. A genetically encodable, protein-encased photosensitizer is one way to achieve this goal. Through a systematic and rational approach involving mutations to a LOV2 protein that binds the chromophore flavin mononucleotide (FMN), we have developed a promising photosensitizer that overcomes many of the problems that affect related systems currently in use. Specifically, by decreasing the extent of hydrogen bonding between FMN and a specific amino acid residue in the local protein environment, we decrease the susceptibility of FMN to undesired photoinitiated electron-transfer reactions that kinetically compete with O(2)(a(1)Δ(g)) production. As a consequence, our protein-encased FMN system produces O(2)(a(1)Δ(g)) with the uniquely large quantum efficiency of 0.25 ± 0.03. We have also quantified other key photophysical parameters that characterize this sensitizer system, including unprecedented H(2)O/D(2)O solvent isotope effects on the O(2)(a(1)Δ(g)) formation kinetics and yields. As such, our results facilitate future systematic developments in this field. PMID:25575190

  8. Genetically encoded far-red fluorescent sensors for caspase-3 activity.

    PubMed

    Zlobovskaya, Olga A; Sergeeva, Tatiana F; Shirmanova, Marina V; Dudenkova, Varvara V; Sharonov, George V; Zagaynova, Elena V; Lukyanov, Konstantin A

    2016-02-01

    Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues. PMID:26842350

  9. Genetically-encoded tools for cAMP probing and modulation in living systems

    PubMed Central

    Paramonov, Valeriy M.; Mamaeva, Veronika; Sahlgren, Cecilia; Rivero-Müller, Adolfo

    2015-01-01

    Intracellular 3′-5′-cyclic adenosine monophosphate (cAMP) is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming—all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells), underpin the ensuing limitations of the conventional cAMP assays: (1) genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; (2) inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control—something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs. PMID:26441653

  10. Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

    PubMed Central

    Han, Zhou; Jin, Lei; Chen, Fuyi; Loturco, Joseph J.; Cohen, Lawrence B.; Bondar, Alexey; Lazar, Josef; Pieribone, Vincent A.

    2014-01-01

    ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins. PMID:25419571

  11. Genetically encoded molecular biosensors to image histone methylation in living animals.

    PubMed

    Sekar, Thillai V; Foygel, Kira; Gelovani, Juri G; Paulmurugan, Ramasamy

    2015-01-20

    Post-translational addition of methyl groups to the amino terminal tails of histone proteins regulates cellular gene expression at various stages of development and the pathogenesis of cellular diseases, including cancer. Several enzymes that modulate these post-translational modifications of histones are promising targets for development of small molecule drugs. However, there is no promising real-time histone methylation detection tool currently available to screen and validate potential small molecule histone methylation modulators in small animal models. With this in mind, we developed genetically encoded molecular biosensors based on the split-enzyme complementation approach for in vitro and in vivo imaging of lysine 9 (H3-K9 sensor) and lysine 27 (H3-K27 sensor) methylation marks of histone 3. These methylation sensors were validated in vitro in HEK293T, HepG2, and HeLa cells. The efficiency of the histone methylation sensor was assessed by employing methyltransferase inhibitors (Bix01294 and UNC0638), demethylase inhibitor (JIB-04), and siRNA silencing at the endogenous histone K9-methyltransferase enzyme level. Furthermore, noninvasive bioluminescence imaging of histone methylation sensors confirmed the potential of these sensors in monitoring histone methylation status in response to histone methyltransferase inhibitors in living animals. Experimental results confirmed that the developed H3-K9 and H3-K27 sensors are specific and sensitive to image the drug-induced histone methylation changes in living animals. These novel histone methylation sensors can facilitate the in vitro screening and in vivo characterization of new histone methyltransferase inhibitors and accelerate the pace of introduction of epigenetic therapies into the clinic. PMID:25506787

  12. The genetically encoded tool set for investigating cAMP: more than the sum of its parts

    PubMed Central

    Patel, Neha; Gold, Matthew G.

    2015-01-01

    Intracellular fluctuations of the second messenger cyclic AMP (cAMP) are regulated with spatial and temporal precision. This regulation is supported by the sophisticated arrangement of cyclases, phosphodiesterases, anchoring proteins, and receptors for cAMP. Discovery of these nuances to cAMP signaling has been facilitated by the development of genetically encodable tools for monitoring and manipulating cAMP and the proteins that support cAMP signaling. In this review, we discuss the state-of-the-art in development of different genetically encoded tools for sensing cAMP and the activity of its primary intracellular receptor protein kinase A (PKA). We introduce sequences for encoding adenylyl cyclases that enable cAMP levels to be artificially elevated within cells. We chart the evolution of sequences for selectively modifying protein–protein interactions that support cAMP signaling, and for driving cAMP sensors and manipulators to different subcellular locations. Importantly, these different genetically encoded tools can be applied synergistically, and we highlight notable instances that take advantage of this property. Finally, we consider prospects for extending the utility of the tool set to support further insights into the role of cAMP in health and disease. PMID:26300778

  13. Carbazole incorporated ratiometric chemosensor for Zn2+

    NASA Astrophysics Data System (ADS)

    Helal, Aasif; Kim, Hong-Seok

    2013-03-01

    An electron donating carbazole incorporated thiazole (3) based Zn2+ selective intrinsic chemosensor has been synthesized and investigated. It was found that electron donating substituents such as methyl and carbazole on chemosensor (1) produce remarkable red shift in emission upon complexation with Zn2+. The sensor shows a selective fluorescence response with Zn2+ over biologically relevant cations (Ca2+, Mg2+, Na+, and K+) and biologically non-relevant cations (Cd2+, In3+ and Ga3+) in an aqueous ethanol system. It also produce an enhancement in the quantum yield and a longer emission wavelength shift on Zn2+ binding with the potential of a ratiometric assay.

  14. A mitochondria-targeted ratiometric two-photon fluorescent probe for biological zinc ions detection.

    PubMed

    Ning, Peng; Jiang, Jiacheng; Li, Longchun; Wang, Shuxin; Yu, Haizhu; Feng, Yan; Zhu, Manzhou; Zhang, Buchang; Yin, Hang; Guo, Qingxiang; Meng, Xiangming

    2016-03-15

    A mitochondria-targeted ratiometric two-photon fluorescent probe (Mito-MPVQ) for biological zinc ions detection was developed based on quinolone platform. Mito-MPVQ showed large red shifts (68 nm) and selective ratiometric signal upon Zn(2+) binding. The ratio of emission intensity (I488 nm/I420 nm) increases dramatically from 0.45 to 3.79 (ca. 8-fold). NMR titration and theoretical calculation confirmed the binding of Mito-MPVQ and Zn(2+). Mito-MPVQ also exhibited large two-photon absorption cross sections (150 GM) at nearly 720 nm and insensitivity to pH within the biologically relevant pH range. Cell imaging indicated that Mito-MPVQ could efficiently located in mitochondria and monitor mitochondrial Zn(2+) under two-photon excitation with low cytotoxicity. PMID:26528806

  15. Quantitative generalized ratiometric fluorescence spectroscopy for turbid media based on probe encapsulated by biologically localized embedding.

    PubMed

    Yan, Xiu-Fang; Chen, Zeng-Ping; Cui, Yin-Yin; Hu, Yuan-Liang; Yu, Ru-Qin

    2016-05-19

    PEBBLE (probe encapsulated by biologically localized embedding) nanosensor encapsulating an intensity-based fluorescence indicator and an inert reference fluorescence dye inside the pores of stable matrix can be used as a generalized wavelength-ratiometric probe. However, the lack of an efficient quantitative model render the choices of inert reference dyes and intensity-based fluorescence indicators used in PEBBLEs based generalized wavelength-ratiometric probes rather limited. In this contribution, an extended quantitative fluorescence model was derived specifically for generalized wavelength-ratiometric probes based on PEBBLE technique (QFMGRP) with a view to simplify the design of PEBBLEs and hence further extend their application potentials. The effectiveness of QFMGRP has been tested on the quantitative determination of free Ca(2+) in both simulated and real turbid media using a Ca(2+) sensitive PEBBLE nanosensor encapsulating Rhod-2 and eosin B inside the micropores of stable polyacrylamide matrix. Experimental results demonstrated that QFMGRP could realize precise and accurate quantification of free Ca(2+) in turbid samples, even though there is serious overlapping between the fluorescence excitation peaks of eosin B and Ca(2+) bound Rhod-2. The average relative predictive error value of QFMGRP for the test simulated turbid samples was 5.9%, about 2-4 times lower than the corresponding values of partial least squares calibration model and the empirical ratiometric model based on the ratio of fluorescence intensities at the excitation peaks of Ca(2+) bound Rhod-2 and eosin B. The recovery rates of QFMGRP for the real and spiked turbid samples varied from 93.1% to 101%, comparable to the corresponding results of atomic absorption spectrometry. PMID:27126788

  16. Targeted silver nanoparticles for ratiometric cell phenotyping.

    PubMed

    Willmore, Anne-Mari A; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N; Ruoslahti, Erkki; Braun, Gary B; Teesalu, Tambet

    2016-04-28

    Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 ± 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 ± 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo. PMID:26646247

  17. Studies of Hematopoietic Cell Differentiation with a Ratiometric and Reversible Sensor of Mitochondrial Reactive Oxygen Species

    PubMed Central

    Kaur, Amandeep; Jankowska, Karolina; Pilgrim, Chelsea; Fraser, Stuart T.

    2016-01-01

    Abstract Aims: Chronic elevations in cellular redox state are known to result in the onset of various pathological conditions, but transient increases in reactive oxygen species (ROS)/reactive nitrogen species (RNS) are necessary for signal transduction and various physiological functions. There is a distinct lack of reversible fluorescent tools that can aid in studying and unraveling the roles of ROS/RNS in physiology and pathology by monitoring the variations in cellular ROS levels over time. In this work, we report the development of ratiometric fluorescent sensors that reversibly respond to changes in mitochondrial redox state. Results: Photophysical studies of the developed flavin–rhodamine redox sensors, flavin–rhodamine redox sensor 1 (FRR1) and flavin–rhodamine redox sensor 2 (FRR2), confirmed the reversible response of the probes upon reduction and re-oxidation over more than five cycles. The ratiometric output of FRR1 and FRR2 remained unaltered in the presence of other possible cellular interferants (metals and pH). Microscopy studies indicated clear mitochondrial localization of both probes, and FRR2 was shown to report the time-dependent increase of mitochondrial ROS levels after lipopolysaccharide stimulation in macrophages. Moreover, it was used to study the variations in mitochondrial redox state in mouse hematopoietic cells at different stages of embryonic development and maturation. Innovation: This study provides the first ratiometric and reversible probes for ROS, targeted to the mitochondria, which reveal variations in mitochondrial ROS levels at different stages of embryonic and adult blood cell production. Conclusions: Our results suggest that with their ratiometric and reversible outputs, FRR1 and FRR2 are valuable tools for the future study of oxidative stress and its implications in physiology and pathology. Antioxid. Redox Signal. 24, 667–679. PMID:26865422

  18. A two-photon ratiometric fluorescent probe enables spatial coordinates determination of intracellular pH.

    PubMed

    Wang, Junjie; Sun, Yuming; Zhang, Weijia; Liu, Yong; Yu, Xiaoqiang; Zhao, Ning

    2014-11-01

    We reported a two-photon ratiometric fluorescent probe for detecting intracellular pH. When excited with 800 nm laser, an optimal output of laser as the routine equipment of two-photon fluorescence microscopy, the two-photon excited fluorescence of this probe showed distinct emission peak shift as large as 109 nm upon the change of pH values in vitro. Very importantly, the experiment results show that this probe has large two-photon absorption cross-section at pH 4.5 at 800 nm of 354 g, which ranks it as one of the best two-photon ratiometric fluorescent pH probes, and its working pH value is between 4.0 and 8.0 which could fit the intracellular pH range. Moreover, utilizing this probe, the two-photon ratiometric fluorescent images in living cells have been obtained, and the spatial coordinates of intracellular pH can be mapped. At the same time, the probe also exhibited selectivity, photostability and membrane permeability. And the photophysical properties of this probe in various solvents indicated that these photophysical properties variations are due to an intramolecular charge transfer process. At last, the imaging depth of the probe in liver biopsy slices was investigated. The experimental results demonstrated the maximum imaging depth can arrive 66 µm in living rat liver tissues. PMID:25127590

  19. Targeted silver nanoparticles for ratiometric cell phenotyping

    NASA Astrophysics Data System (ADS)

    Willmore, Anne-Mari A.; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N.; Ruoslahti, Erkki; Braun, Gary B.; Teesalu, Tambet

    2016-04-01

    Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The

  20. Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation

    PubMed Central

    2014-01-01

    The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology. PMID:25341086

  1. Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

    PubMed

    Mondal, Samsuzzoha; Rakshit, Ananya; Pal, Suranjana; Datta, Ankona

    2016-07-15

    Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling. PMID:27082310

  2. A novel reaction-based colorimetric and ratiometric fluorescent sensor for cyanide anion with a large emission shift and high selectivity.

    PubMed

    Wang, Shaodan; Fei, Xiaoliang; Guo, Jing; Yang, Qingbiao; Li, Yaoxian; Song, Yan

    2016-02-01

    A hybrid carbazole-hemicyanine dye (Cac) has been developed as a novel colorimetric and ratiometric fluorescent sensor for cyanide detection. Upon treatment with cyanide, Cac displayed a remarkable fluorescence ratiometric response, with the emission wavelength displaying a very large emission shift (214 nm). The detection of cyanide was performed via the nucleophilic addition of cyanide anion to the indolium group of the sensor, which resulted in the blocking of the intramolecular charge transfer (ICT) process in the sensor, inducing a ratiometric fluorescence change and simultaneously an obvious color change. Furthermore, competitive anions did not showed any significant changes both in color and emission intensity ratio (I381/I595), indicating the high selectivity of the sensor to CN(-). PMID:26653444

  3. Cu nanoclusters-based ratiometric fluorescence probe for ratiometric and visualization detection of copper ions.

    PubMed

    Liu, Zhi-Chao; Qi, Jian-Wen; Hu, Chun; Zhang, Li; Song, Wei; Liang, Ru-Ping; Qiu, Jian-Ding

    2015-10-01

    Copper is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, sensitive detection of Cu(2+) is very important to prevent over-ingestion, and visual detection is preferred for practical applications. In this work, we developed a simple and environmental friendly approach to synthesize hyperbranched polyethyleneimine-protected copper nanoclusters (hPEI-Cu NCs) with great stability against extreme pH, high ionic strength, thiols etching and light illumination, which were then conjugated to the surface of silica coated CdSe quantum dots (QDs) to design a ratiometric fluorescence probe. In the presence of different amounts of Cu(2+) ions, the fluorescence of Cu NCs can be drastically quenched, while the emission from QDs stayed constant to serve as a reference signal and the color of the probe changed from yellow-green to red, resulting in ratiometric and visualization detection of Cu(2+) ion with high accuracy. The detection limit for Cu(2+) was estimated to be 8.9 nM, much lower than the allowable level of Cu(2+) in drinking water (∼20 μM) set by U.S. Environmental Protection Agency. Additionally, this probe can be also applied for the determination of Cu(2+) ion in complex real water samples. PMID:26454464

  4. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.

    PubMed

    Wu, Peng; Shui, Wenqing; Carlson, Brian L; Hu, Nancy; Rabuka, David; Lee, Julia; Bertozzi, Carolyn R

    2009-03-01

    The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Cotranslational modification of the proteins by FGE produced products bearing a unique aldehyde group. Proteins bearing this "aldehyde tag" were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. We applied the technique to site-specific modification of monoclonal antibodies, the fastest growing class of biopharmaceuticals, as well as membrane-associated and cytosolic proteins expressed in mammalian cells. PMID:19202059

  5. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

    PubMed

    Bedbrook, Claire N; Kato, Mihoko; Ravindra Kumar, Sripriya; Lakshmanan, Anupama; Nath, Ravi D; Sun, Fei; Sternberg, Paul W; Arnold, Frances H; Gradinaru, Viviana

    2015-08-20

    Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering. PMID:26211362

  6. Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.

    PubMed

    Mascali, Florencia C; Ching, H Y Vincent; Rasia, Rodolfo M; Un, Sun; Tabares, Leandro C

    2016-09-01

    Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, in vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E. coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements. PMID:27496179

  7. Ratiometric Fluorescent Polymeric Thermometer for Thermogenesis Investigation in Living Cells.

    PubMed

    Qiao, Juan; Hwang, Yoon-Ho; Chen, Chuan-Fang; Qi, Li; Dong, Ping; Mu, Xiao-Yu; Kim, Dong-Pyo

    2015-10-20

    Intracellular temperature has a fundamental effect on cellular events. Herein, a novel fluorescent polymer ratiometric nanothermometer has been developed based on transferrin protein-stabilized gold nanoclusters as the targeting and fluorescent ratiometric unit and the thermosensitve polymer as the temperature sensing unit. The resultant nanothermometer could feature a high and spontaneous uptake into the HeLa cells and the ratiometric temperature sensing over the physiological temperature range. Moreover, the precise temperature sensing for intracellular heat generation in HeLa cells following calcium ions stress has been achieved. This practical intracellular thermometry could eliminate the interference of the intracellular surrounding environment in cancer cells without a microinjection procedure, which is user-friendly. The prepared new nanothermometer can provide tools for unveiling the intrinsic relationship between the intracellular temperature and ion channel function. PMID:26393404

  8. Flavone-Based ESIPT Ratiometric Chemodosimeter for Detection of Cysteine in Living Cells

    PubMed Central

    2015-01-01

    We have designed and synthesized a novel ratiometric fluorescent chemodosimeter MHF-based ESIPT process for specific detection of cysteine among the biological thiols. The probe MHF shows very weak blue fluorescence under UV excitation. Upon addition of cysteine (Cys), the reaction of Cys with MHF induces acrylate hydrolysis, thereby enabling the ESIPT process to shift the weak blue emission to a strong green emission with about 20-fold enhancement. We utilized 1H NMR spectra to elucidate the fluorescence sensing mechanism. Moreover, the cellular imaging experiment indicated the MHF possessed excellent selectivity, low cytotoxicity, and desirable cell permeability for biological applications. PMID:24571859

  9. Effect of Ca2+ on the Steady-State and Time-Resolved Emission Properties of the Genetically Encoded Fluorescent Sensor CatchER

    PubMed Central

    2015-01-01

    We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ. PMID:24836743

  10. One-pot synthesis of two-sized clusters for ratiometric sensing of Hg2+.

    PubMed

    Chen, Tzu-Heng; Lu, Chi-Yu; Tseng, Wei-Lung

    2013-12-15

    This paper presents a discussion of a one-pot approach for preparing lyszoyme type VI (Lys VI) stabilized clusters, including small (Au7Ag and Au8) and large (Au24Ag) clusters, for ratiometric fluorescence sensing of Hg(2+). Our previous study (Chen and Tseng, Small 8 (2012) 1912) showed the formation of intermediate Au8 clusters in the conversion of Au(+)-Lys VI protein complexes to Au25 clusters. The presence of Ag(+) in the precursor solution slowed this conversion, thereby forming two-sized clusters. With an increase in Ag(+) content, a systematic blue shift in the first exciton absorption and fluorescence peaks indicated the formation of Au-Ag bimetallic clusters. The prepared Ag(+)/Au(3+) molar ratio of 2:8 resulted in the formation of two-sized clusters, with dual emission bands centered at 471 and 613 nm. After these clusters are separated by a membrane filter, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine the composition of Au24Ag clusters. By monitoring the intensity ratio of the two emission wavelengths, the solution consisting of Hg(2+)-insensitive small clusters (Au7Ag and Au8) and Hg(2+)-sensitive Au24Ag clusters exhibited a ratiometric fluorescence response toward Hg(2+), and provided a built-in correction for photobleaching; the limit of detection at a signal-to-noise ratio of three for Hg(2+) was estimated to be 1 nM. This probe was successfully applied to ratiometric fluorescence sensing of Hg(2+) in tap water. PMID:24209338

  11. Ratiometric electrochemical immunoassay based on internal reference value for reproducible and sensitive detection of tumor marker.

    PubMed

    Cai, Xiaohui; Weng, Shaohuang; Guo, Rubin; Lin, Liqing; Chen, Wei; Zheng, Zongfu; Huang, Zhengjun; Lin, Xinhua

    2016-07-15

    A ratiometric assay in an electrochemical immunosensor for tumor marker, herein carcinoembryonic antigen (CEA) was chosen as a model analyte, was developed to improve simplicity and accuracy. The immunosensor was fabricated via the simple expedient way of using Polythionine-gold (PTh-Au) as electrode modified material to be an internal reference signal and K3[Fe(CN)6] in electrolyte as an indicator signal. When the CEA was fixed on the modified electrode via immunoreaction, only the indicator signal sensitively altered; by contrast, the internal reference signal of PTh-Au remained constant at a suitable pH of the electrolyte. The ratio between the alterations of the indicator signal of K3[Fe(CN)6] and the constant internal reference signal can be used to monitor the concentration of CEA reliably, reproducibly, and sensitively. The prepared ratiometric electrochemical immunosensor could detect CEA with good specificity within a wide linear range from 0.005ng/ml to 40ng/ml with a detection limit of 2.2pg/ml. Additionally, experimental results confirm that our proposed method is practical. Thus, this method can expand to recognize and test other protein markers. PMID:26945184

  12. A Genetically Encoded FRET Lactate Sensor and Its Use To Detect the Warburg Effect in Single Cancer Cells

    PubMed Central

    San Martín, Alejandro; Ceballo, Sebastián; Ruminot, Iván; Lerchundi, Rodrigo; Frommer, Wolf B.; Barros, Luis Felipe

    2013-01-01

    Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3–5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single

  13. A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

    PubMed

    Li, Nan; Ramil, Carlo P; Lim, Reyna K V; Lin, Qing

    2015-02-20

    The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry. PMID:25347611

  14. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    NASA Astrophysics Data System (ADS)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  15. A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

    PubMed Central

    Cox, Dianne; Hodgson, Louis

    2014-01-01

    Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

  16. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

    PubMed Central

    Goryashchenko, Alexander S.; Khrenova, Maria G.; Bochkova, Anna A.; Ivashina, Tatiana V.; Vinokurov, Leonid M.; Savitsky, Alexander P.

    2015-01-01

    This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated. PMID:26204836

  17. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

    PubMed

    Schreiber, Andreas; Huber, Matthias C; Cölfen, Helmut; Schiller, Stefan M

    2015-01-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties. PMID:25813537

  18. Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway

    PubMed Central

    Leung, Daisy W.; Otomo, Chinatsu; Chory, Joanne; Rosen, Michael K.

    2008-01-01

    General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs. PMID:18728185

  19. Imaging neuronal responses in slice preparations of vomeronasal organ expressing a genetically encoded calcium sensor.

    PubMed

    Ma, Limei; Haga-Yamanaka, Sachiko; Yu, Qingfeng Elden; Qiu, Qiang; Kim, Sangseong; Yu, C Ron

    2011-01-01

    The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species. These intraspecies signals, the pheromones, as well as signals from some predators, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses. The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations. Third, this method can be combined with molecular cloning techniques to allow receptor identification. Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory

  20. A fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Gao, Feng; Tang, Lijuan; Dai, Lu; Wang, Lun

    2007-06-01

    We have synthesized dual-fluorophore-doped core-shell silica nanoparticles used as ratiometric pH sensor. The nanoparticles were prepared with a reverse microemulsion technique by simultaneously encapsulating two different fluorophores, the pH-sensitive dye fluorescein as a pH indicator and the pH-insensitive dye phenosafranine as an internal reference for fluorescence ratiometric measurement, into silica shell. The nanoparticles prevent the fluorescence dyes leaching from the silica matrix when immersed inside water. The hydrophilic silica shells were made by hydrolysing and polymerizing tetraethoxysilane (TEOS) in water-in-oil microemulsion. The fluorescence intensity ratio of the two dyes varied linearly as a function of pH in the range from 4.0 to 8.0. The sensor was also applied to measure pH of real water samples. The results are in good agreements with that using the conventional glass electrode method. The as-prepared fluorescent nanoparticles showed rapid response, excellent stability and high reproducibility as pH sensors.

  1. eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn2+ Imaging

    PubMed Central

    2015-01-01

    Zn2+ plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn2+ homeostasis and the putative signaling role of Zn2+, various fluorescent sensors have been developed that allow monitoring of Zn2+ concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn2+ sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer and co-workers. Both have been successfully used to measure cytosolic free Zn2+, but distinctly different concentrations have been reported when using these sensors to measure Zn2+ concentrations in the ER and mitochondria. Here, we report the development of a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was created on the surface of the donor and acceptor fluorescent domains. This eZinCh-2 sensor binds Zn2+ with a high affinity that is similar to that of eCALWY-4 (Kd = 1 nM at pH 7.1), while displaying a substantially larger change in emission ratio. eZinCh-2 not only provides an attractive alternative for measuring Zn2+ in the cytosol but was also successfully used for measuring Zn2+ in the ER, mitochondria, and secretory vesicles. Moreover, organelle-targeted eZinCh-2 can also be used in combination with the previously reported redCALWY sensors to allow multicolor imaging of intracellular Zn2+ simultaneously in the cytosol and the ER or mitochondria. PMID:26151333

  2. A selective colorimetric and ratiometric fluorescent probe for hydrogen sulfide.

    PubMed

    Wu, Ming-Yu; Li, Kun; Hou, Ji-Ting; Huang, Zheng; Yu, Xiao-Qi

    2012-10-01

    A reaction-based colorimetric and ratiometric fluorescent probe based on an ICT-strategy for selective detection of H(2)S that exploited the H(2)S-mediated reduction of nitrocompound to amines was explored. And it displayed high selectivity for H(2)S over other relevant reactive sulfur, oxygen, nitrogen species and other anions with more than 120 nm blue shift and the change of emission intensity ratio inducted by H(2)S was over 4750. PMID:22965805

  3. Harnessing a ratiometric fluorescence output from a sensor array.

    PubMed

    Wang, Zhuo; Palacios, Manuel A; Zyryanov, Grigory; Anzenbacher, Pavel

    2008-01-01

    Ratiometric fluorescence-based sensors are widely sought after because they can effectively convert even relatively small changes in optical output into a strong and easy-to-read signal. However, ratiometric sensor molecules are usually difficult to make. We present a proof-of-principle experiment that shows that efficient ratiometric sensing may be achieved by an array of two chromophores, one providing an on-to-off response and the second yielding an off-to-on response in a complementary fashion. In the case that both chromophores emit light of different color, the result is a switching of colors that may be utilized in the same way as from a true ratiometric probe. The chromophore array comprises two sensor elements: i) a polyurethane membrane with embedded N-anthracen-9-yl-methyl-N-7-nitrobenzoxa-[1,2,5]diazo-4-yl-N',N'-dimethylethylenediamine hydrochloride and ii) a membrane with N,N-dimethyl-N'-(9-methylanthracenyl)ethylenediamine. A combination of photoinduced electron transfer (PET) and fluorescence resonance energy transfer (FRET) allows for green-to-blue emission switching in the presence of Zn(II) ions. The sensing experiments carried out with different Zn(II) salts at controlled pH revealed that the degree of color switching in the individual sensor elements depends on both the presence of Zn(II) ions and the counter anion. These results suggest that sensing of both cations and anions may perhaps be extended to different cation-anion pairs. PMID:18688830

  4. Fluorescent Peptide Beacons for the Selective Ratiometric Detection of Heparin.

    PubMed

    Maity, Debabrata; Schmuck, Carsten

    2016-09-01

    Heparin is extensively used as an anticoagulant drug during surgery. Two fluorophore-functionalized cationic oligopeptides HS 1 and HS 2 were developed to monitor heparin ratiometrically in aqueous media. Upon binding to heparin, HS 1 and HS 2 undergo a conformational change from an open form to a folded form, which leads to a distinct change in the fluorescence properties. HS 1 switches from pyrene monomer emission to an excimer emission. For HS 2, a fluorescence resonance energy transfer (FRET) process is enabled between a naphthalene donor and a dansyl acceptor. This method is highly selective for heparin relative to other similar biological analytes such as hyaluronic acid or chondroitin sulfate. HS 1 and HS 2 could also detect heparin ratiometrically in diluted bovine serum. The strong ratiometric emission color change can also be observed by the naked eye. Addition of the polycationic protein protamine releases both HS 1 and HS 2 from their heparin complex, which simultaneously restores pyrene monomer emission for the first case and decreases the FRET process for the latter case, respectively. Dynamic light scattering (DLS) and AFM studies confirm aggregate formation of heparin with HS 1 and HS 2. PMID:27534383

  5. Ion-Switchable Quantum Dot Förster Resonance Energy Transfer Rates in Ratiometric Potassium Sensors.

    PubMed

    Ruckh, Timothy T; Skipwith, Christopher G; Chang, Wendi; Senko, Alexander W; Bulovic, Vladimir; Anikeeva, Polina O; Clark, Heather A

    2016-04-26

    The tools for optically imaging cellular potassium concentrations in real-time are currently limited to a small set of molecular indicator dyes. Quantum dot-based nanosensors are more photostable and tunable than organic indicators, but previous designs have fallen short in size, sensitivity, and selectivity. Here, we introduce a small, sensitive, and selective nanosensor for potassium measurements. A dynamic quencher modulates the fluorescence emitted by two different quantum dot species to produce a ratiometric signal. We characterized the potassium-modulated sensor properties and investigated the photonic interactions within the sensors. The quencher's protonation changes in response to potassium, which modulates its Förster radiative energy transfer rate and the corresponding interaction radii with each quantum dot species. The nanosensors respond to changes in potassium concentrations typical of the cellular environment and thus provide a promising tool for imaging potassium fluxes during biological events. PMID:27089024

  6. Visual and fluorescent detection of tyrosinase activity by using a dual-emission ratiometric fluorescence probe.

    PubMed

    Yan, Xu; Li, Hongxia; Zheng, Weishi; Su, Xingguang

    2015-09-01

    In this work, we designed a dual-emission ratiometric fluorescence probe by hybridizing two differently colored quantum dots (QDs), which possess a built-in correction that eliminates the environmental effects and increases sensor accuracy. Red emissive QDs were embedded in the silica nanoparticle as reference while the green emissive QDs were covalently linked to the silica nanoparticle surface to form ratiometric fluorescence probes (RF-QDs). Dopamine (DA) was then conjugated to the surface of RF-QDs via covalent bonding. The ratiometric fluorescence probe functionalized with dopamine (DA) was highly reactive toward tyrosinase (TYR), which can catalyze the oxidization of DA to dopamine quinine and therefore quenched the fluorescence of the green QDs on the surface of ratiometric fluorescence probe. With the addition of different amounts of TYR, the ratiometric fluorescence intensity of the probe continually varied, leading to color changes from yellow-green to red. So the ratiometric fluorescence probe could be utilized for sensitive and selective detection of TYR activity. There was a good linear relationship between the ratiometric fluorescence intensity and TYR concentration in the range of 0.05-5.0 μg mL(-1), with the detection limit of 0.02 μg mL(-1). Significantly, the ratiometric fluorescence probe has been used to fabricate paper-based test strips for visual detection of TYR activity, which validates the potential on-site application. PMID:26249217

  7. Measuring Phagosome pH by Ratiometric Fluorescence Microscopy.

    PubMed

    Nunes, Paula; Guido, Daniele; Demaurex, Nicolas

    2015-01-01

    Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H(+) is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized

  8. A simple Chalcone-based ratiometric chemosensor for silver ion.

    PubMed

    Velmurugan, K; Suresh, S; Santhoshkumar, S; Saranya, M; Nandhakumar, R

    2016-05-01

    Herein, we report the selective binding of Ag(+) ion by the anthracene-based chalcone receptor 1. Receptor 1 behaves as a selective and sensitive chemosensor for the recognition of Ag(+) over other heavy and transition metal ions without any interference and is capable of detecting the metal ion down to 0.15 × 10(-6) M. Receptor 1 on binding with Ag(+) ions exhibits a ratiometric fluorescence enhancement, which is due to the inhibition of photoinduced electron transfer along with the intramolecular charge transfer mechanism. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26333533

  9. A ratiometric two-photon fluorescent probe for fluoride ion imaging in living cells and zebrafish.

    PubMed

    Hu, Wei; Zeng, Lingyu; Wang, Yanying; Liu, Zhihong; Ye, Xiaoxue; Li, Chunya

    2016-09-21

    Using 6-hydroxyl-quinoline-2-benzothiazole (HQB) as a two-photon fluorophore and tert-butyldiphenylsilyl as a recognition domain for F(-), a ratiometric two-photon fluorescent fluoride probe, QF, was synthesized and fully characterized. QF displays both one- and two-photon ratiometric responses towards fluoride ions in aqueous solution. QF was enabled to detect exogenous fluoride ions in living cells by a ratiometric method. Two-photon microscopic imaging of fluoride ions in living HeLa cells and zebrafish has also been achieved. QF has been demonstrated to be an excellent fluorescent probe with high selectivity, low cytotoxicity and good photostability. PMID:27353376

  10. Single-trial imaging of spikes and synaptic potentials in single neurons in brain slices with genetically encoded hybrid voltage sensor

    PubMed Central

    Ghitani, Nima; Bayguinov, Peter O.; Ma, Yihe

    2014-01-01

    Genetically encoded voltage sensors expand the optogenetics toolkit into the important realm of electrical recording, enabling researchers to study the dynamic activity of complex neural circuits in real time. However, these probes have thus far performed poorly when tested in intact neural circuits. Hybrid voltage sensors (hVOS) enable the imaging of voltage by harnessing the resonant energy transfer that occurs between a genetically encoded component, a membrane-tethered fluorescent protein that serves as a donor, and a small charged molecule, dipicrylamine, which serves as an acceptor. hVOS generates optical signals as a result of voltage-induced changes in donor-acceptor distance. We expressed the hVOS probe in mouse brain by in utero electroporation and in transgenic mice with a neuronal promoter. Under conditions favoring sparse labeling we could visualize single-labeled neurons. hVOS imaging reported electrically evoked fluorescence changes from individual neurons in slices from entorhinal cortex, somatosensory cortex, and hippocampus. These fluorescence signals tracked action potentials in individual neurons in a single trial with excellent temporal fidelity, producing changes that exceeded background noise by as much as 16-fold. Subthreshold synaptic potentials were detected in single trials in multiple distinct cells simultaneously. We followed signal propagation between different cells within one field of view and between dendrites and somata of the same cell. hVOS imaging thus provides a tool for high-resolution recording of electrical activity from genetically targeted cells in intact neuronal circuits. PMID:25411462

  11. Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

    PubMed Central

    Shirmanova, Marina; Yuzhakova, Diana; Snopova, Ludmila; Perelman, Gregory; Serebrovskaya, Ekaterina; Lukyanov, Konstantin; Turchin, Ilya; Subochev, Pavel; Lukyanov, Sergey; Kamensky, Vladislav; Zagaynova, Elena

    2015-01-01

    The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation. PMID:26657001

  12. Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

    PubMed

    Shirmanova, Marina; Yuzhakova, Diana; Snopova, Ludmila; Perelman, Gregory; Serebrovskaya, Ekaterina; Lukyanov, Konstantin; Turchin, Ilya; Subochev, Pavel; Lukyanov, Sergey; Kamensky, Vladislav; Zagaynova, Elena

    2015-01-01

    The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation. PMID:26657001

  13. Single-trial imaging of spikes and synaptic potentials in single neurons in brain slices with genetically encoded hybrid voltage sensor.

    PubMed

    Ghitani, Nima; Bayguinov, Peter O; Ma, Yihe; Jackson, Meyer B

    2015-02-15

    Genetically encoded voltage sensors expand the optogenetics toolkit into the important realm of electrical recording, enabling researchers to study the dynamic activity of complex neural circuits in real time. However, these probes have thus far performed poorly when tested in intact neural circuits. Hybrid voltage sensors (hVOS) enable the imaging of voltage by harnessing the resonant energy transfer that occurs between a genetically encoded component, a membrane-tethered fluorescent protein that serves as a donor, and a small charged molecule, dipicrylamine, which serves as an acceptor. hVOS generates optical signals as a result of voltage-induced changes in donor-acceptor distance. We expressed the hVOS probe in mouse brain by in utero electroporation and in transgenic mice with a neuronal promoter. Under conditions favoring sparse labeling we could visualize single-labeled neurons. hVOS imaging reported electrically evoked fluorescence changes from individual neurons in slices from entorhinal cortex, somatosensory cortex, and hippocampus. These fluorescence signals tracked action potentials in individual neurons in a single trial with excellent temporal fidelity, producing changes that exceeded background noise by as much as 16-fold. Subthreshold synaptic potentials were detected in single trials in multiple distinct cells simultaneously. We followed signal propagation between different cells within one field of view and between dendrites and somata of the same cell. hVOS imaging thus provides a tool for high-resolution recording of electrical activity from genetically targeted cells in intact neuronal circuits. PMID:25411462

  14. Chemical Calcium Indicators

    PubMed Central

    Paredes, R. Madelaine; Etzler, Julie C.; Watts, Lora Talley; Lechleiter, James D.

    2008-01-01

    Our understanding of the underlying mechanisms of Ca2+ signaling as well as our appreciation for its ubiquitous role in cellular processes and has been rapidly advanced, in large part, due to the development of fluorescent Ca2+ indicators. In this chapter, we discuss some of the most common chemical Ca2+ indicators that are widely used for the investigation of intracellular Ca2+ signaling. Advantages, limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. Chemical indicators that are now available allow for intracellular Ca2+ detection over a very large range (<50 nM to >50 μM). Higher affinity indicators can be used to quantify Ca2+ levels in the cytosol while lower affinity indicators can be optimized for measuring Ca2+ in subcellular compartments with higher concentrations. Indicators can be classified into either single wavelength or ratiometric dyes. Both classes require specific lasers, filters, and/or detection methods that are dependent upon their spectral properties and both classes have advantages and limitations. Single wavelength indicators are generally very bright and optimal for Ca2+ detection when more than one fluorophore is being imaging. Ratiometric indicators can be calibrated very precisely and they minimize the most common problems associated with chemical Ca2+ indicators including uneven dye loading, leakage, photobleaching and changes in cell volume. Recent technical advances that permit in vivo Ca2+ measurements will also be discussed. PMID:18929663

  15. Dual-emission of a fluorescent graphene oxide-quantum dot nanohybrid for sensitive and selective visual sensor applications based on ratiometric fluorescence.

    PubMed

    Zhu, Houjuan; Zhang, Wen; Zhang, Kui; Wang, Suhua

    2012-08-10

    A novel nanohybrid ratiometric fluorescence probe comprised of fluorescent graphene oxide and quantum dots (QDs) has been prepared by bringing CdTe QDs of red fluorescence and fluorescent graphene oxide (FGO) of blue fluorescence together through electrostatic attraction and hydrogen bonding interaction between their surface functional groups including carboxyl and amine groups. The nanohybrid ratiometric fluorescence probe exhibits dual emissions at 450 and 650 nm under a single excitation wavelength and shows high sensitivity for the detection of ferrous ions in the presence of H₂O₂. Ferrous ions reacts with H₂O₂ to generate very reactive hydroxyl radicals which possess a strong oxidizing nature and easily capture the electrons from the surfaces of the CdTe QDs, leading to fluorescence quenching of the QDs and no effect on the fluorescence of the graphene oxide, which hence results in a great change of the fluorescence ratio. Moreover, the ratiometric fluorescence probe is not only extremely sensitive to ferrous ions, but is also selective over other biologically relevant metal cations. The changes of fluorescence colour ratios can be used for visual sensing applications for ferrous ions in the presence of hydrogen peroxide, and can also be used for the indication of the existence of hydrogen peroxide. PMID:22797082

  16. DNA-regulated silver nanoclusters for label-free ratiometric fluorescence detection of DNA.

    PubMed

    Liu, Lin; Yang, Qianhui; Lei, Jianping; Xu, Nan; Ju, Huangxian

    2014-11-18

    Two kinds of DNA-regulated Ag nanoclusters were one-pot synthesized on an oligonucleotide, and delicately utilized in the design of a label-free ratiometric fluorescence strategy for DNA detection with simplicity and high sensitivity. PMID:25247781

  17. A Simple and Effective Ratiometric Fluorescent Probe for the Selective Detection of Cysteine and Homocysteine in Aqueous Media.

    PubMed

    Na, Risong; Zhu, Meiqing; Fan, Shisuo; Wang, Zhen; Wu, Xiangwei; Tang, Jun; Liu, Jia; Wang, Yi; Hua, Rimao

    2016-01-01

    Biothiols such as cysteine (Cys) and homocysteine (Hcy) are essential biomolecules participating in molecular and physiological processes in an organism. However, their selective detection remains challenging. In this study, ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5-carboxylate (NL) was synthesized as a ratiometric fluorescent probe for the rapid and selective detection of Cys and Hcy over glutathione (GSH) and other amino acids. The fluorescence intensity of the probe in the presence of Cys/Hcy increased about 3-fold at a concentration of 20 equiv. of the probe, compared with that in the absence of these chemicals in aqueous media. The limits of detection of the fluorescent assay were 0.911 μM and 0.828 μM of Cys and Hcy, respectively. ¹H-NMR and MS analyses indicated that an excited-state intramolecular proton transfer is the mechanism of fluorescence sensing. This ratiometric probe is structurally simple and highly selective. The results suggest that it has useful applications in analytical chemistry and diagnostics. PMID:27527138

  18. FRET ratiometric probes reveal the chiral-sensitive cysteine-dependent H2S production and regulation in living cells

    NASA Astrophysics Data System (ADS)

    Wei, Lv; Yi, Long; Song, Fanbo; Wei, Chao; Wang, Bai-Fan; Xi, Zhen

    2014-04-01

    Hydrogen sulfide (H2S) is an endogenously produced gaseous signalling molecule with multiple biological functions. In order to visualize and quantify the endogenous in situ production of H2S in living cells, here we developed two new sulphide ratiometric probes (SR400 and SR550) based on fluorescence resonance energy transfer (FRET) strategy for live capture of H2S. The FRET-based probes show excellent selectivity toward H2S in a high thiol background under physiological buffer. The probe can be used to in situ visualize cysteine-dependent H2S production in a chiral-sensitive manner in living cells. The ratiometric imaging studies indicated that D-Cys induces more H2S production than that of L-Cys in mitochondria of human embryonic kidney 293 cells (HEK293). The cysteine mimics propargylglycine (PPG) has also been found to inhibit the cysteine-dependent endogenous H2S production in a chiral-sensitive manner in living cells. D-PPG inhibited D-Cys-dependent H2S production more efficiently than L-PPG, while, L-PPG inhibited L-Cys-dependent H2S production more efficiently than D-PPG. Our bioimaging studies support Kimura's discovery of H2S production from D-cysteine in mammalian cells and further highlight the potential of D-cysteine and its derivatives as an alternative strategy for classical H2S-releasing drugs.

  19. Ratiometric fluorescent probe for alkaline phosphatase based on betaine-modified polyethylenimine via excimer/monomer conversion.

    PubMed

    Zheng, Fangyuan; Guo, Sihua; Zeng, Fang; Li, Jun; Wu, Shuizhu

    2014-10-01

    Alkaline phosphatase (ALP) is an important diagnostic indicator for a number of human diseases since abnormal level of ALP is closely related to a variety of pathological processes; hence, the development of convenient and reliable assay methods for monitoring ALP is of great significance for medical sciences as well as biological diagnostics. Herein, we report the first ratiometric fluorescent sensing system for ALP. This sensing system consists of two components: the betaine-modified and positively charged polyethylenimine (PEI) and the negatively charged pyrene derivative containing one ALP-responsive phosphate group (Py-P, an aliphatic phosphate ester). In the absence of ALP, the two-component sensing system shows the excimer's emission of Py-P, since Py-P molecules complex with the positively charged polyelectrolyte via electrostatic interactions, leading to the formation of pyrene excimers. While in the presence of ALP, the phosphate moieties are cleaved from Py-P molecules due to the enzymatic reaction, thereby destroying the electrostatic interactions; as a result, the system displays the monomer emission of Py-P. This assay system is operable in aqueous media with a very low detection limit of 0.1 U/mL. The system is capable of detecting ALP in such biological fluid as serum, and this strategy may provide a new and effective approach for designing ratiometric sensing systems for detecting other biomolecules. PMID:25211600

  20. Quantitative description of RF power-based ratiometric chemical exchange saturation transfer (CEST) pH imaging

    PubMed Central

    Wu, Renhua; Longo, Dario Livio; Aime, Silvio; Sun, Phillip Zhe

    2015-01-01

    Chemical exchange saturation transfer (CEST) MRI holds great promise for imaging pH. However, routine CEST measurement varies not only with pH-dependent chemical exchange rate but also with CEST agent concentration, providing pH-weighted information. Conventional ratiometric CEST imaging normalizes the confounding concentration factor by analyzing the relative CEST effect from different exchangeable groups, requiring CEST agents with multiple chemically distinguishable labile proton sites. Recently, an RF power-based ratiometric CEST MRI approach has been developed for concentration-independent pH MRI using CEST agents with a single exchangeable group. To facilitate quantification and optimization of the new ratiometric analysis, we quantitated RF power-based ratiometric CEST ratio (rCESTR) and derived its signal-to-noise and contrast-to-noise ratio. Using creatine as a representative CEST agent containing a single exchangeable site, our study demonstrated that optimized RF power-based ratiometric analysis provides good pH sensitivity. We showed that rCESTR follows a base-catalyzed exchange relationship with pH independent of creatine concentration. The pH accuracy of RF power-based ratiometric MRI was within 0.15–0.20 pH unit. Furthermore, absolute exchange rate can be obtained from the proposed ratiometric analysis. To summarize, RF power-based ratiometric CEST analysis provides concentration-independent pH-sensitive imaging and complements conventional multiple labile proton groups-based ratiometric CEST analysis. PMID:25807919

  1. A highly sensitive ratiometric fluorescent probe with a large emission shift for imaging endogenous cysteine in living cells.

    PubMed

    Zhu, Baocun; Guo, Bingpeng; Zhao, Yunzhou; Zhang, Bing; Du, Bin

    2014-05-15

    A new design strategy for the construction of ratiometric fluorescent probe with a large emission shift was developed. Based on this strategy, a highly selective and sensitive colorimetric and ratiometic fluorescent probe for cysteine (Cys) with a 117 nm red-shifted emission was synthesized and applied to the ratiometric imaging of endogenous Cys in living cells. PMID:24362081

  2. Ratiometric fluorescence, electrochemiluminescence, and photoelectrochemical chemo/biosensing based on semiconductor quantum dots

    NASA Astrophysics Data System (ADS)

    Wu, Peng; Hou, Xiandeng; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-04-01

    Ratiometric fluorescent sensors, which can provide built-in self-calibration for correction of a variety of analyte-independent factors, have attracted particular attention for analytical sensing and optical imaging with the potential to provide a precise and quantitative analysis. A wide variety of ratiometric sensing probes using small fluorescent molecules have been developed. Compared with organic dyes, exploiting semiconductor quantum dots (QDs) in ratiometric fluorescence sensing is even more intriguing, owing to their unique optical and photophysical properties that offer significant advantages over organic dyes. In this review, the main photophysical mechanism for generating dual-emission from QDs for ratiometry is discussed and categorized in detail. Typically, dual-emission can be obtained either with energy transfer from QDs to dyes or with independent dual fluorophores of QDs and dye/QDs. The recent discovery of intrinsic dual-emission from Mn-doped QDs offers new opportunities for ratiometric sensing. Particularly, the signal transduction of QDs is not restricted to fluorescence, and electrochemiluminescence and photoelectrochemistry from QDs are also promising for sensing, which can be made ratiometric for correction of interferences typically encountered in electrochemistry. All these unique photophysical properties of QDs lead to a new avenue of ratiometry, and the recent progress in this area is addressed and summarized here. Several interesting applications of QD-based ratiometry are presented for the determination of metal ions, temperature, and biomolecules, with specific emphasis on the design principles and photophysical mechanisms of these probes.

  3. Alzheimer’s disease imaging with a novel Tau targeted near infrared ratiometric probe

    PubMed Central

    Kim, Hye-Yeong; Sengupta, Urmi; Shao, Pin; Guerrero-Muñoz, Marcos J; Kayed, Rakez; Bai, Mingfeng

    2013-01-01

    Neurofibrillary tangles (NFTs) have long been recognized as one of the pathological hallmarks in Alzheimer’s disease (AD). Recent studies, however, showed that soluble aggregated Tau species, especially hyperphosphorylated Tau oligomers, which are formed at early stage of AD prior to the formation of NFT, disrupted neural system integration. Unfortunately, little is known about Tau aggregates, and few Tau targeted imaging probe has been reported. Successful development of new imaging methods that can visualize early stages of Tau aggregation specifically will obviously be important for AD imaging, as well as understanding Tau-associated neuropathology of AD. Here, we report the first NIR ratiometric probe, CyDPA2, that targets Tau aggregates. The specificity of CyPDA2 to aggregated Tau was evaluated with in vitro hyperphosphorylated Tau proteins (pTau), as well as ex vivo Tau samples from AD human brain samples and the tauopathy transgenic mouse model, P301L. The characteristic enhancements of absorption ratio and fluorescence intensity in CyDPA2 were observed in a pTau concentration-dependent manner. In addition, fluorescence microscopy and gel staining studies demonstrated CyDPA2-labeled Tau aggregates. These data indicate that CyDPA2 is a promising imaging probe for studying Tau pathology and diagnosing AD at an early stage. PMID:23526074

  4. Ratiometric Signaling of Hypochlorite by the Oxidative Cleavage of Sulfonhydrazide-Based Rhodamine-Dansyl Dyad.

    PubMed

    Lee, Hyo Jin; Cho, Min Jeoung; Chang, Suk-Kyu

    2015-09-01

    A reaction-based probe 1 for hypochlorite signaling was designed by the conjugation of two fluorophores, rhodamine and dansyl moieties, by the reaction of rhodamine B base with dansylhydrazine. Probe 1 exhibited pronounced hypochlorite-selective chromogenic and fluorescent signaling behavior over other oxidants used in practical applications, such as hydrogen peroxide, peracetic acid, and ammonium persulfate, as well as commonly encountered metal ions and anions. Signaling was attributed to the hypochlorite-induced oxidative cleavage of the sulfonhydrazide linkage of the probe. In particular, favorable ratiometric fluorescence signaling was possible by utilizing the emissions of the two fluorophores. A detection limit of 1.13 × 10(-6) M (0.058 ppm) was estimated for the determination of hypochlorite. A paper-based test strip was prepared and was used as a semiquantitative indicator for the presence of hypochlorite in aqueous solutions. The probe was also successfully applied for the determination of hypochlorite in practical tap water samples. PMID:26313428

  5. A colorimetric and ratiometric fluorescent probe for quantification of bovine serum albumin.

    PubMed

    Zeng, Xiaodan; Zhang, Xiaoling; Zhu, Baocun; Jia, Hongying; Li, Yamin; Xue, Juan

    2011-10-01

    A 4-aminonaphthalimide-based ratiometric fluorescent probe 1 employing the internal charge transfer (ICT) mechanism was designed and synthesized to detect bovine serum albumin (BSA). The interaction of 1 and BSA was investigated by fluorescence and UV-vis absorption spectroscopy. Upon treatment with BSA, the probe successfully exhibited a ratiometric fluorescent response at 540 nm and 480 nm. The fluorescent intensity ratio at 540 nm and 480 nm (F(540)/F(480)) increases linearly with BSA concentration in the range of 0-75.0 μg mL(-1) and the detection limit was about 2.4 ng mL(-1). Our strategy is expected to provide a methodology to quantify BSA either by a normal or by a ratiometric and colorimetric way with high sensitivity. PMID:21858298

  6. A Ratiometric Wavelength Measurement Based on a Silicon-on-Insulator Directional Coupler Integrated Device.

    PubMed

    Wang, Pengfei; Hatta, Agus Muhamad; Zhao, Haoyu; Zheng, Jie; Farrell, Gerald; Brambilla, Gilberto

    2015-01-01

    A ratiometric wavelength measurement based on a Silicon-on-Insulator (SOI) integrated device is proposed and designed, which consists of directional couplers acting as two edge filters with opposite spectral responses. The optimal separation distance between two parallel silicon waveguides and the interaction length of the directional coupler are designed to meet the desired spectral response by using local supermodes. The wavelength discrimination ability of the designed ratiometric structure is demonstrated by a beam propagation method numerically and then is verified experimentally. The experimental results have shown a general agreement with the theoretical models. The ratiometric wavelength system demonstrates a resolution of better than 50 pm at a wavelength around 1550 nm with ease of assembly and calibration. PMID:26343668

  7. A Ratiometric Wavelength Measurement Based on a Silicon-on-Insulator Directional Coupler Integrated Device

    PubMed Central

    Wang, Pengfei; Hatta, Agus Muhamad; Zhao, Haoyu; Zheng, Jie; Farrell, Gerald; Brambilla, Gilberto

    2015-01-01

    A ratiometric wavelength measurement based on a Silicon-on-Insulator (SOI) integrated device is proposed and designed, which consists of directional couplers acting as two edge filters with opposite spectral responses. The optimal separation distance between two parallel silicon waveguides and the interaction length of the directional coupler are designed to meet the desired spectral response by using local supermodes. The wavelength discrimination ability of the designed ratiometric structure is demonstrated by a beam propagation method numerically and then is verified experimentally. The experimental results have shown a general agreement with the theoretical models. The ratiometric wavelength system demonstrates a resolution of better than 50 pm at a wavelength around 1550 nm with ease of assembly and calibration. PMID:26343668

  8. Small molecule-based ratiometric fluorescence probes for cations, anions, and biomolecules

    PubMed Central

    Lee, Min Hee

    2014-01-01

    Quantitative determination of specific analytes is essential for a variety of applications ranging from life sciences to environmental monitoring. Optical sensing allows non-invasive measurements within biological milieus, parallel monitoring of multiple samples, and less invasive imaging. Among the optical sensing methods currently being explored, ratiometric fluorescence sensing has received particular attention as a technique with the potential to provide precise and quantitative analyses. Among its advantages are high sensitivity and inherent reliability, which reflect the self-calibration provided by monitoring two (or more) emissions. A wide variety of ratiometric sensing probes using small fluorescent molecules have been developed for sensing, imaging, and biomedical applications. In this research highlight, we provide an overview of the design principles underlying small fluorescent probes that have been applied to the ratiometric detection of various analytes, including cations, anions, and biomolecules in solution and in biological samples. This highlight is designed to be illustrative, not comprehensive. PMID:25286013

  9. Economical wireless optical ratiometric pH sensor

    NASA Astrophysics Data System (ADS)

    Vuppu, Sandeep; Kostov, Yordan; Rao, Govind

    2009-04-01

    The development and application of a portable, wireless fluorescence-based optical pH sensor is presented. The design incorporates the MSP430 microcontroller as the control unit, an RF transceiver for wireless communication, digital filters and amplifiers and a USB-based communication module for data transmission. The pH sensor is based on ratiometric fluorescence detection from pH sensitive dye incorporated in a peel-and-stick patch. The ability of the instrument to detect the pH of the solution with contact only between the sensor patch and the solution makes it partially non-invasive. The instrument also has the ability to transmit data wirelessly, enabling its use in processes that entail stringent temperature control and sterility. The use of the microcontroller makes it a reliable, low-cost and low-power device. The luminous intensity of the light source can be digitally controlled to maximize the sensitivity of the instrument. It has a resolution of 0.05 pH. The sensor is accurate and reversible over the pH range of 6.5-9.

  10. Ratiometric artifact reduction in low power reflective photoplethysmography.

    PubMed

    Patterson, J A C; Guang-Zhong Yang

    2011-08-01

    This paper presents effective signal-processing techniques for the compensation of motion artifacts and ambient light offsets in a reflective photoplethysmography sensor suitable for wearable applications. A ratiometric comparison of infrared (IR) and red absorption characteristics cancels out noise that is multiplicative in nature and amplitude modulation of pulsatile absorption signals enables rejection of additive noise. A low-power, discrete-time pulse-oximeter platform is used to capture IR and red photoplethysmograms so that the data used for analysis have noise levels representative of what a true body sensor network device would experience. The proposed artifact rejection algorithm is designed for real-time implementation with a low-power microcontroller while being robust enough to compensate for varying levels in ambient light as well as reducing the effects of motion-induced artifacts. The performance of the system is illustrated by its ability to extract a typical plethysmogram heart-rate waveform since the sensor is subjected to a range of physical disturbances. PMID:23851947

  11. Ratiometric fluorescent nanosensors for selective detecting cysteine with upconversion luminescence.

    PubMed

    Guan, Yunlong; Qu, Songnan; Li, Bin; Zhang, Liming; Ma, Heping; Zhang, Ligong

    2016-03-15

    Fluorescent sensors based on upconversion (UC) luminescence have been considered as a promising strategy to detect bio-analyte due to their advantages in deep penetration, minimum autofluorescence, and ratiometric fluorescent output. A prototype of nanosensors combined with mesoporous silica coated upconversion nanoparticles (UCNPs) and a fluorescein-based fluorescent probe loaded in pores was therefore designed to detect cysteine (Cys). The silica shell provided loading space for the probe and enabled the nanosensors to disperse in water. In the presence of Cys, the fluorescent probe was transformed into 5(6)-carboxyfluorescein with an emission band centering at 518 nm which was secondarily excited by the light at around 475 nm from NaYF4:Yb(3+), Tm(3+) UCNPs driven by 980 nm near-infrared (NIR) laser. The intensity ratio between green and blue luminescence (I518/I475) grew exponentially with increasing concentrations of Cys over a range of 20-200 μmolL(-1). The response of the nanosensors towards Cys was recognizable with naked eyes by luminescence color change. Evidences suggest that these nanosensors are capable of sensing Cys in aqueous solution and distinguishing Cys from homocysteine (Hcy) with kinetically-controlled selectivity. The system was further employed to detect Cys in human serum and the result was in agreement with it tested by high performance liquid chromatography with acceptable recovery. PMID:26402589

  12. Ratiometric Raman Spectroscopy for Quantification of Protein Oxidative Damage

    PubMed Central

    Jiang, Dongping; Yanney, Michael; Zou, Sige; Sygula, Andrzej

    2009-01-01

    A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement as it requires only one conjugation reaction, a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is ~0.33 pmol carbonyl/measurement. Fluorescence and/or immunoassay based techniques only detect a signal from the labeling molecule and thus yield no structural or quantitative information for the modified protein while the RMRS technique provides for protein identification and protein carbonyl quantification in a single experiment. PMID:19457432

  13. Ratiometric CdSe/ZnS quantum dot protein sensor.

    PubMed

    Tyrakowski, Christina M; Snee, Preston T

    2014-03-01

    We have created a platform for the ratiometric fluorescent sensing of targeted proteins by conjugating conjoined protein binding agent/organic dye ligands to water-soluble, emissive semiconductor quantum dots (QDs). The QD emission is tuned such that it may serve as an energy transfer donor to the dye acceptor. Upon exposure to the target proteins, these analytes bind to the surfaces of the QDs and change the microenvironments of the QD-bound dyes such that the emissive properties of the dyes are perturbed. The resulting alteration in the QD and dye fluorescence spectra creates a readout that is fully quantitative. The advantage of our methodology is that the detection of proteins is very fast as the platform is fully homogeneous, whereas the heterogeneous ELISA assay involves multiple steps with blocking agents and secondary reporters that ultimately complicate the process. The calculated detection limits for the two QD protein-sensing examples reported here are also competitive with the ubiquitous ELISA assay. PMID:24506832

  14. Fluorescence ratiometric classifier for the detection of skin pathologies

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Cosci, Alessandro; Rossari, Susanna; Kapsokalyvas, Dimitrios; Baria, Enrico; Maio, Vincenza; Massi, Daniela; De Giorgi, Vincenzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-07-01

    Detection of pre-malignant lesions in skin could help in reducing the 5 year patient mortality rates and greatly advancing the quality of life. Current gold standard for the detection of skin pathologies is a tissue biopsy and followed by a series of steps before it is examined under a light microscope by a pathologist. The disadvantage with this method is its invasiveness. Light based biomedical point spectroscopic techniques offers an adjunct technique to invasive tissue pathology. In this context, we have implemented a simple multiplexed ratiometric approach (F470/F560 and F510/F580) based on fluorescence at two excitation wavelengths 378 nm and 445 nm respectively. The emission profile at these excitation wavelengths showed a shift towards the longer wavelengths for melanoma when compared with normal and nevus. At both excitation wavelengths, we observed an increased intensity ratios for normal, followed by nevus and melanoma. This intensity ratios provide a good diagnostic capability in differentiating normal, nevus and melanocytic skin lesions. This method could be applied in vivo because of the simplicity involved in discriminating normal and pathological skin tissues.

  15. A Genetically-Encoded YFP Sensor with Enhanced Chloride Sensitivity, Photostability and Reduced pH Interference Demonstrates Augmented Transmembrane Chloride Movement by Gerbil Prestin (SLC26a5)

    PubMed Central

    Zhong, Sheng; Navaratnam, Dhasakumar; Santos-Sacchi, Joseph

    2014-01-01

    Background Chloride is the major anion in cells, with many diseases arising from disordered Cl− regulation. For the non-invasive investigation of Cl− flux, YFP-H148Q and its derivatives chameleon and Cl-Sensor previously were introduced as genetically encoded chloride indicators. Neither the Cl− sensitivity nor the pH-susceptibility of these modifications to YFP is optimal for precise measurements of Cl− under physiological conditions. Furthermore, the relatively poor photostability of YFP derivatives hinders their application for dynamic and quantitative Cl− measurements. Dynamic and accurate measurement of physiological concentrations of chloride would significantly affect our ability to study effects of chloride on cellular events. Methodology/Principal Findings In this study, we developed a series of YFP derivatives to remove pH interference, increase photostability and enhance chloride sensitivity. The final product, EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP), has a chloride Kd of 14 mM and pKa of 5.9. The bleach time constant of 175 seconds is over 15-fold greater than wild-type EYFP. We have used the sensor fused to the transmembrane protein prestin (gerbil prestin, SLC26a5), and shown for the first time physiological (mM) chloride flux in HEK cells expressing this protein. This modified fluorescent protein will facilitate investigations of dynamics of chloride ions and their mediation of cell function. Conclusions Modifications to YFP (EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP) results in a photostable fluorescent protein that allows measurement of physiological changes in chloride concentration while remaining minimally affected by changes in pH. PMID:24901231

  16. Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mt00257e Click here for additional data file.

    PubMed Central

    Hessels, Anne M.; Taylor, Kathryn M.

    2016-01-01

    The Zn2+-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn2+ from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn2+ release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn2+ concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn2+, eZinCh-2 (K d = 1 nM at pH 7.1) and eCALWY-4 (K d = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn2+ and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn2+ in both cytosol and ER, suggesting that Zn2+ was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn2+ levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn2+, nor in experiments in which cytosolic and ER Zn2+ were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF–ionomycin treatment does not result in significant changes in cytosolic Zn2+ levels as a result from Zn2+ release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn2+ dyes. PMID:26739447

  17. The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.

    PubMed

    Ching, H Y Vincent; Mascali, Florencia C; Bertrand, Hélène C; Bruch, Eduardo M; Demay-Drouhard, Paul; Rasia, Rodolfo M; Policar, Clotilde; Tabares, Leandro C; Un, Sun

    2016-03-17

    A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 μM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements. PMID:26938795

  18. Design of modular "plug-and-play" expression platforms derived from natural riboswitches for engineering novel genetically encodable RNA regulatory devices.

    PubMed

    Trausch, Jeremiah J; Batey, Robert T

    2015-01-01

    Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA leaders and regulating their expression. These regulatory elements are generally composed of two domains: an aptamer that binds a specific effector molecule and an expression platform that informs the transcriptional or translational machinery. While it was long established that riboswitch aptamers are modular and portable, capable of directing different output domains including ribozymes, switches, and fluorophore-binding modules, the same has not been demonstrated until recently for expression platforms. We have engineered and validated a set of expression platforms that regulate transcription through a secondary structural switch that can host a variety of different aptamers, including those derived through in vitro selection methods, to create novel chimeric riboswitches. These synthetic switches are capable of a highly specific regulatory response both in vitro and in vivo. Here we present the methodology for the design and engineering of chimeric switches using biological expression platforms. PMID:25605380

  19. Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as Ratiometric Biological Probes and Organelle-Selective Bioimaging Reagents.

    PubMed

    Zhang, Kenneth Yin; Liu, Hua-Wei; Tang, Man-Chung; Choi, Alex Wing-Tat; Zhu, Nianyong; Wei, Xi-Guang; Lau, Kai-Chung; Lo, Kenneth Kam-Wing

    2015-07-01

    In this Article, we present a series of cyclometalated iridium(III) polypyridine complexes of the formula [Ir(N^C)2(N^N)](PF6) that showed dual emission under ambient conditions. The structures of the cyclometalating and diimine ligands were changed systematically to investigate the effects of the substituents on the dual-emission properties of the complexes. On the basis of the photophysical data, the high-energy (HE) and low-energy (LE) emission features of the complexes were assigned to triplet intraligand ((3)IL) and triplet charge-transfer ((3)CT) excited states, respectively. Time-dependent density functional theory (TD-DFT) calculations supported these assignments and indicated that the dual emission resulted from the interruption of the communication between the higher-lying (3)IL and the lower-lying (3)CT states by a triplet amine-to-ligand charge-transfer ((3)NLCT) state. Also, the avidin-binding properties of the biotin complexes were studied by emission titrations, and the results showed that the dual-emissive complexes can be utilized as ratiometric probes for avidin. Additionally, all the complexes exhibited efficient cellular uptake by live HeLa cells. The MTT and Annexin V assays confirmed that no cell death and early apoptosis occurred during the cell imaging experiments. Interestingly, laser-scanning confocal microscopy revealed that the complexes were selectively localized on the cell membrane, mitochondria, or both, depending on the nature of the substituents of the ligands. The results of this work will contribute to the future development of dual-emissive transition metal complexes as ratiometric probes and organelle-selective bioimaging reagents. PMID:26087119

  20. Target catalyzed hairpin assembly for constructing a ratiometric electrochemical aptasensor.

    PubMed

    Gao, Fenglei; Qian, Yong; Zhang, Lei; Dai, Shizhen; Lan, Yanfei; Zhang, Yu; Du, Lili; Tang, Daoquan

    2015-09-15

    In this paper, we develop a novel dual-signaling amplified aptasensor for protein detection via target-catalyzed hairpin assembly. Thrombin was chosen as a model target. This aptasensor contains two DNA hairpins termed as H1 and H2. H1, which is modified at its 3' ends with a methylene blue (MB), consists of the aptamer sequence of human thrombin. Meanwhile, H2 which is modified at its 3' ends with a ferrocene (Fc), is partially complementary to H1. Upon the addition of target protein, it can facilitate the opening of the hairpin structure of H1 and thus accelerate the hybridization between H1 and H2, the target protein can be displaced from hairpin H1 by hairpin H2 through a process similar to DNA branch migration. The released target found another H1 to trigger the cycle, resulting in the multiplication of the Fc confined near the GE surface and MB away from the GE surface. When IFc/IMB is used as the response signal for quantitative determination of thrombin, the detection limit (41 fM) is much lower than that by using either MB or Fc alone. This new dual-signaling aptasensor is readily regenerated and shows good response toward the target. Furthermore, this amplified aptasensor shows high selectivity toward its target protein. The clever combination of the functional DNA hairpin and the novel device achieved a ratiometric electrochemical aptasensor, which could be used as a simple, sensitive high repeatability and selective platform for target protein detection. PMID:25897885

  1. Magnetic and fluorescent core-shell nanoparticles for ratiometric pH sensing

    NASA Astrophysics Data System (ADS)

    Lapresta-Fernández, Alejandro; Doussineau, Tristan; Dutz, Silvio; Steiniger, Frank; Moro, Artur J.; Mohr, Gerhard J.

    2011-10-01

    This paper describes the preparation of nanoparticles composed of a magnetic core surrounded by two successive silica shells embedding two fluorophores, showing uniform nanoparticle size (50-60 nm in diameter) and shape, which allow ratiometric pH measurements in the pH range 5-8. Uncoated iron oxide magnetic nanoparticles (~10 nm in diameter) were formed by the coprecipitation reaction of ferrous and ferric salts. Then, they were added to a water-in-oil microemulsion where the hydrophilic silica shells were obtained through hydrolysis and condensation of tetraethoxyorthosilicate together with the corresponding silylated dye derivatives—a sulforhodamine was embedded in the inner silica shell and used as the reference dye while a pH-sensitive fluorescein was incorporated in the outer shell as the pH indicator. The magnetic nanoparticles were characterized using vibrating sample magnetometry, dynamic light scattering, transmission electron microscopy, x-ray diffraction and Fourier transform infrared spectroscopy. The relationship between the analytical parameter, that is, the ratio of fluorescence between the sensing and reference dyes versus the pH was adjusted to a sigmoidal fit using a Boltzmann type equation giving an apparent pKa value of 6.8. The fluorescence intensity of the reference dye did not change significantly (~3.0%) on modifying the pH of the nanoparticle dispersion. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% of horse serum, indicating that there are no significant statistical differences at a 95% confidence level.

  2. Preassembly-driven ratiometric sensing of H2PO4(-) anions in organic and aqueous environments.

    PubMed

    Gong, Wei-tao; Na, Duo; Fang, Le; Mehdi, Hassan; Ning, Gui-ling

    2015-02-21

    Gemini surfactant-like receptor is designed and synthesized. The special preassembly phenomenon of in a nonpolar solvent facilitates the novel ratiometric fluorescence sensing of H2PO4(-)via an anion-induced reassembly process in organic solvents and an anion-induced disassembly process in water. PMID:25563510

  3. A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity.

    PubMed

    Mallick, Suman; Chandra, Falguni; Koner, Apurba L

    2016-02-01

    An ultrasensitive ratiometric fluorescent sensor made of an N,N-dimethylaminonaphthalene anhydride moiety for detection of aliphatic primary amines is reported. Biogenic amines at nanomolar concentration is detected with the additional ability to discriminate between primary, secondary and tertiary amines by using both UV-Visible and fluorescence spectroscopy. PMID:26734688

  4. Croconaine rotaxane for acid activated photothermal heating and ratiometric photoacoustic imaging of acidic pH.

    PubMed

    Guha, Samit; Shaw, Gillian Karen; Mitcham, Trevor M; Bouchard, Richard R; Smith, Bradley D

    2016-01-01

    Absorption of 808 nm laser light by liposomes containing a pH sensitive, near-infrared croconaine rotaxane dye increases dramatically in weak acid. A stealth liposome composition permits acid activated, photothermal heating and also acts as an effective nanoparticle probe for ratiometric photoacoustic imaging of acidic pH in deep sample locations, including a living mouse. PMID:26502996

  5. Naphthylamine-rhodamine-based ratiometric fluorescent probe for the determination of Pd2+ ions.

    PubMed

    Sun, Shiguo; Qiao, Bo; Jiang, Na; Wang, Jitao; Zhang, Si; Peng, Xiaojun

    2014-02-21

    A naphthylamine-rhodamine hybrid ratiometric and colorimetric fluorescent probe (RN) was designed and synthesized. RN can identify Pd(2+) ions with high selectivity and sensitivity. Furthermore, the probe can be used to monitor Pd(2+) ions in live mice by fluorescence imaging. PMID:24483148

  6. Ratiometric detection of enzyme turnover and flavin reduction using rare-earth upconverting phosphors.

    PubMed

    Harvey, Peter; Oakland, Chloë; Driscoll, Max D; Hay, Sam; Natrajan, Louise S

    2014-04-14

    Gd4O2S:Yb:Tm rare-earth upconversion phosphors have been utilised to monitor the redox behaviour of flavin mononucleotide and report on the turnover of a flavo-protein, (pentaerythritol tetranitrate reductase). The presence of two bands separated by over 300 nm in the UCP emission spectra allows ratiometric signalling of these processes with high sensitivity. PMID:24531569

  7. A FRET-based ratiometric fluorescent aptasensor for rapid and onsite visual detection of ochratoxin A.

    PubMed

    Qian, Jing; Wang, Kan; Wang, Chengquan; Hua, Mengjuan; Yang, Zhenting; Liu, Qian; Mao, Hanping; Wang, Kun

    2015-11-01

    A color change observable by the naked eye to indicate the content of an analyte is considered to be the most conceivable way of various sensing protocols. By taking advantage of the Förster resonance energy transfer (FRET) principles, we herein designed a dual-emission ratiometric fluorescent aptasensor for ochratoxin A (OTA) detection via a dual mode of fluorescent sensing and onsite visual screening. Amino group-modified OTA's aptamer was firstly labeled with the green-emitting CdTe quantum dots (gQDs) donor. The red-emitting CdTe QDs (rQDs) which were wrapped in the silica sphere could serve as the reference signal, while the gold nanoparticle (AuNP) acceptors were attached on the silica surface to bind with the thiolated complementary DNA (cDNA). The hybridization reaction between the aptamer and the cDNA brought gQD-AuNP pair close enough, thereby making the FRET occur in the aptasensor fabrication, while the subsequent fluorescence recovery induced by OTA was obtained in the detection procedure. Based on the red background of the wrapped rQDs, the aptasensor in response to increasing OTA displayed a distinguishable color change from red to yellow-green, which could be conveniently readout in solution even by the naked eye. Since the bioconjugations used as the aptasensor can be produced at large scale, this method can be used for in situ, rapid, or high-throughput OTA detection after only an incubation step in a homogeneous mode. We believe that this novel aptasensing strategy provides not only a promising method for OTA detection but also a universal model for detecting diverse targets by changing the corresponding aptamer. PMID:26396995

  8. Ratiometric imaging of gastrodermal lipid bodies in coral-dinoflagellate endosymbiosis

    NASA Astrophysics Data System (ADS)

    Luo, Y.-J.; Wang, L.-H.; Chen, W.-N. U.; Peng, S.-E.; Tzen, J. T.-C.; Hsiao, Y.-Y.; Huang, H.-J.; Fang, L.-S.; Chen, C.-S.

    2009-03-01

    Cnidaria-dinoflagellate endosymbiosis is the phenomenon of autotrophic symbionts living inside the gastrodermal cells of their animal hosts. The molecular mechanism that regulates this association remains unclear. Using quantitative microscopy, we now provide evidence that the dynamic lipid changes in gastrodermal “lipid bodies” (LBs) reflect the symbiotic status of the host cell and its symbiont in the hermatypic coral Euphyllia glabrescens. By dual-emission ratiometric imaging with a solvatochromic fluorescent probe, Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one), we showed that the in situ distribution of polar versus neutral lipids in LBs in living gastrodermal cells and symbionts can be analyzed. The ratio of Nile red fluorescence at red (R) versus green (G) wavelength region (i.e., R/G ratio) correlated with the relative molar ratio of polar (P) versus neutral (NP) lipids (i.e., P/NP ratio). The R/G ratio in host LBs increased after bleaching, indicating a decrease in neutral lipid accumulation in gastrodermal cells. On the other hand, neutral lipid accumulation inside the symbiont LBs resulted in gradual decreases of the R/G ratio as a result of bleaching. In comparison with the bleaching event, there was no relative lipid concentration change in host LBs under continual light or dark treatments as shown by insignificant R/G ratio shift. Patterns of R/G ratio shift in symbiont LBs were also different between corals undergoing bleaching and continual light/dark treatment. In the latter, there was little lipid accumulation in symbionts, with no resulting R/G ratio decrease. These results, demonstrating that the symbiotic status positively correlated with morphological and compositional changes of lipid bodies, not only highlight the pivotal role of LBs, but also implicate an involvement of lipid trafficking in regulating the endosymbiosis.

  9. A versatile approach for ratiometric time-resolved read-out of colorimetric chemosensors using broadband phosphors as secondary emitters.

    PubMed

    Borisov, Sergey M; Klimant, Ingo

    2013-07-17

    A new approach for referencing of colorimetric chemosensors is described. The sensing materials rely on combination of absorption-based indicators and inorganic phosphors. Chromium(III)-activated yttrium aluminum borate and gadolinium aluminum borate were chosen to illustrate the new sensing scheme due to their spectral properties and high chemical and photochemical stability. The ratiometric luminescence read-out becomes possible due to the overlap of at least one form of the indicator with broadband emission (650-900 nm) or excitation (400-700 nm) of the phosphor. Long luminescence decay time of the phosphors (80-150 μs) allows for complete elimination of background fluorescence originating from the media, optical components or the indicator. The versatile scheme enables robust read-out of numerous colorimetric chemosensors and probes. Examples of sensing pH (using a BF2-chelated tetraarylazadipyrromethene dye as an indicator) and carbon dioxide (a triphenylmethane dye as an indicator) are provided. It is also demonstrated that temperature can be accessed via luminescence decay time of the phosphor to enable compensation of the sensors for temperature effects. PMID:23830442

  10. Cd(II)-terpyridine-based complex as a ratiometric fluorescent probe for pyrophosphate detection in solution and as an imaging agent in living cells.

    PubMed

    Jiao, Shu-Yan; Li, Kun; Zhang, Wei; Liu, Yan-Hong; Huang, Zeng; Yu, Xiao-Qi

    2015-01-21

    The terpyridine anthracene ligand was synthesized and characterized. is a ratiometric fluorescent probe for Cd(2+) with a recognition mechanism based on intramolecular charge transfer (ICT). An complex was isolated, and its structure was established using single-crystal XRD. The complex was able to serve as a novel reversible chemosensing ensemble to allow ratiometric response to pyrophosphate (PPi) in aqueous media. Moreover, the fluorescence imaging in living cells from these two emission channels suggested that was a ratiometric probe for Cd(2+), and the in situ generated complex was also a ratiometric ensemble for PPi detection in living cells. PMID:25421139

  11. A real-time colorimetric and ratiometric fluorescent probe for sulfite.

    PubMed

    Wu, Ming-Yu; He, Ting; Li, Kun; Wu, Ming-Bo; Huang, Zheng; Yu, Xiao-Qi

    2013-05-21

    A real-time colorimetric and ratiometric fluorescent probe based on modulating the intramolecular charge transfer (ICT) of the coumarin platform for selective detection of sulfite is presented. This reaction based probe utilized the Michael addition to the dicyano-vinyl group with the detection limit of 5.8 × 10(-5) M. The probe displayed a high selectivity for sulfite over other anions and reactive sulfur especially for biothiols including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), with about 100 nm blue shift and more than 230 times intensity ratios change of the emission spectrum. Meanwhile, it could be easily observed that the probe for sulfite changes from red to pale yellow by the naked eye, and from red to blue under UV lamp immediately after the sulfite is added. To the best of our knowledge, it is the fastest response probe for sulfite ever reported, which could give a colorimetric and ratiometric fluorescent response instantly. PMID:23563108

  12. An effective colorimetric and ratiometric fluorescent probe based FRET with a large Stokes shift for bisulfite.

    PubMed

    Wu, Wen-Li; Wang, Zhao-Yang; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

    2016-01-01

    Bisulfite plays crucial roles in diverse physiological processes. Therefore, the efficient detection of bisulfite is very important. In this study, we report a colorimetric and ratiometric fluorescent probe (CPT) with a large Stokes shift (162 nm) for bisulfite (HSO3(-)) based FRET mechanism. The probe can quantitatively detect HSO3(-) with low detection limit (45 nM) and high specificity over other common anions and biothiols. A nucleophilic addition reaction was proposed for the sensing mechanism, which was confirmed by HRMS spectra. The test strips of the probe were made and used easily. Moreover, probe CPT was used to ratiometric fluorescent imaging of exogenous and endogenous HSO3(-) in living cells. PMID:27137791

  13. A simple levulinate-based ratiometric fluorescent probe for sulfite with a large emission shift.

    PubMed

    Liu, Caiyun; Wu, Huifang; Yang, Wen; Zhang, Xiaoling

    2014-01-01

    A simple 4-hydroxynaphthalimide-derived colorimetric and ratiometric fluorescent probe (1) containing a receptor of levulinate moiety was designed and synthesized to monitor sulfite. Probe 1 could quantificationally detect sulfite by a ratiometric fluorescence spectroscopy method with high selectivity and sensitivity. Specially, probe 1 exhibited a 100 nm red-shifted absorption spectrum along with the color changes from colorless to yellow, and 103 nm red-shifted emission spectra upon the addition of sulfite. Thus, 1 can serve as a "naked-eye" probe for sulfite. Further, the recognition mechanism of probe 1 for sulfite was confirmed using nuclear magnetic resonance and electrospray ionization mass spectrometry. Also, the preliminary practical application demonstrated that our proposed probe provided a promising method for the determination of sulfite. PMID:24813958

  14. A Ratiometric Luminescent Switch Based on Platinum Complexes Tethered to a Crown-Ether Scaffold.

    PubMed

    Sinn, Stephan; Biedermann, Frank; Vishe, Mahesh; Aliprandi, Alessandro; Besnard, Céline; Lacour, Jérôme; De Cola, Luisa

    2016-06-17

    A ratiometric chemosensor for potassium is reported, based on phosphorescent dinuclear cyclometalated Pt(II) complexes featuring a cis-crown ether as the cation-recognition unit. The metal complexes are blue luminescent in a non-aggregated state but become strongly orange emissive when in a close physical proximity, as is the case when the macrocycle is in the folded state. Upon binding of the cation, unfolding occurs, resulting in a pronounced change in the emission properties (e.g. emission wavelength), which can be used for ratiometric sensing applications. The reversibility of the binding was confirmed by competitive titration experiments with unsubstituted 18-crown-6; the system shows supramolecular switching behavior. PMID:26918952

  15. An integrated high-performance ratio-metric wavelength measurement device on glass

    NASA Astrophysics Data System (ADS)

    Wang, Gencheng; Yang, Bing; Shen, Ao; Pei, Chongyang; Yang, Longzhi; Yu, Hui; Jiang, Xiaoqing; Li, Yubo; Hao, Yinlei; Yang, Jianyi

    2015-10-01

    The measurable wavelength range and the resolution of the ratio-metric wavelength monitor are limited by each other in a conventional structure. To solve this problem we designed and fabricated a high-performance integrated double ratio-metric wavelength measurement device on glass by the method of ion-exchange. It consists of four unbalanced Mach-Zehnder interferometers (MZIs) to form a rough wavelength measurement with a wide range and a fine wavelength measurement with high resolution. The highest measured resolution can reach 10 pm in a 1.6 nm-wide wavelength range for the fine wavelength measurement together with a 45 nm-wide wavelength range for the rough measurement. By heating the unbalanced MZI, the performance of the fine wavelength monitor can be improved.

  16. A near-Infrared Fluorescent Chemodosimeter for Ratiometric Detecting Fluoride Based on Desilylation Reaction.

    PubMed

    Xie, Puhui; Guo, Fengqi; Gao, Guangqin; Fan, Wei; Yang, Guoyu; Xie, Lixia

    2016-09-01

    A new chemodosimeter based on dicyanomethylene-4H-chromene chromophore (probe 1) was developed as a ratiometric fluorescent probe in near-infrared range for F(-) with good selectivity in acetonitrile. Probe 1 could be used to directly visualize F(-) by the naked eye and showed more than 621-fold fluorescence enhancement at 715 nm upon reaction with F(-) upon excitation at 625 nm. The recognition of probe 1 to fluoride was featured by F(-)-induced red-shifts of both absorption (185 nm) and fluorescence peaks (132 nm) based on internal charge transfer (ICT) in acetonitrile. The desilylation reaction of 1 by F(-) was proposed for its dual absorption and emission ratiometric detection of fluoride. PMID:27365125

  17. A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range.

    PubMed

    Xue, Lin; Prifti, Efthymia; Johnsson, Kai

    2016-04-27

    We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics. PMID:27071001

  18. Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

    PubMed

    Rana, Subinoy; Elci, S Gokhan; Mout, Rubul; Singla, Arvind K; Yazdani, Mahdieh; Bender, Markus; Bajaj, Avinash; Saha, Krishnendu; Bunz, Uwe H F; Jirik, Frank R; Rotello, Vincent M

    2016-04-01

    Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling. PMID:26967961

  19. An effective colorimetric and ratiometric fluorescent probe based FRET with a large Stokes shift for bisulfite

    PubMed Central

    Wu, Wen-Li; Wang, Zhao-Yang; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

    2016-01-01

    Bisulfite plays crucial roles in diverse physiological processes. Therefore, the efficient detection of bisulfite is very important. In this study, we report a colorimetric and ratiometric fluorescent probe (CPT) with a large Stokes shift (162 nm) for bisulfite (HSO3−) based FRET mechanism. The probe can quantitatively detect HSO3− with low detection limit (45 nM) and high specificity over other common anions and biothiols. A nucleophilic addition reaction was proposed for the sensing mechanism, which was confirmed by HRMS spectra. The test strips of the probe were made and used easily. Moreover, probe CPT was used to ratiometric fluorescent imaging of exogenous and endogenous HSO3− in living cells. PMID:27137791

  20. A simple ratiometric and colorimetric chemosensor for the selective detection of fluoride in DMSO buffered solution

    NASA Astrophysics Data System (ADS)

    Niu, Hu; Shu, Qinghai; Jin, Shaohua; Li, Bingjun; Zhu, Jiaping; Li, Lijie; Chen, Shusen

    2016-01-01

    A derivative of squaramide (cyclobuta[b]quinoxaline-1, 2(3H, 8H)-dione) has been synthesized for the ratiometric and colorimetric sensing of F- in aqueous solution in competitive fashion. With F-, probe 1 showed a highly selective naked-eye detectable color change along with a characteristic UV-Vis absorbance over other tested ions, which probably originates from the deprotonation occurred between 1 and F-, as proved by the 1H NMR titration experiments and DFT calculations.

  1. Stimulus and Network Dynamics Collide in a Ratiometric Model of the Antennal Lobe Macroglomerular Complex

    PubMed Central

    Chong, Kwok Ying; Capurro, Alberto; Karout, Salah; Pearce, Timothy Charles

    2012-01-01

    Time is considered to be an important encoding dimension in olfaction, as neural populations generate odour-specific spatiotemporal responses to constant stimuli. However, during pheromone mediated anemotactic search insects must discriminate specific ratios of blend components from rapidly time varying input. The dynamics intrinsic to olfactory processing and those of naturalistic stimuli can therefore potentially collide, thereby confounding ratiometric information. In this paper we use a computational model of the macroglomerular complex of the insect antennal lobe to study the impact on ratiometric information of this potential collision between network and stimulus dynamics. We show that the model exhibits two different dynamical regimes depending upon the connectivity pattern between inhibitory interneurons (that we refer to as fixed point attractor and limit cycle attractor), which both generate ratio-specific trajectories in the projection neuron output population that are reminiscent of temporal patterning and periodic hyperpolarisation observed in olfactory antennal lobe neurons. We compare the performance of the two corresponding population codes for reporting ratiometric blend information to higher centres of the insect brain. Our key finding is that whilst the dynamically rich limit cycle attractor spatiotemporal code is faster and more efficient in transmitting blend information under certain conditions it is also more prone to interference between network and stimulus dynamics, thus degrading ratiometric information under naturalistic input conditions. Our results suggest that rich intrinsically generated network dynamics can provide a powerful means of encoding multidimensional stimuli with high accuracy and efficiency, but only when isolated from stimulus dynamics. This interference between temporal dynamics of the stimulus and temporal patterns of neural activity constitutes a real challenge that must be successfully solved by the nervous system

  2. A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A.

    PubMed

    Dai, Zi-Ru; Ge, Guang-Bo; Feng, Lei; Ning, Jing; Hu, Liang-Hai; Jin, Qiang; Wang, Dan-Dan; Lv, Xia; Dou, Tong-Yi; Cui, Jing-Nan; Yang, Ling

    2015-11-18

    Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems. PMID:26488456

  3. Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions.

    PubMed

    Ma, Victor Pui-Yan; Liu, Yang; Blanchfield, Lori; Su, Hanquan; Evavold, Brian D; Salaita, Khalid

    2016-07-13

    Short-range communication between cells is required for the survival of multicellular organisms. One mechanism of chemical signaling between adjacent cells employs surface displayed ligands and receptors that only bind when two cells make physical contact. Ligand-receptor complexes that form at the cell-cell junction and physically bridge two cells likely experience mechanical forces. A fundamental challenge in this area pertains to mapping the mechanical forces experienced by ligand-receptor complexes within such a fluid intermembrane junction. Herein, we describe the development of ratiometric tension probes for direct imaging of receptor tension, clustering, and lateral transport within a model cell-cell junction. These probes employ two fluorescent reporters that quantify both the ligand density and the ligand tension and thus generate a tension signal independent of clustering. As a proof-of-concept, we applied the ratiometric tension probes to map the forces experienced by the T-cell receptor (TCR) during activation and showed the first direct evidence that the TCR-ligand complex experiences sustained pN forces within a fluid membrane junction. We envision that the ratiometric tension probes will be broadly useful for investigating mechanotransduction in juxtacrine signaling pathways. PMID:27192323

  4. Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

    PubMed

    Fu, Hongxia; Li, Yanru; Sun, Lingbo; He, Pan; Duan, Xinrui

    2015-11-17

    Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules. PMID:26501208

  5. Ratiometric Optical Temperature Sensor Using Two Fluorescent Dyes Dissolved in an Ionic Liquid Encapsulated by Parylene Film

    PubMed Central

    Kan, Tetsuo; Aoki, Hironori; Binh-Khiem, Nguyen; Matsumoto, Kiyoshi; Shimoyama, Isao

    2013-01-01

    A temperature sensor that uses temperature-sensitive fluorescent dyes is developed. The droplet sensor has a diameter of 40 μm and uses 1 g/L of Rhodamine B (RhB) and 0.5 g/L of Rhodamine 110 (Rh110), which are fluorescent dyes that are dissolved in an ionic liquid (1-ethyl-3-methylimidazolium ethyl sulfate) to function as temperature indicators. This ionic liquid is encapsulated using vacuum Parylene film deposition (which is known as the Parylene-on-liquid-deposition (PoLD) method). The droplet is sealed by the chemically stable and impermeable Parylene film, which prevents the dye from interacting with the molecules in the solution and keeps the volume and concentration of the fluorescent material fixed. The two fluorescent dyes enable the temperature to be measured ratiometrically such that the droplet sensor can be used in various applications, such as the wireless temperature measurement of microregions. The sensor can measure the temperature of such microregions with an accuracy of 1.9 °C, a precision of 3.7 °C, and a fluorescence intensity change sensitivity of 1.0%/K. The sensor can measure temperatures at different sensor depths in water, ranging from 0 to 850 μm. The droplet sensor is fabricated using microelectromechanical system (MEMS) technology and is highly applicable to lab-on-a-chip devices. PMID:23535716

  6. A novel ratiometric two-photon fluorescent probe for imaging of Pd(2+) ions in living cells and tissues.

    PubMed

    Zhou, Liyi; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd(2+) in biosystems, herein, we reported a ratiometric two-photon fluorescent probe, termed as Np-Pd, which based on a D-π-A-structure two-photon fluorophore of the naphthalimide derivative and deprotection of aryl propargyl ethers by palladium species. The probe Np-Pd displayed a more than 25-fold enhancement towards palladium species with high sensitivity and selectivity. Additionally, the probe Np-Pd was further used for fluorescence imaging of Pd(2+) ions in living cells and tissues under two-photon excitation (820nm), which showed large tissue-imaging depth (19.6-184.6μm), and a high resolution for ratiometric imaging. PMID:27203231

  7. A novel ratiometric two-photon fluorescent probe for imaging of Pd2 + ions in living cells and tissues

    NASA Astrophysics Data System (ADS)

    Zhou, Liyi; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd2 + in biosystems, herein, we reported a ratiometric two-photon fluorescent probe, termed as Np-Pd, which based on a D-π-A-structure two-photon fluorophore of the naphthalimide derivative and deprotection of aryl propargyl ethers by palladium species. The probe Np-Pd displayed a more than 25-fold enhancement towards palladium species with high sensitivity and selectivity. Additionally, the probe Np-Pd was further used for fluorescence imaging of Pd2 + ions in living cells and tissues under two-photon excitation (820 nm), which showed large tissue-imaging depth (19.6-184.6 μm), and a high resolution for ratiometric imaging.

  8. Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells.

    PubMed

    Wang, Xuzhe; Zhou, Li; Qiang, Fei; Wang, Feiyi; Wang, Rui; Zhao, Chunchang

    2016-03-10

    A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4-10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells. PMID:26893093

  9. A Three-Photon Active Organic Fluorophore for Deep Tissue Ratiometric Imaging of Intracellular Divalent Zinc.

    PubMed

    Philips, Divya Susan; Sreejith, Sivaramapanicker; He, Tingchao; Menon, Nishanth Venugopal; Anees, Palapuravan; Mathew, Jomon; Sajikumar, Sreedharan; Kang, Yuejun; Stuparu, Mihaiela Corina; Sun, Handong; Zhao, Yanli; Ajayaghosh, Ayyappanpillai

    2016-05-20

    Deep tissue bioimaging with three-photon (3P) excitation using near-infrared (NIR) light in the second IR window (1.0-1.4 μm) could provide high resolution images with an improved signal-to-noise ratio. Herein, we report a photostable and nontoxic 3P excitable donor-π-acceptor system (GMP) having 3P cross-section (σ3 ) of 1.78×10(-80)  cm(6)  s(2)  photon(-2) and action cross-section (σ3 η3 ) of 2.31×10(-81)  cm(6)  s(2)  photon(-2) , which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn(2+) binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ3 of 1.85×10(-80)  cm(6)  s(2)  photon(-2) and σ3 η3 of 3.33×10(-81)  cm(6)  s(2)  photon(-2) . The application of this probe is demonstrated for ratiometric 3P imaging of Zn(2+) in vitro using HuH-7 cell lines. Furthermore, the Zn(2+) concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn(2+) ion imaging. PMID:26991763

  10. Terbium(III)/gold nanocluster conjugates: the development of a novel ratiometric fluorescent probe for mercury(II) and a paper-based visual sensor.

    PubMed

    Qi, Yan-Xia; Zhang, Min; Zhu, Anwei; Shi, Guoyue

    2015-08-21

    In this work, a novel ratiometric fluorescent probe was developed for rapid, highly accurate, sensitive and selective detection of mercury(II) (Hg(2+)) based on terbium(III)/gold nanocluster conjugates (Tb(3+)/BSA-AuNCs), in which bovine serum albumin capped gold nanoclusters (BSA-AuNCs) acted as the signal indicator and terbium(III) (Tb(3+)) was used as the build-in reference. Our proposed ratiometric fluorescent probe exhibited unique specificity toward Hg(2+) against other common environmentally and biologically important metal ions, and had high accuracy and sensitivity with a low detection limit of 1 nM. In addition, our proposed probe was effectively employed to detect Hg(2+) in the biological samples from the artificial Hg(2+)-infected rats. More significantly, an appealing paper-based visual sensor for Hg(2+) was designed by using filter paper embedded with Tb(3+)/BSA-AuNC conjugates, and we have further demonstrated its feasibility for facile fluorescent sensing of Hg(2+) in a visual format, in which only a handheld UV lamp is used. In the presence of Hg(2+), the paper-based visual sensor, illuminated by a handheld UV lamp, would undergo a distinct fluorescence color change from red to green, which can be readily observed with naked eyes even in trace Hg(2+) concentrations. The Tb(3+)/BSA-AuNC-derived paper-based visual sensor is cost-effective, portable, disposable and easy-to-use. This work unveiled a facile approach for accurate, sensitive and selective measuring of Hg(2+) with self-calibration. PMID:26140286

  11. A Sensitive Ratiometric Fluorescent Sensor for Zinc(II) with High Selectivity

    PubMed Central

    Lv, Yuanyuan; Cao, Mingda; Li, Jiakai; Wang, Junbo

    2013-01-01

    A new fluorescent Zn2+ chemosensor (P1) based on a functionalized porphyrin was synthesized and characterized. P1 displayed dramatic ratiometric variations in absorption and fluorescent emission spectra upon exposure to Zn2+ due to the formation of a 1:1 Zn2+/P1 complex. The sensor also exhibited high selectivity and sensitivity toward Zn2+ over other common metal ions in the physiological pH range with a detection limit of 1.8 μM. The sensor showed fast response times and excellent reproducibility, thus confirming its potential applicability as a fluorescent sensor for Zn2+ sensing. PMID:23467028

  12. An Elaborate Supramolecular Assembly for a Smart Nanodevice for Ratiometric Molecular Recognition and Logic Gates.

    PubMed

    Xie, Yu-Jie; Wu, Wen-Yu; Chen, Hao; Li, Xiang; Zhang, Hao-Li; Liu, Liang-Liang; Shao, Xing-Xin; Shan, Chang-Fu; Liu, Wei-Sheng; Tang, Yu

    2016-06-01

    Ingenious approaches to supramolecular assembly for fabricating smart nanodevices is one of the more significant topics in nanomaterials research. Herein, by using surface quaternized cationic carbon dots (CDots) as the assembly and fluorescence platform, anionic sulfonatocalix[4]arene with modifiable lower and upper rims as a connector, as well as in situ coordination of Tb(3+) ions, we propose an elaborate supramolecular assembly strategy for the facile fabrication of a multifunctional nanodevice. The dynamic equilibrium characteristics of the supramolecular interaction can eventually endow this nanodevice with functions of fluorescent ratiometric molecular recognition and as a nano-logic gate with two output channels. PMID:27106796

  13. Small quinolinium-based enzymatic probes via blue-to-red ratiometric fluorescence.

    PubMed

    Wang, Pan; Du, Jiajun; Liu, Huijing; Bi, Guoqiang; Zhang, Guoqing

    2016-02-01

    A small fluorescence ratiometric probe consisting of a single dye species, N-methyl-6-hydroxyquinolinium (MHQ), and coupled enzymatic substrates, exhibits a dramatic colour change (deep blue to red) and possesses a huge response ratio (over 2000 fold) upon specific recognition of target enzymes. Such dramatic responses are attributed to the excited-state proton transfer processes of MHQ molecules in water. Here the detection of β-galactosidase and porcine pancreatic lipase is successfully demonstrated and this class of molecules has the potential to be developed as a "naked-eye" probe in vitro. PMID:26788553

  14. A cationic fluorescent polymeric thermometer for the ratiometric sensing of intracellular temperature.

    PubMed

    Uchiyama, Seiichi; Tsuji, Toshikazu; Ikado, Kumiko; Yoshida, Aruto; Kawamoto, Kyoko; Hayashi, Teruyuki; Inada, Noriko

    2015-07-01

    We developed new cationic fluorescent polymeric thermometers containing both benzothiadiazole and BODIPY units as an environment-sensitive fluorophore and as a reference fluorophore, respectively. The temperature-dependent fluorescence spectra of the thermometers enabled us to perform highly sensitive and practical ratiometric temperature sensing inside living mammalian cells. Intracellular temperatures of non-adherent MOLT-4 (human acute lymphoblastic leukaemia) and adherent HEK293T (human embryonic kidney) cells could be monitored with high temperature resolutions (0.01-1.0 °C) using the new cationic fluorescent polymeric thermometer. PMID:25988198

  15. Dual core quantum dots for highly quantitative ratiometric detection of trypsin activity in cystic fibrosis patients

    NASA Astrophysics Data System (ADS)

    Castelló Serrano, Iván; Stoica, Georgiana; Matas Adams, Alba; Palomares, Emilio

    2014-10-01

    We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help.We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for

  16. Preparation of europium-quantum dots and europium-fluorescein composite nanoparticles available for ratiometric luminescent detection of metal ions

    NASA Astrophysics Data System (ADS)

    Dong, Haitao; Liu, Yan; Wang, Dandan; Zhang, Wenzhu; Ye, Zhiqiang; Wang, Guilan; Yuan, Jingli

    2010-10-01

    The silica-encapsulated luminescent lanthanide nanoparticles have been developed for the selective tagging of a wide range of important targets in recent years, however, they are mainly limited to europium and terbium compounds. In this work, two types of europium-containing dual-luminophore silica nanoparticles, silica-encapsulated CdTe quantum dots (CdTe QDs)-BHHCT-Eu3 + complex nanoparticles and BHHCT-Eu3 + surface-bound silica-encapsulated fluorescein isothiocyanate (FITC) nanoparticles (BHHCT: 4, 4'-bis(1'', 1'', 1'', 2'', 2'', 3'', 3''-heptafluoro-4'', 6''-hexanedion-6''-yl)chlorosulfo-o-terphenyl), were successfully prepared using a water-in-oil (W/O) reverse microemulsion method. The results of transmission electron microscopy and luminescence spectroscopy characterizations indicate that the two types of nanoparticles are all monodisperse, spherical and uniform in size (~50 nm in diameter), and have well-resolved and stable dual luminescence emission properties. The CdTe QDs-BHHCT-Eu3 + nanoparticles can be excited at 365 nm to give dual-emission peaks at 535 and 610 nm, and the FITC-BHHCT-Eu3 + nanoparticles can be excited at 335 nm to give dual-emission peaks at 515 and 610 nm. The luminescence response investigations of the nanoparticles to different metal ions indicate that the new nanoparticles can be used as ratiometric luminescent sensing probes for the selective detection of Cu2 + and Fe2 + ions, respectively. The performance of the nanoparticle probe for metal ion detection was investigated.

  17. Rapid and ratiometric detection of hypochlorite with real application in tap water: molecules to low cost devices (TLC sticks).

    PubMed

    Goswami, Shyamaprosad; Manna, Abhishek; Paul, Sima; Quah, Ching Kheng; Fun, Hoong-Kun

    2013-12-25

    We have designed a chemodosimeter DPNO (weak fluorescence) which can be oxidized to HPNO (strong blue fluorescence) by OCl(-) with high selectivity and sensitivity in a ratiometric approach with a noticeably lower detection limit. The sensor could be useful for the detection of hypochlorites in tap water. PMID:24185489

  18. A Ratiometric Sensor Using Single Chirality Near-Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring.

    PubMed

    Giraldo, Juan P; Landry, Markita P; Kwak, Seon-Yeong; Jain, Rishabh M; Wong, Min Hao; Iverson, Nicole M; Ben-Naim, Micha; Strano, Michael S

    2015-08-26

    Advances in the separation and functionalization of single walled carbon nanotubes (SWCNT) by their electronic type have enabled the development of ratiometric fluorescent SWCNT sensors for the first time. Herein, single chirality SWCNT are independently functionalized to recognize either nitric oxide (NO), hydrogen peroxide (H(2)O(2)), or no analyte (remaining invariant) to create optical sensor responses from the ratio of distinct emission peaks. This ratiometric approach provides a measure of analyte concentration, invariant to the absolute intensity emitted from the sensors and hence, more stable to external noise and detection geometry. Two distinct ratiometric sensors are demonstrated: one version for H(2)O(2), the other for NO, each using 7,6 emission, and each containing an invariant 6,5 emission wavelength. To functionalize these sensors from SWCNT isolated from the gel separation technique, a method for rapid and efficient coating exchange of single chirality sodium dodecyl sulfate-SWCNT is introduced. As a proof of concept, spatial and temporal patterns of the ratio sensor response to H(2)O(2) and, separately, NO, are monitored in leaves of living plants in real time. This ratiometric optical sensing platform can enable the detection of trace analytes in complex environments such as strongly scattering media and biological tissues. PMID:25981520

  19. Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

    PubMed

    Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

    2016-08-01

    Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels. PMID:27334762

  20. Dual excitation ratiometric fluorescent pH sensor for noninvasive bioprocess monitoring: development and application.

    PubMed

    Kermis, Haley R; Kostov, Yordan; Harms, Peter; Rao, Govind

    2002-01-01

    The development and application of a fluorescent excitation-ratiometric, noninvasive pH sensor for continuous on-line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion-exchange resin, which was subsequently entrapped into a proton-permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6-9. The sensor responded rapidly (<9 min) and reversibly to changes in the solution pH with high precision. The sterilizable HPTS sensor was used for on-line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off-line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed-loop feedback control systems. PMID:12363356

  1. Dimerization of Organic Dyes on Luminescent Gold Nanoparticles for Ratiometric pH Sensing.

    PubMed

    Sun, Shasha; Ning, Xuhui; Zhang, Greg; Wang, Yen-Chung; Peng, Chuanqi; Zheng, Jie

    2016-02-01

    Synergistic effects arising from the conjugation of organic dyes onto non-luminescent metal nanoparticles (NPs) have greatly broadened their applications in both imaging and sensing. Herein, we report that conjugation of a well-known pH-insensitive dye, tetramethyl-rhodamine (TAMRA), to pH-insensitive luminescent gold nanoparticles (AuNPs) can lead to an ultrasmall nanoindicator that can fluorescently report local pH in a ratiometric way. Such synergy originated from the dimerization of TAMRA on AuNPs, of which geometry was very sensitive to surface charges of the AuNPs and can be reversely modulated through protonation of surrounding glutathione ligands. Not limited to pH-insensitive dyes, this pH-dependent dimerization can also enhance the pH sensitivity of fluorescein, a well-known pH-sensitive dye, within a larger pH range, opening up a new pathway to design ultrasmall fluorescent ratiometric nanoindicators with tunable wavelengths and pH response ranges. PMID:26748538

  2. A ratiometric theranostic probe for tumor targeting therapy and self-therapeutic monitoring.

    PubMed

    Li, Shi-Ying; Cheng, Hong; Xie, Bo-Ru; Qiu, Wen-Xiu; Song, Li-Lin; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2016-10-01

    Feedback imaging-guided precise photodynamic therapy (PDT) can facilitate the development of personalized medicine. In this work, a Förster resonance energy transfer (FRET) based theranostic probe was fabricated for simultaneous tumor targeting PDT and ratiometric imaging of the therapeutic effect. The theranostic probe (designated as P-PpIX) was comprised of a targeting moiety, a caspase-3 responsive linker, a FRET fluorophore pair and a photosensitizer. It was found that P-PpIX exhibited low intrinsic background fluorescence due to the high FRET quenching efficiency. The Arg-Gly-Asp (RGD) targeting moiety allowed P-PpIX to selectively accumulate in αvβ3 integrin overexpressed tumor cells. Upon photo irradiation, the PDT effect of P-PpIX could induce cell death with apoptosis related mechanism, and the activated caspase-3 would subsequently cleave the Asp-Glu-Val-Asp (DEVD) peptide sequence to terminate the intramolecular FRET process. The activated caspase-3 expression and the real time therapeutic efficacy could be precisely assessed in situ by the fluorescence intensity ratio of the released 5(6)-carboxylfluorescein (FAM, reporter fluorescence) and protoporphyrin IX (PpIX, internal reference fluorescence). This novel ratiometric theranostic probe could provide the real-time feedback for precise PDT. PMID:27475726

  3. Polymeric Nanoparticles with Precise Ratiometric Control over Drug Loading for Combination Therapy

    PubMed Central

    Aryal, Santosh; Hu, Che-Ming Jack; Zhang, Liangfang

    2011-01-01

    We report a novel approach for nanoparticle-based combination chemotherapy by concurrently incorporating two different types of drugs into a single polymeric nanoparticle with ratiometric control over the loading of the two drugs. By adapting metal alkoxide chemistry, we synthesize highly hydrophobic drug-poly-l-lactide (drug-PLA) conjugates, of which the polymer has the same chain length while the drug may differ. These drug-polymer conjugates are then encapsulated into lipid-coated polymeric nanoparticles through a single-step nanoprecipication method. Using doxorubicin (DOX) and camptothecin (CPT) as two model chemotherapy drugs, various ratios of DOX-PLA and CPT-PLA conjugates are loaded into the nanoparticles with over 90% loading efficiency. The resulting nanoparticles are uniform in size, size distribution and surface charge. The loading yield of DOX and CPT in the particles can be precisely controlled by simply adjusting the DOX-PLA:CPT-PLA molar ratio. Cellular cytotoxicity results show that the dual-drug loaded nanoparticles are superior to the corresponding cocktail mixtures of single-drug loaded nanoparticles. This dual-drug delivery approach offers a solution to the long-standing challenge in ratiometric control over the loading of different types of drugs onto the same drug delivery vehicle. We expect that this approach can be exploited for many types of chemotherapeutic agents containing hydroxyl groups and thus enable co-delivery of various drug combinations for combinatorial treatments of diseases. PMID:21696189

  4. Ratiometric QD-FRET Sensing of Aqueous H2S in Vitro.

    PubMed

    Shamirian, Armen; Samareh Afsari, Hamid; Wu, Donghui; Miller, Lawrence W; Snee, Preston T

    2016-06-01

    We report a platform for the ratiometric fluorescent sensing of endogenously generated gaseous transmitter H2S in its aqueous form (bisulfide or hydrogen sulfide anion) based on the alteration of Förster resonance energy transfer from an emissive semiconductor quantum dot (QD) donor to a dithiol-linked organic dye acceptor. The disulfide bridge between the two chromophores is cleaved upon exposure to bisulfide, resulting in termination of FRET as the dye diffuses away from the QD. This results in enhanced QD emission and dye quenching. The resulting ratiometric response can be correlated quantitatively to the concentration of bisulfide and was found to have a detection limit as low as 1.36 ± 0.03 μM. The potential for use in biological applications was demonstrated by measuring the response of the QD-based FRET sensor microinjected into live HeLa cells upon extracellular exposure to bisulfide. The methodology used here is built upon a highly multifunctional platform that offers numerous advantages, such as low detection limit, enhanced photochemical stability, and sensing ability within a biological milieu. PMID:27156947

  5. An efficient ratiometric fluorescent probe for tracking dynamic changes in lysosomal pH.

    PubMed

    Wang, Qianqian; Zhou, Liyi; Qiu, Liping; Lu, Danqing; Wu, Yongxiang; Zhang, Xiao-Bing

    2015-08-21

    Lysosomes are acidic organelles (approximately pH 4.5-5.5) and tracking the changes in lysosomal pH is of great biological importance. To address this issue, quite a few of fluorescent probes have been developed. However, few of these probes can realize the tracking of dynamic changes in lysosomal pH. Herein, we report a new lysosome-targeted ratiometric fluorescent probe (FR-Lys) by hybridizing morpholine with a xanthane derivative and an o-hydroxy benzoxazole group. In this probe, the morpholine group serves as a targeting unit for lysosome, the xanthane derivative exhibits a pH-modulated open/close reaction of the spirocycle, while the o-hydroxy benzoxazole moiety shows a pH modulated excited-state intramolecular proton transfer (ESIPT) process. Such a design affords the probe a ratiometric fluorescence response towards pH with pH values ranging from 4.0 to 6.3. The response of the probe to pH was fast and reversible with high selectivity. Moreover, this probe possesses further advantages such as easy synthesis, high photostability and low cytotoxicity. These features are favorable for tracking dynamic pH changes in biosystems. It was then applied for dynamic imaging pH changes in lysosomes with satisfactory results. PMID:26107774

  6. Raman and infra-red microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis.

    PubMed

    Kumar, Srividya; Verma, Taru; Mukherjee, Ria; Ariese, Freek; Somasundaram, Kumaravel; Umapathy, Siva

    2016-03-29

    Biomolecular structure elucidation is one of the major techniques for studying the basic processes of life. These processes get modulated, hindered or altered due to various causes like diseases, which is why biomolecular analysis and imaging play an important role in diagnosis, treatment prognosis and monitoring. Vibrational spectroscopy (IR and Raman), which is a molecular bond specific technique, can assist the researcher in chemical structure interpretation. Based on the combination with microscopy, vibrational microspectroscopy is currently emerging as an important tool for biomedical research, with a spatial resolution at the cellular and sub-cellular level. These techniques offer various advantages, enabling label-free, biomolecular fingerprinting in the native state. However, the complexity involved in deciphering the required information from a spectrum hampered their entry into the clinic. Today with the advent of automated algorithms, vibrational microspectroscopy excels in the field of spectropathology. However, researchers should be aware of how quantification based on absolute band intensities may be affected by instrumental parameters, sample thickness, water content, substrate backgrounds and other possible artefacts. In this review these practical issues and their effects on the quantification of biomolecules will be discussed in detail. In many cases ratiometric analysis can help to circumvent these problems and enable the quantitative study of biological samples, including ratiometric imaging in 1D, 2D and 3D. We provide an extensive overview from the recent scientific literature on IR and Raman band ratios used for studying biological systems and for disease diagnosis and treatment prognosis. PMID:26497386

  7. Ratiometric fluorescence chemosensor based on tyrosine derivatives for monitoring mercury ions in aqueous solutions.

    PubMed

    Thirupathi, Ponnaboina; Saritha née Gudelli, Ponnaboina; Lee, Keun-Hyeung

    2014-09-28

    Ratiometric fluorescent chemosensors 1 and 2 were synthesized based on tyrosine amino acid derivatives with a pyrene fluorophore. 1 and 2 showed high selectivity for Hg(II) ions among 13 metal ions in aqueous solutions. Both 1 and 2 sensitively detected Hg(II) ions in aqueous solutions by ratiometric response without interference of any of the other tested metal ions including Cu(II), Cd(II), Pb(II), and Ag(I) ions. 1 and 2 had tight binding affinities (5.72 × 10(13) M(−2), 1.15 × 10(13) M(−2)) for Hg(II) with nano-molar detection limits. The binding mode was characterized with the help of organic spectroscopic data, which revealed that the methoxyphenyl moieties of 1 and 2 played a vital role in the coordination of Hg(II). The deprotonation of the sulfonamide group is not a critical process for the binding of mercury ions. The methoxyphenyl moiety, sulfonamide group, and the C-terminal amide moiety of 1 and 2 as ligands for Hg(II) played crucial roles in the stabilization of the 2:1 complexes. PMID:25092444

  8. Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

    PubMed

    Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-01-01

    Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences. PMID:26695234

  9. A Fiber Optic Ammonia Sensor Using a Universal pH Indicator

    PubMed Central

    Rodríguez, Adolfo J.; Zamarreño, Carlos R.; Matías, Ignacio R.; Arregui, Francisco. J.; Domínguez Cruz, Rene F.; May-Arrioja, Daniel. A.

    2014-01-01

    A universal pH indicator is used to fabricate a fiber optic ammonia sensor. The advantage of this pH indicator is that it exhibits sensitivity to ammonia over a broad wavelength range. This provides a differential response, with a valley around 500 nm and a peak around 650 nm, which allows us to perform ratiometric measurements. The ratiometric measurements provide not only an enhanced signal, but can also eliminate any external disturbance due to humidity or temperature fluctuations. In addition, the indicator is embedded in a hydrophobic and gas permeable polyurethane film named Tecoflex®. The film provides additional advantages to the sensor, such as operation in dry environments, efficient transport of the element to be measured to the sensitive area of the sensor, and prevent leakage or detachment of the indicator. The combination of the universal pH indicator and Tecoflex® film provides a reliable and robust fiber optic ammonia sensor. PMID:24583969

  10. A cyclization-induced emission enhancement (CIEE)-based ratiometric fluorogenic and chromogenic probe for the facile detection of a nerve agent simulant DCP.

    PubMed

    Mahapatra, Ajit Kumar; Maiti, Kalipada; Manna, Saikat Kumar; Maji, Rajkishor; Mondal, Sanchita; Das Mukhopadhyay, Chitrangada; Sahoo, Prithidipa; Mandal, Debasish

    2015-06-14

    The first ratiometric fluorescent probe for the detection of a nerve agent simulant was developed based on tandem phosphorylation and intramolecular cyclization, by which high sensitivity as well as large emission shift could be achieved. PMID:25980383