The Experts below are selected from a list of 5115 Experts worldwide ranked by ideXlab platform
Markita P. Landry - One of the best experts on this subject based on the ideXlab platform.
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high throughput evolution of near infrared serotonin Nanosensors
bioRxiv, 2019Co-Authors: Sanghwa Jeong, Darwin Yang, Abraham G Beyene, Anneliese M M Gest, Markita P. LandryAbstract:ABSTRACT Release and reuptake of neuromodulator serotonin, 5-HT, is central to mood regulation and neuropsychiatric disorders, whereby imaging serotonin is of fundamental importance to study the brain’s serotonin signaling system. We introduce a reversible near-infrared nanosensor for serotonin (nIRHT), for which synthetic molecular recognition toward serotonin is systematically evolved from ssDNA-carbon nanotube constructs generated from large libraries of 6.9 × 1010 unique ssDNA sequences. nIRHT produces a ∼200% fluorescence enhancement upon exposure to serotonin with a Kd = 6.3 µM affinity. nIRHT shows selective responsivity towards serotonin over serotonin analogs, metabolites, and receptor-targeting drugs, and a 5-fold increased affinity for serotonin over dopamine. Further, nIRHT can be introduced into the brain extracellular space in acute slice, and can be used to image exogenous serotonin reversibly. Our results suggest evolution of Nanosensors could be generically implemented to rapidly develop other neuromodulator probes, and that these probes can image neuromodulator dynamics at spatiotemporal scales compatible with endogenous neuromodulation.
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electrostatic assemblies of single walled carbon nanotubes and sequence tunable peptoid polymers detect a lectin protein and its target sugars
Nano Letters, 2019Co-Authors: Linda Chio, Markita P. Landry, Ian R Mcfarlane, Jae Hong Kim, Ronald N. Zuckermann, Jackson Travis Del Bonisodonnell, Mark KlineAbstract:A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent Nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNTs) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin's target sugars, suggesting the lectin protein remains active and selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs and also demonstrate these assemblies can serve as optical Nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.
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Electrostatic Assemblies of Single-Walled Carbon Nanotubes and Sequence-Tunable Peptoid Polymers Detect a Lectin Protein and Its Target Sugars
2019Co-Authors: Linda Chio, Ian R Mcfarlane, Jackson Travis Del Bonis-o’donnell, Mark A. Kline, Jae Hong Kim, Ronald N. Zuckermann, Markita P. LandryAbstract:A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent Nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNTs) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin’s target sugars, suggesting the lectin protein remains active and selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs and also demonstrate these assemblies can serve as optical Nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates
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electrostatic assemblies of single walled carbon nanotubes and sequence tunable peptoid polymers detect a lectin protein and its target sugars
bioRxiv, 2018Co-Authors: Linda Chio, Markita P. Landry, Ian R Mcfarlane, Jae Hong Kim, Ronald N. Zuckermann, Jackson Travis Del Bonisodonnell, Mark KlineAbstract:A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally-occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent Nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNT) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions, and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can selectively detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. This WGA protein nanosensor is characterized with near-infrared spectroscopy and microscopy to study protein-nanosensor interaction parameters. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin conjugate sugars, suggesting the lectin protein selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs, and also demonstrate these assemblies can serve as optical Nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.
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Chemometric Approaches for Developing Infrared Nanosensors To Image Anthracyclines.
Biochemistry, 2018Co-Authors: Jackson Travis Del Bonis-o'donnell, Rebecca L. Pinals, Sanghwa Jeong, Ami Thakrar, Russell D. Wolfinger, Markita P. LandryAbstract:Generation, identification, and validation of optical probes to image molecular targets in a biological milieu remain a challenge. Synthetic molecular recognition approaches leveraging the intrinsic near-infrared fluorescence of single-walled carbon nanotubes are promising for long-term biochemical imaging in tissues. However, generation of Nanosensors for selective imaging of molecular targets requires a heuristic approach. Here, we present a chemometric platform for rapidly screening libraries of candidate single-walled carbon nanotube Nanosensors against biochemical analytes to quantify the fluorescence response to small molecules, including vitamins, neurotransmitters, and chemotherapeutics. We further show this method can be applied to identify biochemical analytes that selectively modulate the intrinsic near-infrared fluorescence of candidate Nanosensors. Chemometric analysis thus enables identification of nanosensor–analyte “hits” and also nanosensor fluorescence signaling modalities such as wavele...
Ian R Mcfarlane - One of the best experts on this subject based on the ideXlab platform.
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electrostatic assemblies of single walled carbon nanotubes and sequence tunable peptoid polymers detect a lectin protein and its target sugars
Nano Letters, 2019Co-Authors: Linda Chio, Markita P. Landry, Ian R Mcfarlane, Jae Hong Kim, Ronald N. Zuckermann, Jackson Travis Del Bonisodonnell, Mark KlineAbstract:A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent Nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNTs) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin's target sugars, suggesting the lectin protein remains active and selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs and also demonstrate these assemblies can serve as optical Nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.
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Electrostatic Assemblies of Single-Walled Carbon Nanotubes and Sequence-Tunable Peptoid Polymers Detect a Lectin Protein and Its Target Sugars
2019Co-Authors: Linda Chio, Ian R Mcfarlane, Jackson Travis Del Bonis-o’donnell, Mark A. Kline, Jae Hong Kim, Ronald N. Zuckermann, Markita P. LandryAbstract:A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent Nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNTs) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin’s target sugars, suggesting the lectin protein remains active and selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs and also demonstrate these assemblies can serve as optical Nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates
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electrostatic assemblies of single walled carbon nanotubes and sequence tunable peptoid polymers detect a lectin protein and its target sugars
bioRxiv, 2018Co-Authors: Linda Chio, Markita P. Landry, Ian R Mcfarlane, Jae Hong Kim, Ronald N. Zuckermann, Jackson Travis Del Bonisodonnell, Mark KlineAbstract:A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally-occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent Nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNT) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions, and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can selectively detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. This WGA protein nanosensor is characterized with near-infrared spectroscopy and microscopy to study protein-nanosensor interaction parameters. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin conjugate sugars, suggesting the lectin protein selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs, and also demonstrate these assemblies can serve as optical Nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.
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dual near infrared two photon microscopy for deep tissue dopamine nanosensor imaging
Advanced Functional Materials, 2017Co-Authors: Jackson Travis Del Bonisodonnell, Markita P. Landry, Abraham G Beyene, Ian R Mcfarlane, Ralph H Page, Eric G TindallAbstract:A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically relevant context of the intact brain for which photons must traverse the cranium, skin, and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, this study describes optimal excitation and emission wavelengths for through-cranium imaging, and demonstrates that near-infrared emissive Nanosensors can be photoexcited using a two-photon 1560 nm excitation source. Dopamine-sensitive Nanosensors can undergo two-photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. The two-photon absorption cross-section and quantum yield of dopamine Nanosensors are further calculated, and a two-photon power law relationship for the nanosensor excitation process is confirmed. Finally, the improved image quality of the Nanosensors embedded 2-mm-deep into a brain-mimetic tissue phantom is shown, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under two-photon excitation. The approach overcomes traditional limitations in deep-tissue fluorescence microscopy, and can enable neurotransmitter imaging in the biologically relevant milieu of the intact and living brain.
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stochastic simulation of dopamine neuromodulation for implementation of fluorescent neurochemical probes in the striatal extracellular space
ACS Chemical Neuroscience, 2017Co-Authors: Abraham G Beyene, Rebecca L. Pinals, Ian R Mcfarlane, Markita P. LandryAbstract:Imaging the dynamic behavior of neuromodulatory neurotransmitters in the extracelluar space that arise from individual quantal release events would constitute a major advance in neurochemical imaging. Spatial and temporal resolution of these highly stochastic neuromodulatory events requires concurrent advances in the chemical development of optical Nanosensors selective for neuromodulators in concert with advances in imaging methodologies to capture millisecond neurotransmitter release. Herein, we develop and implement a stochastic model to describe dopamine dynamics in the extracellular space (ECS) of the brain dorsal striatum to guide the design and implementation of fluorescent neurochemical probes that record neurotransmitter dynamics in the ECS. Our model is developed from first-principles and simulates release, diffusion, and reuptake of dopamine in a 3D simulation volume of striatal tissue. We find that in vivo imaging of neuromodulation requires simultaneous optimization of dopamine nanosensor rev...
Tza-huei Wang - One of the best experts on this subject based on the ideXlab platform.
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single quantum dot based dna nanosensor
Nature Materials, 2005Co-Authors: Chun-yang Zhang, Marcos Kuroki, Tza-huei WangAbstract:Rapid and highly sensitive detection of DNA is critical in diagnosing genetic diseases. Conventional approaches often rely on cumbersome, semi-quantitative amplification of target DNA to improve detection sensitivity. In addition, most DNA detection systems (microarrays, for example), regardless of their need for target amplification, require separation of unhybridized DNA strands from hybridized stands immobilized on a solid substrate, and are thereby complicated by solution–surface binding kinetics1,2. Here, we report an ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET) capable of detecting low concentrations of DNA in a separation-free format. This system uses quantum dots (QDs)3,4,5 linked to DNA probes to capture DNA targets. The target strand binds to a dye-labelled reporter strand thus forming a FRET donor–acceptor ensemble. The QD also functions as a concentrator that amplifies the target signal by confining several targets in a nanoscale domain. Unbound Nanosensors produce near-zero background fluorescence, but on binding to even a small amount of target DNA (∼50 copies or less) they generate a very distinct FRET signal. A nanosensor-based oligonucleotide ligation assay has been demonstrated to successfully detect a point mutation6 typical of some ovarian tumours in clinical samples.
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Single-quantum-dot-based DNA nanosensor.
Nature materials, 2005Co-Authors: Chun-yang Zhang, Hsin-chih Yeh, Marcos T Kuroki, Tza-huei WangAbstract:Rapid and highly sensitive detection of DNA is critical in diagnosing genetic diseases. Conventional approaches often rely on cumbersome, semi-quantitative amplification of target DNA to improve detection sensitivity. In addition, most DNA detection systems (microarrays, for example), regardless of their need for target amplification, require separation of unhybridized DNA strands from hybridized stands immobilized on a solid substrate, and are thereby complicated by solution-surface binding kinetics. Here, we report an ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET) capable of detecting low concentrations of DNA in a separation-free format. This system uses quantum dots (QDs) linked to DNA probes to capture DNA targets. The target strand binds to a dye-labelled reporter strand thus forming a FRET donor-acceptor ensemble. The QD also functions as a concentrator that amplifies the target signal by confining several targets in a nanoscale domain. Unbound Nanosensors produce near-zero background fluorescence, but on binding to even a small amount of target DNA (approximately 50 copies or less) they generate a very distinct FRET signal. A nanosensor-based oligonucleotide ligation assay has been demonstrated to successfully detect a point mutation typical of some ovarian tumours in clinical samples.
Tedrick Thomas Salim Lew - One of the best experts on this subject based on the ideXlab platform.
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nanosensor detection of synthetic auxins in planta using corona phase molecular recognition
ACS Sensors, 2021Co-Authors: Mervin Chunyi Ang, Tedrick Thomas Salim Lew, Niha Dhar, Duc Thinh Khong, Minkyung Park, Sreelatha Sarangapani, Jianqiao Cui, Aniket DehadraiAbstract:Synthetic auxins such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) have been extensively used in plant tissue cultures and as herbicides because they are chemically more stable and potent than most endogenous auxins. A tool for rapid in planta detection of these compounds will enhance our knowledge about hormone distribution and signaling and facilitate more efficient usage of synthetic auxins in agriculture. In this work, we show the development of real-time and nondestructive in planta NAA and 2,4-D Nanosensors based on the concept of corona phase molecular recognition (CoPhMoRe), to replace the current state-of-the-art sensing methods that are destructive and laborious. By designing a library of cationic polymers wrapped around single-walled carbon nanotubes with general affinity for chemical moieties displayed on auxins and its derivatives, we developed selective sensors for these synthetic auxins, with a particularly large quenching response to NAA (46%) and a turn-on response to 2,4-D (51%). The NAA and 2,4-D Nanosensors are demonstrated in planta across several plant species including spinach, Arabidopsis thaliana (A. thaliana), Brassica rapa subsp. chinensis (pak choi), and Oryza sativa (rice) grown in various media, including soil, hydroponic, and plant tissue culture media. After 5 h of 2,4-D supplementation to the hydroponic medium, 2,4-D is seen to accumulate in susceptible dicotyledon pak choi leaves, while no uptake is observed in tolerant monocotyledon rice leaves. As such, the 2,4-D nanosensor had demonstrated its capability for rapid testing of herbicide susceptibility and could help elucidate the mechanisms of 2,4-D transport and the basis for herbicide resistance in crops. The success of the CoPhMoRe technique for measuring these challenging plant hormones holds tremendous potential to advance the plant biology study.
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cellular lensing and near infrared fluorescent nanosensor arrays to enable chemical efflux cytometry
Nature Communications, 2021Co-Authors: Sooyeon Cho, Xun Gong, Volodymyr B Koman, Matthias Kuehne, Sun Jin Moon, Manki Son, Tedrick Thomas Salim Lew, Pavlo Gordiichuk, Xiaojia JinAbstract:Nanosensors have proven to be powerful tools to monitor single cells, achieving spatiotemporal precision even at molecular level. However, there has not been way of extending this approach to statistically relevant numbers of living cells. Herein, we design and fabricate nanosensor array in microfluidics that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). nIR fluorescent carbon nanotube array is integrated along microfluidic channel through which flowing cells is guided. We can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR profiles and extract rich information. This unique biophotonic waveguide allows for quantified cross-correlation of biomolecular information with various physical properties and creates label-free chemical cytometer for cellular heterogeneity measurement. As an example, the NCC can profile the immune heterogeneities of human monocyte populations at attomolar sensitivity in completely non-destructive and real-time manner with rate of ~600 cells/hr, highest range demonstrated to date for state-of-the-art chemical cytometry.
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real time detection of wound induced h2o2 signalling waves in plants with optical Nanosensors
Nature plants, 2020Co-Authors: Seonyeong Kwak, Volodymyr B Koman, Tedrick Thomas Salim Lew, Pavlo Gordiichuk, Minkyung Park, Kevin S Silmore, Jun Sung SeoAbstract:Decoding wound signalling in plants is critical for understanding various aspects of plant sciences, from pest resistance to secondary metabolite and phytohormone biosynthesis. The plant defence responses are known to primarily involve NADPH-oxidase-mediated H2O2 and Ca2+ signalling pathways, which propagate across long distances through the plant vasculature and tissues. Using non-destructive optical Nanosensors, we find that the H2O2 concentration profile post-wounding follows a logistic waveform for six plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa) and Arabidopsis thaliana, ranked in order of wave speed from 0.44 to 3.10 cm min−1. The H2O2 wave tracks the concomitant surface potential wave measured electrochemically. We show that the plant RbohD glutamate-receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the wound-induced H2O2 wave. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in plants. A novel optical sensor for hydrogen peroxide, based on DNA-wrapped single wall carbon nanotubes, is developed and validated in plants. It can be used in non-model species to study the kinetics of systemic H2O2 propagation in response to wounding.
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Real-time detection of wound-induced H_2O_2 signalling waves in plants with optical Nanosensors
Nature Plants, 2020Co-Authors: Tedrick Thomas Salim Lew, Seonyeong Kwak, Volodymyr B Koman, Pavlo Gordiichuk, Mervin Chunyi Ang, Duc Thinh Khong, Minkyung Park, Kevin S Silmore, Jun Sung Seo, Michael A LeeAbstract:A novel optical sensor for hydrogen peroxide, based on DNA-wrapped single wall carbon nanotubes, is developed and validated in plants. It can be used in non-model species to study the kinetics of systemic H_2O_2 propagation in response to wounding. Decoding wound signalling in plants is critical for understanding various aspects of plant sciences, from pest resistance to secondary metabolite and phytohormone biosynthesis. The plant defence responses are known to primarily involve NADPH-oxidase-mediated H_2O_2 and Ca^2+ signalling pathways, which propagate across long distances through the plant vasculature and tissues. Using non-destructive optical Nanosensors, we find that the H_2O_2 concentration profile post-wounding follows a logistic waveform for six plant species: lettuce ( Lactuca sativa ), arugula ( Eruca sativa ), spinach ( Spinacia oleracea ), strawberry blite ( Blitum capitatum ), sorrel ( Rumex acetosa ) and Arabidopsis thaliana , ranked in order of wave speed from 0.44 to 3.10 cm min^−1. The H_2O_2 wave tracks the concomitant surface potential wave measured electrochemically. We show that the plant RbohD glutamate-receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the wound-induced H_2O_2 wave. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in plants.
Wolf B Frommer - One of the best experts on this subject based on the ideXlab platform.
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glut1 and glut9 as major contributors to glucose influx in hepg2 cells identified by a high sensitivity intramolecular fret glucose sensor
Biochimica et Biophysica Acta, 2008Co-Authors: Hitomi Takanaga, Bhavna Chaudhuri, Wolf B FrommerAbstract:Abstract Genetically encoded FRET glucose Nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05–11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes as well, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens.
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conversion of a putative agrobacterium sugar binding protein into a fret sensor with high selectivity for sucrose
Journal of Biological Chemistry, 2006Co-Authors: Ida Lager, Sylvie Lalonde, Loren L Looger, Melanie Hilpert, Wolf B FrommerAbstract:Abstract Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes. Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose Nanosensors. Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 μm affinity. This FLIPsuc-4μ protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose. Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4μ for sucrose, maltose, and glucose. Mutant Nanosensors showed up to 50- and 11-fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells.
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rapid metabolism of glucose detected with fret glucose Nanosensors in epidermal cells and intact roots of arabidopsis rna silencing mutants
The Plant Cell, 2006Co-Authors: Karen Deuschle, Bhavna Chaudhuri, Ida Lager, Sylvie Lalonde, Sakiko Okumoto, Wolf B FrommerAbstract:Genetically encoded glucose Nanosensors have been used to measure steady state glucose levels in mammalian cytosol, nuclei, and endoplasmic reticulum. Unfortunately, the same Nanosensors in Arabidopsis thaliana transformants manifested transgene silencing and undetectable fluorescence resonance energy transfer changes. Expressing Nanosensors in sgs3 and rdr6 transgene silencing mutants eliminated silencing and resulted in high fluorescence levels. To measure glucose changes over a wide range (nanomolar to millimolar), Nanosensors with higher signal-to-noise ratios were expressed in these mutants. Perfusion of leaf epidermis with glucose led to concentration-dependent ratio changes for Nanosensors with in vitro K d values of 600 μM (FLIPglu-600μΔ13) and 3.2 mM (FLIPglu-3.2mΔ13), but one with 170 nM K d (FLIPglu-170nΔ13) showed no response. In intact roots, FLIPglu-3.2mΔ13 gave no response, whereas FLIPglu-600μΔ13, FLIPglu-2μΔ13, and FLIPglu-170nΔ13 all responded to glucose. These results demonstrate that cytosolic steady state glucose levels depend on external supply in both leaves and roots, but under the conditions tested they are lower in root versus epidermal and guard cells. Without photosynthesis and external supply, cytosolic glucose can decrease to
