Wang Y.,BioTechPlex Corporation |
Wang Y.,Cytoptics Corporation |
Mao H.,BioTechPlex Corporation |
Mao H.,Cytoptics Corporation |
And 2 more authors.
Nanotechnology | Year: 2010
We have synthesized a chloride sensing quantum dots (QD) nanosensor, Cl-QD, for the dynamic measurements of chloride ion concentration in the millimolar range, a sensitivity that is applicable to most physiological intracellular chloride ion concentration ([Cl-]i) measurements in epithelial cells. The Cl-QD is synthesized by conjugating an anion receptor, 1-(2-mercapto-ethyl)-3-phenyl-thiourea (MEPTU) to a water soluble CdSe/ZnS QD at an emission wavelength of 620 nm. Upon binding of chloride ions to the Cl-QD, a photo-induced electron transfer mechanism caused the fluorescence of the QD to quench. This resulted in an inversely proportional relationship between the chloride ion concentration and the fluorescence intensity of the Cl-QD. We have utilized this Cl-QD to measure [Cl-]i in T84 and CF-PAC cultured cells, with either the C1C-2 or CFTR chloride channels being manipulated by pharmacological chloride channel activators and inhibitors. Activations of C1C-2 and CFTR chloride channels in T84 by the respective lubiprostone and genistein caused predictive increases in the fluorescence of the Cl-QD, i.e., a decrease of [Cl-]i. Conversely, glibenclamide, a chloride channel inhibitor, applied to the CF-PAC cells caused a predictable decrease in the fluorescence of Cl-QD due to the increase of [Cl-]i. These are the first data in using QD-based chloride ion sensors for dynamic measurements of intracellular chloride ion concentrations in epithelial cells. © IOP Publishing Ltd.
Wang Y.,Cytoptics Corporation |
Mao H.,Cytoptics Corporation |
Wong L.B.,Cytoptics Corporation
Talanta | Year: 2011
We have developed a Na-quantum dot (QD) nanosensor for [Na+]i measurements. Using this Na-QD, we determined the dynamic physiological responses of [Na+]i in nonexcitable human HEK-293F cells and excitable primary rat cardiac myocytes by pharmacologically manipulating the membrane permeability to Na+, the Na-K-2Cl cotransporter, and the Na+/H+ antiporter. These data suggest that the mechanisms of [Na+]i homeostasis can now be elucidated with this novel Na-QD nanosensor. This could have a broad impact on Na+ channel drug discovery. © 2011 Elsevier B.V. All rights reserved.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.03M | Year: 2011
DESCRIPTION (provided by applicant): Ion channel drug discovery has been hampered by the lack of cell-based high throughput screening (HTS) assay to physiologically and pharmacologically characterize compound activities. Cytoptics Corporation (Cytoptics) proposes to develop a new generation of chloride ion and pH sensing luminous quantum dots (QD) indicators to measure intracellular chloride ion concentrations, [Cl-]i, and pHi for fluorescence microscopy and cell-based high throughput screening (HTS) targeting GI drug discovery. These fluorescence microscopy and cell-based HTS assays utilize the size-tunable, non- overlapping emission wavelengths, ion-selective QD to measure [Cl-]i and pHi simultaneously in T84WT /T84C1C2 as well as in HEK293WT/HEK293C1C-2 cells. Using the quantum dots (QD) as Forester Resonance Energy Transfer (FRET) donors and the respective Cl- and H+ chemical sensors conjugated to the QD as acceptors, these two nanosensors, Cl-QD and pH-QD, quench their fluorescence intensities of the QDaccording to the concentrations of the Cl- and H+ bound to their respective Cl- and H+ sensors. We have accomplished all the proposed Phase I objectives, namely, 1) synthesis and spectral characterization of chloride ion sensing luminous QD, Cl-QD; 2) development of a loading protocol of the Cl-QD into T84 cells; and 3) measurements of the physio-pharmacological [Cl-]i responses in T84WT and T84C1C-2 cells using the Cl- QDTM. In Phase II, Cytoptics will incorporate and extend the accomplishments of Phase Ito produce six assays to target the cell-based GI drug target discovery market. They are (1) the Cl-QDTM assay for fluorescence microscopy; (2) the Cl-QDTM assay for cell- based HTS; (3) the pH-QD assay for fluorescence microscopy; (4) the pH-QD assay forcell-based HTS; (5) the Cl-QDTM and pH-QD integrated assay for cell-based HTS; and (6) the Cl-QDTM and pH-QD integrated assay targeting C1C-2 channels using the stable T84C1C-2 and HEK293C1C-2 cells for GI drug discovery. These novel assays address the critical needs of cell-based HTS drug discovery and will also broadly impact drug discovery research in ionic signal transduction. PUBLIC HEALTH RELEVANCE: Chloride channels and proton transporters are the two predominant drug discovery gastrointestinal (GI) target sites for the treatments of diarrhea, constipation and gastric reflux. Cytoptics Corporation proposes to develop a new generation of chloride ion and pH sensing indicators (Cl-QD and pH-QD, respectively) to simultaneously measure the dynamicsof the intracellular chloride ion concentrations ([Cl-]i) and pH (pHi) in epithelial cells for the first time. These nanosensors will be utilized to interrogate the Cl-channels and H+-transporters as direct physiological and pharmacological assays for GIdrug discovery. Using the quantum dots (QD) with different emission wavelengths for these two nanosensors, Cl-QD and pH-QD emit separate colors with their non-interfering fluorescence intensities varied according to their respective concentrations of Cl- and H+, Cytoptics will produce six assays utilizing these two novel QD-based nanosensors to target the cell-based GI HTS drug discovery market. These novel assays will also provide unparallel insights in the ionic cellular signal transduction mechanisms that heretofore not been possible.
Our proprietary technologies allow the detection and analysis of weak optical signals emitted by cells, particularly those embedded in noise. They can be used to detect and follow in real time, with millisecond resolution, multiple simultaneous signals that result from fluorescent and chemiluminescent cellular components ...
Cell-Based Fluorescent Microscopy & HTS Cytoptics provides to industry, academia and government, the evaluation of targeted agents using novel technologies and cell lines for ionic mechanism-dependent drug discovery, validation and safety pharmacology. Please send inquiry ...
News & Events June 2012. Cytoptics' published article "Na+ sensing quantum dots for cell-based screening of intracellular Na+ concentrations ([Na+]i) ...
Quantum-Dots Nanobiosensors for Fluorescent Microscopy & Cell-Based HTS 1) Cl-QD (TM) -- The chloride indicator, Cl-QD, is a new generation of chloride indicator based on quantum dots technology. It is highly quantum efficient ...
Strategic Alliances: Cytoptics is actively pursuing alliance opportunities, with the ultimate goal of synergistically creating substantial increases in value for both the partner and itself. Cytoptics diverse set of core competencies gives our company the unique ability to develop the following:- 1) advanced fluoresence based enabling technologies and optolectronic systems 2) A new generation of ion and biomarker indicators 3) Genetic Modified ion channel stable cell lines for drug discovery If you are interested in finding out how collaborating with Cytoptics could benefit your organization, please contact Dr Lid Wong at 858-695-8035