Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides

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

A Sensitive Water-Soluble Reversible Optical Probe for Hg²⁺ Detection

We report the serendipitous discovery of an optical mercury sensor while trying to develop a water-soluble manganese probe. The sensor is based on a pentaaza macrocycle conjugated to a hemicyanine dye. The pentaaza macrocycle earlier designed in our group was used to develop photoinduced electron transfer (PET)-based “turn-on” fluorescent sensors for manganese. In an attempt to increase the water-solubility of the manganese sensors we changed the dye from BODIPY to hemicyanine. The resultant molecule <b>qHCM</b> afforded a distinct reversible change in the absorption features and a concomitant visible color change upon binding to Hg²⁺ ions, leading to a highly water-soluble mercury sensor with a 10 ppb detection limit. The molecule acts as a reversible “ON–OFF” fluorescent sensor for Hg²⁺ with a 35 times decrease in the emission intensity in the presence of 1 equiv of Hg²⁺ ions. We have demonstrated the applicability of the probe for detecting Hg²⁺ ions in living cells and in live zebrafish larvae using confocal fluorescence microscopy with visible excitation. High selectivity and sensitivity toward Hg²⁺ detection make <b>qHCM</b> an attractive probe for detecting Hg²⁺ in contaminated water sources, which is a major environmental toxicity concern. We have scrutinized the altered metal-ion selectivity of the probe using density functional theory (DFT) and time-dependent DFT calculations, which show that a PET-based metal-sensing scheme is not operational in <b>qHCM</b>. ¹H NMR studies and DFT calculations indicate that Hg²⁺ ions coordinate to oxygen-donor atoms from both the chromophore and macrocycle, leading to sensitive mercury detection

A Sensitive Water-Soluble Reversible Optical Probe for Hg²⁺ Detection

We report the serendipitous discovery of an optical mercury sensor while trying to develop a water-soluble manganese probe. The sensor is based on a pentaaza macrocycle conjugated to a hemicyanine dye. The pentaaza macrocycle earlier designed in our group was used to develop photoinduced electron transfer (PET)-based “turn-on” fluorescent sensors for manganese. In an attempt to increase the water-solubility of the manganese sensors we changed the dye from BODIPY to hemicyanine. The resultant molecule <b>qHCM</b> afforded a distinct reversible change in the absorption features and a concomitant visible color change upon binding to Hg²⁺ ions, leading to a highly water-soluble mercury sensor with a 10 ppb detection limit. The molecule acts as a reversible “ON–OFF” fluorescent sensor for Hg²⁺ with a 35 times decrease in the emission intensity in the presence of 1 equiv of Hg²⁺ ions. We have demonstrated the applicability of the probe for detecting Hg²⁺ ions in living cells and in live zebrafish larvae using confocal fluorescence microscopy with visible excitation. High selectivity and sensitivity toward Hg²⁺ detection make <b>qHCM</b> an attractive probe for detecting Hg²⁺ in contaminated water sources, which is a major environmental toxicity concern. We have scrutinized the altered metal-ion selectivity of the probe using density functional theory (DFT) and time-dependent DFT calculations, which show that a PET-based metal-sensing scheme is not operational in <b>qHCM</b>. ¹H NMR studies and DFT calculations indicate that Hg²⁺ ions coordinate to oxygen-donor atoms from both the chromophore and macrocycle, leading to sensitive mercury detection

A Sensitive Water-Soluble Reversible Optical Probe for Hg²⁺ Detection

We report the serendipitous discovery of an optical mercury sensor while trying to develop a water-soluble manganese probe. The sensor is based on a pentaaza macrocycle conjugated to a hemicyanine dye. The pentaaza macrocycle earlier designed in our group was used to develop photoinduced electron transfer (PET)-based “turn-on” fluorescent sensors for manganese. In an attempt to increase the water-solubility of the manganese sensors we changed the dye from BODIPY to hemicyanine. The resultant molecule <b>qHCM</b> afforded a distinct reversible change in the absorption features and a concomitant visible color change upon binding to Hg²⁺ ions, leading to a highly water-soluble mercury sensor with a 10 ppb detection limit. The molecule acts as a reversible “ON–OFF” fluorescent sensor for Hg²⁺ with a 35 times decrease in the emission intensity in the presence of 1 equiv of Hg²⁺ ions. We have demonstrated the applicability of the probe for detecting Hg²⁺ ions in living cells and in live zebrafish larvae using confocal fluorescence microscopy with visible excitation. High selectivity and sensitivity toward Hg²⁺ detection make <b>qHCM</b> an attractive probe for detecting Hg²⁺ in contaminated water sources, which is a major environmental toxicity concern. We have scrutinized the altered metal-ion selectivity of the probe using density functional theory (DFT) and time-dependent DFT calculations, which show that a PET-based metal-sensing scheme is not operational in <b>qHCM</b>. ¹H NMR studies and DFT calculations indicate that Hg²⁺ ions coordinate to oxygen-donor atoms from both the chromophore and macrocycle, leading to sensitive mercury detection

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added <i>ortho</i>-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λ_ex = 401 nm and λ_em = 490 nm for serotonin; λ_ex = 446 nm and λ_em = 557 nm for dopamine; and λ_ex = 446 nm and λ_em = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of <i>in vitro</i> solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells