Synthesis and Characterization of a New Red-Emitting Ca<sup>2+</sup> Indicator, Calcium Ruby

Gaillard, Stéphane; Yakovlev, A. V.; Luccardini, Camilla; Oheim, Martin; Feltz, and Anne; Mallet, Jean‐Maurice

doi:10.1021/ol070648h

Cited by 30 publications

(41 citation statements)

References 19 publications

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“…13 Azidemodified CalciumRuby-Cl (CaRbCl) dye, structure shown in Figure 1, was synthesized as described in reference. 32 Peptide 2 was covalently labeled with the azido-CaRbCl using the Cu , and 20 mM ascorbic acid, using 0.1 M Tris buffer pH 8.0 to yield a total volume of 150 uL. The mixture was reacted overnight at room temperature (ϳ20 h) and peptide 2-CaRubCl conjugate was purified using mini Ni-NTA agarose resin columns and desalted with an OPC.…”

Section: Methodsmentioning

confidence: 99%

“…), 32 dried in the speed-vac, and stored at Ϫ20°C until assembled on the QDs. Absorption spectra were collected on an Agilent Technologies 8453 UVϪvisible spectrophotometer (Santa Clara, CA).…”

mentioning

confidence: 99%

“…Fitting the curve with a Hill function in SigmaPlot derived the apparent dissociation constant (K d ) for this system. 32 A similar procedure was used for assaying BaCl 2 · 2H 2 O, MnCl 2 · 4H 2 O, MgCl 2 , NaCl, and KCl salt effects on the sensor. For comparison purposes, the ratios of acceptor/donor area were normalized with the native unexposed sensor ratio set to a value of 1.…”

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confidence: 99%

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Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Prasuhn¹,

et al. 2010

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The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD’s surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis−Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide−alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor−acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.

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Section: Methodsmentioning

confidence: 99%

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Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Prasuhn¹,

et al. 2010

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“…Attempts to solve this problem by the simultaneous imaging of fluo-3 and fura-red [24] have enjoyed limited success. This situation has prompted the development of new probes with improved characteristics such as ICPBC [25], calcium ruby [26, 27], CaSiR1 [28] and KFCA [29]. As they have become available only recently, very few examples of their applications have been described.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Hyrc

Minta²,

Escamilla³

et al. 2013

Cell Calcium

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Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (~780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd=0.49±0.07 μM; ACR-1) or low affinity (Kd=6.65±0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd =0.38±0.02 nM) or Mg2+ (Kd ~5 mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=6.31±0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

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“…[9][10][11][12][13] These fluorescent indicators, in general, present a combination of an organic fluorophore part and a Ca 2＋ -chelating moiety, and then resemble a rhodamine-like or fluorescein-like indicator. 14 The most widely used Ca 2＋ indicators, such as Quin-2, Fura-2, Indo-1, Fluo-3, Rhod-2 and Fluo-4, all incorporate a Ca 2＋ -chelating moiety, that usually presents 1,2-bis(2-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). 15 The affinity of the BAPTA moiety towards Ca 2＋ can be tuned by introduction of some groups (increased by electron substituents and decreased by electron withdrawing ones).…”

Section: ＋mentioning

confidence: 99%

Synthesis, Characterization and Biological Activities of a New Fluorescent Indicator for the Intracellular Calcium Ions

Lei

et al. 2009

Chin. J. Chem.

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A novel calcium-selective fluorescent indicator Fluo-3M AM was synthesized by introduction of a methyl group into the Ca 2＋ -chelating moiety and adequately characterized by spectral methods ( 1 H NMR, GC-MS, IR and MALDI-TOF MS). Meanwhile, its fluorescence spectra and some biological activities have been also studied. The results indicate that the new fluorescent indicator has relatively high affinity to calcium and a strong fluorescence signal, which should be useful for biomedical researchers to investigate the effects of calcium ions in biosystems.

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Synthesis and Characterization of a New Red-Emitting Ca²⁺ Indicator, Calcium Ruby

Cited by 30 publications

References 19 publications

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Synthesis, Characterization and Biological Activities of a New Fluorescent Indicator for the Intracellular Calcium Ions

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Synthesis and Characterization of a New Red-Emitting Ca2+ Indicator, Calcium Ruby

Cited by 30 publications

References 19 publications

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Synthesis, Characterization and Biological Activities of a New Fluorescent Indicator for the Intracellular Calcium Ions

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Synthesis and Characterization of a New Red-Emitting Ca²⁺ Indicator, Calcium Ruby