A ratiometric fluorescent viscosity sensor

The development of a dual probe that provides ratiometric measurements of fluid viscosity is described. The design is based on coupling of a primary fluorophore with viscosity-independent fluorescence emission (blue unit) with a secondary fluorophore that exhibits viscosity-sensitive fluorescent emission quantum yield (red unit). Excitation of the secondary fluorophore can be achieved via Resonance Energy Transfer. The ratio of the fluorescence emission of these fluorophores provides an accurate, ratiometric measurement of solvent viscosity.     

Molecular rotors: synthesis and evaluation as viscosity sensors

It has been shown that compounds containing the p-N,N-dialkylaminobenzylidene cyanoacetate motif can serve as fluorescent non-mechanical viscosity sensors. These compounds, referred to as molecular rotors, belong to a class of fluorescent probes that are known to form twisted intramolecular charge-transfer complexes in the excited state. In this study we present the synthesis and spectroscopic characterization of these compounds as viscosity sensors. The effects of the molecular structure and electronic density of these rotors to the emission wavelength, fluorescence intensity, and viscosity sensitivity are discussed.     

Neutral cuprous complexes as ratiometric oxygen gas sensors

Four neutral mononuclear Cu(I) complexes, [Cu(pyin)(PPh(3))(2)] (1a), [Cu(pyin)(DPEphos)] (1b), [Cu(quin)(PPh(3))(2)] (2a) and [Cu(quin)(DPEphos)] (2b) (Hpyin = 2-(2-pyridyl)indole, Hquin = 2-(2-quinolyl)indole and DPEphos = bis(2-(diphenylphosphino)phenyl)ether) have been synthesized. X-Ray crystal structure analysis revealed that the central Cu(I) ion in all complexes is in a distorted tetrahedral coordination environment. All four complexes display the typical metal-to-ligand charge transfer (MLCT) absorption band at 371, 363, 413 and 402 nm, respectively. No emission was observed from any complexes in the solid state due to triplet-triplet annihilation. However, the complexes show unusual dual-emission originating from intraligand charge-transfer (ILCT) and MLCT transitions, when dispersed in a rigid matrix (e.g. PMMA) or in frozen CH(2)Cl(2). The oxidation potential of Cu(I)/Cu(II) in these neutral complexes, ～0.5 V (vs. Ag/AgCl), is lower than those of cationic Cu(I) complexes. Films containing 10 wt% of these complexes in PMMA shows ratiometric fluorescent oxygen gas sensing property with a response ratio of 0.3-3.2 and response time of 3-4 s. Complex 2b acts as a ratiometric oxygen gas sensor with good reversibility through energy and electron transfer mechanisms under the loss of a counteranion.     

Microparticle ratiometric oxygen sensors utilizing near-infrared emitting quantum dots

Luminescent sensors incorporating two luminophores, an indicator and a reference, offer many advantages over intensity measurements from sensors made with one indicator dye. Quantum dots have yet to be widely employed as insensitive reference luminophores in such systems. This work describes the use of near-infrared emitting quantum dots in conjunction with a long-lifetime platinum(II) porphyrin phosphor in a microsphere-based, ratiometric oxygen sensor. The process for self-assembly of the nanocomposite system was developed, and the response and photostability of the prototypes were investigated. Results indicate the sensors possess excellent sensitivity (K(SV) = 0.00826 μM(-1)) at oxygen concentrations below 300 μM and were resistant to photobleaching. The sensor luminophores displayed minimal spectral overlap and little interference from excitation light, preventing the need for optical filters. A reversible photoenhancement of the quantum dot signal was also observed when exposed for extended periods of time. This work demonstrates the advantages of incorporating long-wavelength quantum dots into ratiometric intensity sensing schemes and highlights some key limitations that must be considered in their use.     

Cyclometalated iridium-coumarin ratiometric oxygen sensors: improved signal resolution and tunable dynamic ranges

In this work we introduce a new series of ratiometric oxygen sensors based on phosphorescent cyclometalated iridium centers partnered with organic coumarin fluorophores. Three different cyclometalating ligands and two different pyridyl-containing coumarin types were used to prepare six target complexes with tunable excited-state energies. Three of the complexes display dual emission, with fluorescence arising from the coumarin ligand, and phosphorescence from either the cyclometalated iridium center or the coumarin. These dual-emitting complexes function as ratiometric oxygen sensors, with the phosphorescence quenched under O₂ while fluorescence is unaffected. The use of blue-fluorescent coumarins results in good signal resolution between fluorescence and phosphorescence. Moreover, the sensitivity and dynamic range, measured with Stern–Volmer analysis, can be tuned two orders of magnitude by virtue of our ability to synthetically control the triplet excited-state ordering. The complex with cyclometalated iridium ³MLCT phosphorescence operates under hyperoxic conditions, whereas the two complexes with coumarin-centered phosphorescence are sensitive to very low levels of O₂ and function as hypoxic sensors.

Cyclometalated iridium(iii) coumarin complexes with improved signal resolution for ratiometric oxygen sensing are described. Dynamic ranges are tunable over >2 orders of magnitude.     

Colorimetric and ratiometric sensors derivated from natural building blocks for fluoride ion detection

Three novel colorimetric and ratiometric probes (SH-1~3) for fluoride ion detection were designed and synthesized from nature small molecules. Obvious yellow-to-orange color change of these probes in the THF was achieved only in presence of F^? among the eight anions (F^?, Cl^?, Br^?, I^?, H₂PO₄^?, HSO₄^?, CH₃COO^–, ClO₄^?), along with the emission shifting from green to orange red. These three probes are 1:1 complexed with fluoride ions, with complexation constant of around 0.1 × 10⁴ M^?1. The detection limit of probes SH-1~3 reached as low as around 1 μM. ¹H NMR titration study suggested that the fluoride ion induced deprotonation of the probe through hydrogen bonding interaction between amino group of probe and fluoride ion.     

Synthesis of ratiometric fluorescent nanoparticles for sensing oxygen

A facile reprecipitation-encapsulation method is used for the preparation of ratiometric fluorescent nanoparticles (NPs) for sensing intracellular oxygen. The surface of the NPs is modified in-situ with poly-L-lysine, which renders good biocompatibility and enables easy internalization into living cells. The sensor NPs contain a red fluorescent probe whose fluorescence is sensitive to oxygen with a quenching response of 77 % on going from nitrogen saturation to oxygen saturation, and a reference dye giving a green signal that acts as an oxygen-independent reference. The ratio of the two emissions serves as the analytical information and is sensitive to dissolved oxygen in the 0–43?ppm concentration range. When incorporated into cells, the ratio of the signals increases by 400?% on going from oxygen-saturated to oxygen-free environment.

Synthesis, characterization, and evaluation of rhodamine based sensors for nerve gas mimics

Six new rhodamine-B based compounds were synthesized (1-6) and used as fluorescent turn-ON sensors for diethyl chlorophosphate (DCP) in aqueous media at pH 7.0. Compound 1 and 3 gave high fluorescent enhancement with DCP compared to the other compounds. Very high selectivity and sensitivity were observed as these compounds did not show significant fluorescent enhancement with dimethyl methylphosphonate (DMMP), HCl, and metal ions, such as Na⁺, K⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺. Depending on the way the sensor is presented, results are instantaneous or observed over some minutes.     

Surface immobilization of catalytic beacons based on ratiometric fluorescent DNAzyme sensors: a systematic study

DNAzyme-based catalytic beacons have the potential for sensing a large number of relevant analytes. Thus, a systematic investigation of factors affecting their performance when immobilized into gold-coated nanocapillary array membranes (NCAMs) was undertaken. Enzyme immobilization times were varied to determine that as little as 15 min was sufficient for ratiometric detection of Pb2+-specific activity, while immobilization density saturated after 1.5 h. Immobilization of the DNAzymes into NCAMs with 600 nm pore size resulted in higher immobilization efficiency and higher enzymatic activity than that with 200 nm pore size. A poly-T linker length between the tethering thiol and first oligonucleotide, used to extend the DNAzyme above the backfilling mercaptohexanol (MCH) monolayer, had no effect on DNAzyme activity. The backfilling method of immobilization, involving backfilling followed by hybridization, was found most effective for DNAzyme activity compared to immobilization of hybridized DNAzyme complex (a 67% loss of activity) or concurrent enzyme and MCH immobilization (75% loss of activity). The backfilling MCH monolayer provided approximately 3.5 times increase in activity compared to DNAzyme assembled without MCH, and was over 5 times more active than shorter and longer backfilling molecules tested. The immobilized DNAzyme retained its optimized performance at 50 mM NaCl. Finally, the generalized immobilization and ratiometric procedure was employed for a uranyl-specific DNAzyme with 25 +/- 15 times increase in ratio observed. These findings form a firm basis on which practical applications of catalytic beacons can be realized, including sensors for both Pb2+ and UO22+ ions.     

Fluorescent peptide-based sensors for the ratiometric detection of nanomolar concentration of heparin in aqueous solutions and in serum

New fluorescent peptide-based sensors (1–3) for monitoring heparin in serum sample were synthesized using short peptides (1∼3mer) as a receptor. The peptide-based sensors (2 and 3) showed a sensitive ratiometric response to heparin both in aqueous buffered solution (10 mM HEPES, pH 7.4) and in 2% human serum sample by increase of excimer emission of pyrene at 480 nm and concomitant decrease of monomer emission of pyrene at 376 nm, whereas the peptide-based sensor 1 showed a turn off response only by decrease of monomer emission at 376 nm. 2 and 3 exhibited excellent selectivity toward heparin among various anions and competitors of heparin including chondroitin 4-sulfate (ChS) and hyaluronic acid (HA). Peptide-based sensor 3 showed a more sensitive response to heparin than 2. The detection limit of 3 was determined as 36 pM (R² = 0.998) for heparin in aqueous solution and 204 pM (R² = 0.999) for heparin in aqueous solutions containing 2% human serum. The peptide-based sensors, 2 and 3 provided a practical and potential tool for the detection and quantification of heparin in real biological samples.     

Synthesis and evaluation of bipendant-armed (oligo)thiophene crown ether derivatives as new chemical sensors

Three new (oligo)thiophene bipendant-armed ligands 2a-c, derived from 2-(aminomethyl)-15-crown-5, have been synthesized and characterized. Compounds 2a-c were prepared by reductive amination of the corresponding macrocycle with formyl thiophene derivatives 1a-c in the presence of NaBH(OAc)₃ in fair to good yields. The photophysical properties of ligands 2a-c were studied and they were also evaluated as chemosensors in the presence of Na(I), Ag(I), Pd(II) and Hg(II) cations in acetonitrile solution.     

Zinc(II)-selective ratiometric fluorescent sensors based on inhibition of excited-state intramolecular proton transfer

To develop a zinc(II)-selective emission ratiometric probe suitable for biological applications, we explored the cation-induced inhibition of excited-state intramolecular proton transfer (ESIPT) with a series of 2-(2'-benzenesulfonamidophenyl)benzimidazole derivatives. In the absence of Zn(II) at neutral pH, the fluorophores undergo ESIPT to yield a highly Stokes' shifted emission from the proton-transfer tautomer. Coordination of Zn(II) inhibits the ESIPT process and yields a significant hypsochromic shift of the fluorescence emission maximum. Whereas the paramagnetic metal cations Cu(II), Fe(II), Ni(II), Co(II), and Mn(II) result in fluorescence quenching, the emission response is not altered by millimolar concentrations of Ca(II) or Mg(II), rendering the sensors selective for Zn(II) among all biologically important metal cations. Due to the modular architecture of the fluorophore, the Zn(II) binding affinity can be readily tuned by implementing simple structural modifications. The synthesized probes are suitable to gauge free Zn(II) concentrations in the micromolar to picomolar range under physiological conditions.     

The rhodafluor family. An initial study of potential ratiometric fluorescent sensors for Zn2+

A new class of ratiometric Zn(2+) sensors that employ a hybrid fluorescein and rhodamine fluorophore has been designed, and two members of the rhodafluor family of sensors, RF1 and RF2, have been synthesized. The preparation of RF1 (9-(o-carboxyphenyl)-2-chloro-6-[bis(2-pyridylmethyl)amino]-3-xanthanone, Rhodafluor-1), uses conventional synthetic methods. Elaboration of the RF1 synthesis in an effort to enhance the Zn(2+) affinity was unsuccessful, so palladium-catalyzed aryl amination was applied to prepare RF2 (1-[9'-(o-carboxyphenyl)-6'-amino-2'-chloro-3'-xanthanone]-4,10-(diethyl)-7-(2-pyridylmethyl)-1,4,7,10-tetraazacyclododecane, Rhodafluor-2). The key step in the synthesis of RF2 is coupling of a triprotected tetraazamacrocycle (cyclen) to 3-bromoanisidine. RF2 binds Zn(2+) with a dissociation constant of 13.5 microM accompanied by an approximately 50% increase in quantum yield. Although only small shifts in absorption wavelength were observed, because protonation of the amino nitrogen atoms of the macrocycle prevents the uncomplexed sensor from adopting the desired mesomer, the intensity doubling makes the probe of value for immediate application in situations where our previous tight binding (<1 nM) sensors are inadequate.     

Solution studies of trimetallic lanthanide luminescent anion sensors: towards ratiometric sensing using an internal reference channel

Luminescent europium and terbium complexes and a mixed Eu(III)-Tb(III) complex were prepared, each with three macrocycles coordinating to a single lanthanide ion to form a trimetallic system, and can be used for the ratiometric sensing of anions in the case of the mixed complex.     

Synthesis of new, BODIPY-based sensors and labels

New, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye based thiol-reactive fluorescent label, fluorescent amino acid, and fluoroionophore compounds with 540-560 nm emission are described. Combination of a BODIPY dye with a nitronyl nitroxide or an imino nitroxide or a bifunctional pyrroline nitroxide furnished a nitric oxide, a redox sensitive molecule and a double (spin and fluorescence) label, respectively.     

Synthesis and photophysical evaluation of charge neutral thiourea or urea based fluorescent PET sensors for bis-carboxylates and pyrophosphate

The synthesis of the fluorescent photoinduced electron transfer (PET) chemosensors 1-3 for bis-anions such as bis-carboxylates and pyrophosphate in organic solvents is described herein. These sensors are based on the receptor-spacer-fluorophore-spacer-receptor motif where the receptors are charge neutral aromatic thiourea or urea receptors and the fluorophore is anthracene. The anion recognition was evaluated using 1H NMR as well as absorption and fluorescence spectroscopy in DMSO. For simple anions such as acetate or fluoride, the recognition was shown to be through hydrogen bonding of the corresponding anion to the receptors. This gave rise to only minor changes in the absorption spectra, but significant changes in the fluorescence emission spectra, which was substantially (70-95%) quenched. Analysis of these recognition events implied a 1 : 2 (sensor : anion) binding and ideal PET behaviour for ions such as AcO- and H2PO4-. For F-, the luminescent quenching indicated a 1 : 1 binding, but we deduced that this was due more to complete quenching of the excited state after the addition of one equivalent of the anion. For all of the anions, the quenching contributed to enhanced efficiency of PET from the receptors to the excited state of the fluorophore. In the case of the bis-anions (ambient), such as di-carboxylates, similar fluorescence quenching was observed. However, here either a 1 : 1 or a 1 : 2 binding was observed depending on the length of the spacer separating the two carboxylate moieties and the nature of the receptor. Whereas both pyrophosphate and malonate gave rise to a 1 : 1 binding, glutarate gave rise to approximately 1 : 2 binding for the thiourea sensors 1 and 2. However, for the urea based sensor 3, the binding was found to be 1 : 1 for all the bis-anions. For such a 1 : 1 binding we propose that the anion most likely bridges the fluorophore moiety. This was also evident from the 1H NMR (DMSO-d6) spectrum where the anthracene resonances were significantly affected. By simply modifying the electronic structure of the receptor, the sensitivity of the recognition process could also be modified; e.g. compound 1, bearing the trifluoromethyl substituent, showed stronger binding to the bis-anions than 2, which possessed a simple phenyl moiety.     

双量子点纳米复合物NO比率荧光探针的合成与应用

通过静电吸附作用,合成了CdSe@SiO_2-CdTe双量子点的纳米复合物.一氧化氮(NO)与CdTe量子点表面Cd离子结合形成Cd-NO复合物,引起CdTe量子点荧光猝灭,而不影响CdSe量子点的荧光.当NO浓度在0.1~2.2μmol/L之间变化时,该探针荧光强度比值I_(603)/I_(532)符合线性关系(R=-0.995 4),从而实现对NO的定量检测.     

Design, synthesis and evaluation of ratiometric probes for hydrogencarbonate based on europium emission

A series of cationic, zwitterionic and anionic macrocyclic europium complexes has been prepared incorporating a N or C- linked acridone chromophore that allows sensitisation of Eu emission following excitation at 390-410 nm. Each of these complexes selectively binds bicarbonate at physiological pH and reversible binding is signalled by a change in the form and relative intensity of the Eu emission spectrum. Affinity for bicarbonate is regulated by overall complex charge and falls within the range required for intracellular or extracellular analyses. Monitoring the ratio of the intensity of Eu emission at up to three wavelengths, e.g. 618/588 or 618/702 nm allows the solution concentration of bicarbonate to be deduced in a background of competing anions such as lactate, citrate and phosphate. Preliminary screens reveal the complexes to be non-toxic to NIH-3T3 cells and to be taken inside the cell, encouraging further study.     

BODIPY-derived ratiometric fluorescent sensors: pH-regulated aggregation-induced emission and imaging application in cellular acidification triggered by crystalline silica exposure

Modification of classic fluorophore to possess the emission transitions between aggregation-induced emission (AIE) and intrinsic emission offers reliable approach to the design of ratiometric fluorescent sensors. In this study, a proton acceptor benzimidazole was integrated with BODIPY to form three compounds, BBI-1/2/3, which demonstrated the AIE (~595 nm, I_agg) in neutral aqueous medium and intrinsic BODIPY emission (~510 nm, I_int) in acidic medium. All the three showed the ratiometric pH sensing behavior in a dual excitation/dual emission mode, yet BBI-3 displayed still the dual emission ratiometric pH sensing ability. The pH-dependent emission ratio I_int/I_agg of the three were fully reversible, and no interference was observed from normal abundant chemical species in live cells. Their different pK_a (BBI-1, pK_a 4.4; BBI-2, pK_a 2.7; BBI-3, pKa 3.6) suggested that the substituents on benzimidazole moiety were essential to govern their functioning pH range. The ratiometric imaging of BBI-1 in A549 cells provided an effective intracellular pH (pHi) calibration formula corresponding to emission ratio of I_int/I_agg. Ratiometric pH_i imaging in A549 cells upon small particle exposure confirmed the particle-induced cellular acidification with this formula. Both particle size and the chemical nature of the particle contribute to the observed acidification effect. The synchronization of lysosome disruption to cellular acidification in A549 cells upon crystalline silica exposure was directly observed for the first time with BBI-1, showing the potential application of BBI-1 in the study of silicosis and other related diseases. This study demonstrated that endowing fluorophore with AIE/intrinsic emission transition could be a promising strategy for ratiometric sensor design.