A high-performance fluorosensor for pH measurements between 6 and 9

This study presents a high-performance ratiometric pH optode based on the fluorophore 6,8-dihydroxypyrene-1,3-disulfonic acid (DHPDS). The two pH-sensitive terminal hydroxy groups of DHPDS facilitated dual excitation/dual emission (F₁: λ_1,ex = 420 nm, λ_1,em = 462 nm; F₂: λ_2,ex = 470 nm, λ_2,em = 498 nm) properties for ratiometric (R_F1,F2 = F₁/F₂) normalization of sensor signal. The sensor demonstrated an exponentially decreasing ratiometric response with increasing pH, with a linear correlation (R² = 0.9936) between ¹⁰log(R_F1,F2) and pH within the pH interval 6-9. Precision determined as the IUPAC pooled standard deviation for the pH values 6.00, 7.01 and 9.01, was 0.0057 pH units for the fluorosensor and 0.0054 for a commercially available pH electrode used for comparison. Between the end-points of calibration at pH 7.01, the precision of the sensor was 0.0037 pH units. Effects from changes in ionic strength (I_tot, 10-700 mM) were more pronounced for the electrode, with a linear (R² = 0.9976) increase in response (δE/δpH) with increasing I_tot. The DHPDS-based fluorosensor, however, retained sensitivity (δ¹⁰log(R_F1,F2)/δpH = 0.8024 ± 0.0145), though with an overall increase in ratiometric signal with increasing I_tot. The preserved sensitivity despite changes in ionic strength was possibly a consequence from the dual photo-acidic properties of DHPDS. Analytical characteristics of immobilized DHPDS therefore not only facilitated high-performance measurements over a wide pH range, but also opened for straightforward simultaneous measurements of pH and ionic strength.     

Ratiometric detection of pH fluctuation in mitochondria with a new fluorescein/cyanine hybrid sensor

The homeostasis of mitochondrial pH (pH_m) is crucial in cell physiology. Developing small-molecular fluorescent sensors for the ratiometric detection of pH_m fluctuation is highly demanded yet challenging. A ratiometric pH sensor, Mito-pH, was constructed by integrating a pH-sensitive FITC fluorophore with a pH-insensitive hemicyanine group. The hemicyanine group also acts as the mitochondria targeting group due to its lipophilic cationic nature. Besides its ability to target mitochondria, this sensor provides two ratiometric pH sensing modes, the dual excitation/dual emission mode (D_ex/D_em) and dual excitation (D_ex) mode, and its linear and reversible ratiometric response range from pH 6.15 to 8.38 makes this sensor suitable for the practical tracking of pH_m fluctuation in live cells. With this sensor, stimulated pH_m fluctuation has been successfully tracked in a ratiometric manner via both fluorescence imaging and flow cytometry.     

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.     

A fluorescence ratiometric sensor for hypochlorite based on a novel dual-fluorophore response approach

A fluorescence ratiometric sensor for OCl⁻ has been developed based on a novel dual fluorophore response approach. The sensor molecule contains a coumarin fluorophore and a rhodamine fluorophore, and the two fluorophores are directly linked to an OCl⁻ recognition group. The structure of the sensor was characterized by ESI-MS, NMR, and X-ray crystallographic analysis. Upon treatment with OCl⁻, both fluorophores in the sensor responded simultaneously at two separate optical windows, with large enhancement of the fluorescence ratio (I₅₇₈/I₅₀₁) from 0.01 to 39.55. The fluorescence ratios for the sensor showed a good linearity with the concentration of OCl⁻ in the range of 0.2–40 μM and the detection limits is 0.024 μM (S N⁻¹ = 3). Investigation of reaction products indicated that the sensor reaction with OCl⁻ produced two new fluorescent molecules, which were responsible for the fluorescence changes in two optical windows. In addition, the sensor showed high selectivity to OCl⁻ over other reactive oxygen species, reactive nitrogen species, cations, and anions. The sensor has also been successfully applied to detection of OCl⁻ in natural water samples with satisfactory recovery.     

Ratiometric fluorescence-based dissolved carbon dioxide sensor for use in environmental monitoring applications

The focus of this work is on the development and characterisation of a fluorescence-based ratiometric sol–gel-derived dissolved carbon dioxide (dCO₂) sensor for use in environmental monitoring applications. Fluorescence-based dCO₂ sensors are attractive as they facilitate the development of portable and low-cost systems that can be easily deployed outside the laboratory environment. The sensor developed for this work exploits a pH fluorescent dye 1-hydroxypyrene-3,6,8-trisulfonic acid, ion-paired with cetyltrimethylammonium bromide (HPTS-IP), which has been entrapped in a hybrid sol–gel-based matrix derived from n-propyltriethoxysilane along with the liphophilic organic base. The sensor spot deposited on a cover slip has been interrogated with a robust, ratiometric optical probe that combines effective fluorescence excitation and detection and thus facilitates the production of a highly sensitive sensor system using low-cost optoelectronic components. The probe design involves the use of dual-LED excitation in order to facilitate ratiometric operation and uses a silicon PIN photodiode. HPTS-IP exhibits two pH-dependent changes in excitation bands, which allows for dual excitation ratiometric detection as an indirect measure of the dCO₂. Such measurements are insensitive to changes in dye concentration, leaching and photobleaching of the fluorophore and instrument fluctuations unlike unreferenced fluorescence intensity measurements. The performance of the sensor system is characterised by a high degree of repeatability, reversibility and stability. Calculated limit of detection for the sensor was 35 ppb. The sensor probe was used to monitor dCO₂ levels in a laboratory-based aquatic habitat, and the expected diurnal pattern was clearly visible. The influence of temperature, biofouling and photobleaching on sensor performance has been also investigated.     

Development of ratiometric fluorescent probe for zinc ion based on indole fluorophore

A water-soluble ratiometric fluorescent probe ZID-1 has been developed on the basis of an internal charge transfer (ICT) mechanism. Upon complexation with Zn²⁺ under physiological conditions, ZID-1 exhibits a significant blue shift of 77 nm in the emission spectrum. The fluorescent behavior of ZID-1 suggests that the pyridyl group incorporated into the fluorophore coordinates the metal ion as the fourth ligand and affords an appropriate binding affinity (K_d = 17.1 nM) for the intracellular imaging of Zn²⁺.     

A ratiometric photoelectrochemical microsensor based on a small-molecule organic semiconductor for reliable in vivo analysis

Photoelectrochemical (PEC) sensing has been developing quickly in recent years, while its in vivo application is still in the infancy. The complexity of biological environments poses a high challenge to the specificity and reliability of PEC sensing. We herein proposed the concept of small-molecule organic semiconductor (SMOS)-based ratiometric PEC sensing making use of the structural flexibility as well as readily tunable energy band of SMOS. Xanthene skeleton-based CyOH was prepared as a photoactive molecule, and its absorption band and corresponding PEC output can be modulated by an intramolecular charge transfer process. As such, the target mediated shift of absorption offered the opportunity to construct a ratiometric PEC sensor. A proof-of-concept probe CyOThiols was synthesized and assembled on a Ti wire electrode (TiWE) to prepare a highly selective microsensor for thiols. Under two monochromatic laser excitation (808 nm and 750 nm), CyOThiols/TiWE offered a ratiometric signal (j₈₀₈/j₇₅₀), which exhibited pronounced capacity to offset the disturbance of environmental factors, guaranteeing its reliability for application in vivo. The ratiometric PEC sensor achieved the observation of bio-thiol release induced by cytotoxic edema and fluctuations of thiols in drug-induced epilepsy in living rat brains.

The first small-molecule organic semiconductor-based ratiometric photoelectrochemical sensor was proposed, which exhibited pronounced selectivity and capacity to offset environmental disturbance, guaranteeing its reliability for in vivo analysis.     

Dual‐fluorophore Raspberry‐like Nanohybrids for Ratiometric pH Sensing

We report on the development of raspberry‐like silica structures formed by the adsorption of 8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS)@silica nanoparticles (NPs) on rhodamine B isothiocyanate (RBTIC)@silica NPs for ratiometric fluorescence‐based pH sensing. To overcome the well‐known problem of dye leaching which occurs during encapsulation of anionic HPTS dye in silica NPs, we utilized a polyelectrolyte‐assisted incorporation of the anionic HPTS. The morphological and optical characterization of the as‐synthesized dye‐doped NPs and the resulting nanohybrids were carried out. The pH‐sensitive dye, HPTS, incorporated in the HPTS‐doped silica NPs provided a pH‐dependent fluorescence response while the RBITC‐doped silica provided the reference signal for ratiometric sensing. We evaluated the effectiveness of the nanohybrids for pH sensing; the ratio of the fluorescence emission intensity at 510 nm and 583 nm at excitation wavelengths of 454 nm and 555 nm, respectively. The results showed a dynamic response in the acidic pH range. With this approach, nanohybrids containing different dyes or receptors could be developed for multifunctioning and multiplexing applications.     

5-氨基-1,10-菲咯啉：可异构化杂环螯合荧光载体作为比例计量型锌离子荧光探针的一种原型

5-amino-l,10-phenanthroline （5-AP）, as a tautomeric heterocyclic aromatic chelating fluorophore （THACF）, can sense Zn^2＋ selectively by shifting emission from 495 to 564 nm upon Zn^2＋ addition in ethanol. The ratiometric fluorescent sensing behavior has been correlated to the tautomerization of 5-AP affected by solvents and metal chelation. The strategy using THACF as ratiometric fluorescent sensor for Zn^2＋ not only simplifies the synthetic procedure but also gives a promising alternative for Zn^2＋ ratiometric fluorescent sensor design.     

Esterase-activated two-fluorophore system for ratiometric sensing of biological zinc(II)

Intracellular ester hydrolysis by cytosolic esterases is a common strategy used to trap fluorescent sensors within the cell. We have prepared analogues of Zinpyr-1 (ZP1), an intensity-based fluorescent sensor for Zn2+, that are linked via an amido-ester or diester moiety to a calibrating fluorophore, coumarin 343. These compounds, designated Coumazin-1 and -2, are nonpolar and are quenched by intramolecular interactions between the two fluorophores. Esterase-catalyzed hydrolysis generates a Zn2+-sensitive ZP1-like fluorophore and a Zn2+-insensitive coumarin as a calibrating fluorophore. Upon excitation of the fluorophores, coumarin 343 emission relays information concerning sensor concentration whereas ZP1 emission indicates the relative concentration of Zn2+-bound sensor. This approach enables intracellular monitoring of total sensor concentration and provides a ratiometric system for sensing biological zinc ion.     

Tailoring the sensitivity and spectral properties of a chromoreactand for the detection of amines and alcohols

The chromoreactand N,N-dioctylamino-4′-trifluoroacetyl-2′-nitroazobenzene (CR-546) is sensitive to amines and alcohols when dissolved in plasticised PVC. The chromoreactand provides an improvement over related reactands in that a nitro group meta to the trifluoroacetyl group enhances the chemical reactivity and, consequently, the sensitivity by a factor of 20 compared to the derivative without nitro group. Furthermore, the absorbance is shifted by around 40 nm to longer wavelengths, making the sensor membranes compatible with the green LED as a light source for a miniaturised sensor device. The sensor membrane responds to aqueous 1-propylamine in the 0.5-50 mM concentration range and exhibits a detection limit of 0.1 mM. Response times are in the range of 5-15 min.A fluorescent ethanol-sensitive membrane is obtained by dissolving the inert fluorophore N,N′-bis(1-hexylheptyl)perylene-3,4:9,10-bis(dicarboximide) together with CR-546 and the catalyst tridodecylmethylammonium chloride in the polymer matrix. The trifluoroacetyl form of CR-546 with its absorbance at around 560 nm overlaps the emission of the fluorophore. Increasing ethanol concentrations cause decreasing absorbance at 560 nm and consequently, increasing luminescence of the fluorophore. The resulting layer has been evaluated for its detection of ethanol via changes in luminescence intensity. The calibration is linear in the range from 1 to 15% (v/v) ethanol with a limit of detection of 0.1% (v/v). Response times are in the range from 20 to 40 min for both forward and reverse reaction.     

Ratiometric fluorescent detection of Cu(II) in semi-aqueous solution using a two-fluorophore approach

Schiff base sensor 1, containing naphthalene and naphthalimide fluorophores with separate and distinct emission wavelengths, showed good selectivity for Cu(II) over other tested physiological and environmentally important cations through changes in its fluorescence spectra in THF/H₂O (9:1) HEPES buffered solution. By taking the ratiometric change of the emissions at 435 nm (naphthalene-Schiff base) and 510 nm (naphthalimide) good linearity was observed in the 0-10 μM range. The enhancement of the 435 nm emission upon binding Cu²⁺ was attributed to a prevention of the rapid CN isomerisation that otherwise leads to non-radiative decay, while the quenching of the naphthalimide emission was attributed to electron transfer between the excited naphthalimide fluorophore and the redox active Cu²⁺.     

Chemoselective Alteration of Fluorophore Scaffolds as a Strategy for the Development of Ratiometric Chemodosimeters

Ratiometric sensors generally couple binding events or chemical reactions at a distal site to changes in the fluorescence of a core fluorophore scaffold. However, such approaches are often hindered by spectral overlap of the product and reactant species. We provide a strategy to design ratiometric sensors that display dramatic spectral shifts by leveraging the chemoselective reactivity of novel functional groups inserted within fluorophore scaffolds. As a proof‐of‐principle, fluorophores containing a borinate ( RF₆₂₀ ) or silanediol ( SiOH2R ) functionality at the bridging position of the xanthene ring system are developed as endogenous H₂O₂ sensors. Both these fluorophores display far‐red to near‐infrared excitation and emission prior to reaction. Upon oxidation by H₂O₂ both sensors are chemically converted to tetramethylrhodamine, producing significant (≥66 nm) blue‐shifts in excitation and emission maxima. This work provides a new concept for the development of ratiometric probes.     

An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations

A mitochondria-targeted fluorescent sensor (Mito-ST), constructed by integrating a sulfamoylbenzoxadiazole fluorophore with a phosphonium group, displays the specific Zn(2+)-induced hypsochromic shifts of both excitation (69 nm) and emission (35 nm) maxima. Its ratiometric Zn(2+) imaging ability via dual excitation mode has been applied in MCF-7 cells to clarify the different behaviours of mitochondrial Zn(2+) release stimulated by H(2)O(2) and SNOC.     

A cataluminescence gas sensor based on nanosized V2O5 for tert-butyl mercaptan

This work proposed a gas sensor for the determination of tert-butyl mercaptan, one of the highly toxic volatile sulfur compounds, which was based on cataluminescence emission during its catalytic oxidation on the surface of nanosized V₂O₅. The cataluminescence characteristics and the optimum conditions, including the morphology of sensing material, the wavelength of cataluminescence emission, the oxygen flow rate and working temperature were investigated in detail. Under the optimized conditions, the calibration curve of the relative cataluminescence intensity versus the concentration of tert-butyl mercaptan vapor was made, with the linear range of 5.6-196 μg mL⁻¹ and the detection limit of 0.5 μg mL⁻¹ (S/N = 3). The relative standard deviation (R.S.D.) (n = 5) of relative cataluminescence intensity for 84 μg mL⁻¹ tert-butyl mercaptan was 3.6%. There is no or weak response to some common substances, such as formic acid, alcohol (methanol, ethanol, propanol, isopropanol, n-butanol, isoamyl alcohol), o-dichlorobenzene, acetonitrile, ethyl acetate, aldehyde (formaldehyde, acetaldehyde and propanal), 1,2-dichloroethane and ammonia. Furthermore, the proposed sensor was successfully used for determining tert-butyl mercaptan in four artificial samples, with a good recovery. The results demonstrated that the proposed gas sensor had a promising capability for the tert-butyl mercaptan in routine monitoring.     

A colorimetric and ratiometric fluorescent turn-on fluoride chemodosimeter and application in live cell imaging: high selectivity via specific SiO cleavage in semi aqueous media and prompt recovery of ESIPT along with the X-ray structures

A ratiometric fluorescent turn-on probe for fluoride ion, based on modulation of the excited-state intramolecular proton transfer (ESIPT) process by chemodosimetric desilylation pathway is reported. The probe SNBT (silyl protected hydroxynaphthalene benzothiazole moiety) shows a significant increase of ratiometric absorption band at 440 nm and emission band at 477 nm by the deprotection of fluoride mediated silyl bond cleavage in CH₃CN–H₂O (8/2, v/v, 25 °C). The test strips based on SNBT and F⁻ are fabricated, which can act as a convenient and efficient F⁻ test kits. Furthermore, the biological application shows that it can be very useful as a selective fluoride probe in the fluorescence imaging of living cells.     

Rapid method for the preparation of a robust optical pH sensor

A simple and rapid method for the preparation of a fluorescence-based optical pH sensor is described. The sensor is based on excitation ratiometric detection of a methacryloyl-modified analog of the well-known fluorescent pH indicator dye, 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS). The modified dye, 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS), is similar in structure and function to HPTS. However, unlike HPTS, the presence of the methacrylate moiety allows MA-HPDS to participate as a comonomer in a free radical polymerization reaction. Covalent immobilization by this method is simple, as the preparation of the modified dye is accomplished in a single reaction step and its subsequent reaction with a comonomer results in simultaneous immobilization and purification. Sensors were prepared by copolymerization of the MA-HPDS with poly(ethylene glycol) diacrylate. Minimal leaching of the immobilized dye was observed from the HPDS-PEG matrix. The copolymer of HPDS and PEG-DA is fully autoclavable. The sensor is useful over the pH range of 6-9, with excellent reproducibility. Ionic strength effects on the apparent pKa of the immobilized dye are small and predictable.     

Hybrid supraparticles of carbon dots/porphyrin for multifunctional tongue-mimic sensors

Supraparticles（SPs）, such as assembly of inorganic components with organic, have made tremendous attention in biochemical analysis, which represents a novel but challenging research orientation. Herein, a single-SPs multifunctional fluorescent sensor array has been developed for high-throughput detection of heavy metal ions in biofluids, which is based on an inorganic/organic hybrid SPs consisting of carbon dots（CDs） and an easily available porphyrin [5,10,15,20-tetra（4-carboxyphenyl）porphyrin（T...     

Wavelength encoded analytical imaging and fiber optic sensing with pH sensitive CdTe quantum dots

CdTe quantum dots (QDs), capped with mercaptopropionic acid (MPA), were synthesized and the variation of their fluorescence properties (steady state and lifetime) with pH was assessed in solution and when immobilized in a sol-gel host. Three different sizes of CdTe QDs with excited state lifetimes ranging from 42 to 48 ns and with emission maximum at 540 nm (QD₅₄₀), 580 nm (QD₅₈₀) and 625 nm (QD₆₂₅) were selected. The solution pH affects the maximum emission wavelength (shifts to higher wavelengths of 23, 24 and 27 nm for QD₅₄₀, QD₅₈₀ and QD₆₂₅, respectively), the excited state lifetime and the fluorescence intensity in a reversible way. Linearization of the maximum emission wavelength variation with the pH allows the estimation of an apparent ionization constant (pK_a) for each QD: 6.5 ± 0.1 (QD₅₄₀), 6.1 ± 0.5 (QD₅₈₀) and 5.4 ± 0.3 (QD₆₂₅). The variation of the QDs fluorescence properties was further explored using confocal laser scanning microscopy allowing the implementation of a new calibration method for pH imaging in solution. QDs were successfully immobilized on the tip of an optical fiber by dip-coating using sol-gel procedure. The immobilized QDs showed a similar pH behaviour to the one observed in solution and an apparent lifetime of 80, 68 and 99 ns, respectively. The proposed QDs based methodology can be successfully used to monitor pH using wavelength encoded data in imaging and fiber optic sensing applications.