A fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles

We have synthesized dual-fluorophore-doped core-shell silica nanoparticles used as ratiometric pH sensor. The nanoparticles were prepared with a reverse microemulsion technique by simultaneously encapsulating two different fluorophores, the pH-sensitive dye fluorescein as a pH indicator and the pH-insensitive dye phenosafranine as an internal reference for fluorescence ratiometric measurement, into silica shell. The nanoparticles prevent the fluorescence dyes leaching from the silica matrix when immersed inside water. The hydrophilic silica shells were made by hydrolysing and polymerizing tetraethoxysilane (TEOS) in water-in-oil microemulsion. The fluorescence intensity ratio of the two dyes varied linearly as a function of pH in the range from 4.0 to 8.0. The sensor was also applied to measure pH of real water samples. The results are in good agreements with that using the conventional glass electrode method. The as-prepared fluorescent nanoparticles showed rapid response, excellent stability and high reproducibility as pH sensors.     

Fluorescence ratiometric pH sensor prepared from covalently immobilized porphyrin and benzothioxanthene

A fluorescence ratiometric sensor for pH determination is described in this paper. The sensor incorporated the pH-sensitive dye meso-5,10,15,20-tetra-(4-allyloxyphenyl)porphyrin (TAPP) as an indicator and a pH-insensitive dye N-(2-methacryloxyethyl)benzo[k,l]thioxanthene-3,4-dicarboximide (MBTD), a benzothioxanthene derivative, as a reference for fluorescence ratiometric measurement. To prevent leakage of the dyes, both were photocopolymerized with acrylamide, hydroxyethyl methacrylate, and triethylene glycol dimethacrylate on the silanized glass surface. The reproducibility and response time of the prepared sensor were sufficient. Most common coexisting inorganic ions and organic compounds did not interfere with pH sensing. In the acidic pH range from 1.5 to 5.0 the fluorescence intensity ratio of the two dyes varied linearly as a function of pH. The sensing membrane was found to have a lifetime of at least one month. The sensor was applied to the analysis of waste water and artificial samples.     

Proton "off-on" behaviour of methylpiperazinyl derivative of naphthalimide: a pH sensor based on fluorescence enhancement

In a search for new type pH sensing fluorophores, the possibility of using the proton "off-on" switch behaviour of naphthalimide derivatives for optical pH sensor preparation has been explored. A new compound, N-allyl-4-(4[prime or minute]-methyl-piperazinyl)-1,8-naphthalimide (AMPN), was synthesized. The enhancement of fluorescence of AMPN with the increase of hydrogen ion concentration is based on arresting photo-induced electron transfer to the naphthalimide fluorophore from aliphatic amine group after its protonation. The Stokes Shift of the proposed type of pH sensing fluorophore is significantly larger than that of the fluorescein counterparts. To avoid the leakage of the fluorophore, AMPN was photo-copolymerized with 2-hydroxyethyl methacrylate and acrylamide on the glass surface. The fluorescence intensity of membrane contacted with a pH 3.50 buffer is 4.7 times of that for pH 12.00 buffer solutions. The proposed pH sensor is not susceptible to ionic strength and shows good selectivity, repeatability and short response time. The membrane shows a good stability with a lifetime over two months. The sensor can be used for the determination of pH in the range of pH 4.5-9.0, without interference of most commonly co-existing inorganic ions and some organic species. The sensor has been applied to the analysis of urine samples.     

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.     

Synthesis and evaluation of self-calibrating ratiometric viscosity sensors

We describe the design, synthesis and fluorescent profile of a family of self-calibrating dyes that provide ratiometric measurements of fluid viscosity. The design is based on covalently linking a primary fluorophore (reference) that displays a viscosity-independent fluorescence emission with a secondary fluorophore (sensor) that exhibits a viscosity-sensitive fluorescence emission. Characterization of fluorescent properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. The chemical structures of both fluorophores and the linker length have been evaluated in order to optimize the overall brightness and sensitivity of the viscosity measurements. We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model.     

Magnetic core-shell fluorescent pH ratiometric nanosensor using a Stöber coating method

We describe the use of a modified St?ber method for coating maghemite (γ-Fe(2)O(3)) nanocrystals with silica shells in order to built magnetic fluorescent sensor nanoparticles in the 50-70nm diameter range. In detail, the magnetic cores were coated by two successive silica shells embedding two fluorophores (two different silylated dye derivatives), which allows for ratiometric pH-measurements in the pH range 5-8. Silica coated magnetic nanoparticles were prepared using maghemite nanocrystals as cores (5-10nm in diameter) coated by tetraethoxyorthosilicate via hydrolysis/condensation in ethanol, catalyzed by ammonia. In the inner shell was covalently attached a sulforhodamine B, which was used as a reference dye; while a pH-sensitive fluorescein was incorporated into the outer shell. Once synthesized, the particles were characterized in terms of morphology, size, composition and magnetization, using dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD) and vibrating sample magnetometry (VSM). TEM analysis showed the nanoparticles to be very uniform in size. Wide-angle X-ray diffractograms showed, for uncoated as well as coated nanoparticles, typical peaks for the spinel structure of maghemite at the same diffraction angle, with no structural changes after coating. When using VSM, we obtained the magnetization curves of the resulting nanoparticles and the typical magnetization parameters as saturation magnetization (M(s)), coercivity (H(c)), and remanent magnetization (M(r)). The dual-dye doped magnetic-silica nanoparticles showed a satisfactory magnetization that could be suitable for nanoparticle separation and localized concentration of them. Changes in fluorescence intensity of the pH indicator in the different pH buffered solutions were observed within few seconds indicating an easy accessibility of the embedded dye by protons through the pores of the silica shell. The relationship between the ratio in fluorescence (sensor/reference dyes) and pH was adjusted to a sigmoidal fit using a Boltzmann type equation. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% (w/v) of horse serum added, indicating that there are no significant statistical differences at a 95% confidence level.     

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.     

A ratiometric fluorescent chemosensor for iron: discrimination of Fe2+ and Fe3+ and living cell application

A newly designed probe, 6-thiophen-2-yl-5,6-dihydrobenzo[4,5]imidazo-[1,2-c] quinazoline (HL(1)) behaves as a highly selective ratiometric fluorescent sensor for Fe(2+) at pH 4.0-5.0 and Fe(3+) at pH 6.5-8.0 in acetonitrile-HEPES buffer (1/4) (v/v) medium. A decrease in fluorescence at 412 nm and increase in fluorescence at 472 nm with an isoemissive point at 436 nm with the addition of Fe(2+) salt solution is due to the formation of mononuclear Fe(2+) complex [Fe(II)(HL)(ClO(4))(2)(CH(3)CN)(2)] (1) in acetonitrile-HEPES buffer (100 mM, 1/4, v/v) at pH 4.5 and a decrease in fluorescence at 412 nm and increase in fluorescence at 482 nm with an isoemissive point at 445 nm during titration by Fe(3+) salt due to the formation of binary Fe(3+) complex, [Fe(III)(L)(2)(ClO(4))(H(2)O)] (2) with co-solvent at biological pH 7.4 have been established. Binding constants (K(a)) in the solution state were calculated to be 3.88 × 10(5) M(-1) for Fe(2+) and 0.21 × 10(3) M(-1/2) for Fe(3+) and ratiometric detection limits for Fe(2+) and Fe(3+) were found to be 2.0 μM and 3.5 μM, respectively. The probe is a "naked eye" chemosensor for two states of iron. Theoretical calculations were studied to establish the configurations of probe-iron complexes. The sensor is efficient for detecting Fe(3+)in vitro by developing a good image of the biological organelles.     

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.     

Development of FRET-based dual-excitation ratiometric fluorescent pH probes and their photocaged derivatives

Dual-excitation ratiometric fluorescent probes allow the measurement of fluorescence intensities at two excitation wavelengths, which should provide a built-in correction for environmental effects. However, most of the small-molecule dual-excitation ratiometric probes that have been reported thus far have shown rather limited separation between the excitation wavelengths (20-70 nm) and/or a very small molar absorption coefficient at one of the excitation wavelengths. These shortcomings can lead to cross-excitation and thus to errors in the measurement of fluorescence intensities and ratios. Herein, we report a FRET-based molecular strategy for the construction of small-molecule dual-excitation ratiometric probes in which the donor and acceptor excitation bands exhibit large separations between the excitation wavelengths and comparable excitation intensities, which is highly desirable for determining the fluorescence intensities and signal ratios with high accuracy. Based on this strategy, we created a coumarin-rhodamine FRET platform that was then employed to develop the first class of FRET-based dual-excitation ratiometric pH probes that have two well-resolved excitation bands (excitation separations>160 nm) and comparable excitation intensities. In addition, these pH probes may be considered as in a kind of "secured ratioing mode". As a further application of these pH probes, the dual-excitation ratiometric pH probes were transformed into the first examples of photocaged dual-excitation ratiometric pH probes to improve the spatiotemporal resolution. It is expected that the modular nature of our FRET-based molecular strategy should render it applicable to other small-molecule dual-dye energy-transfer systems based on diverse fluorescent dyes for the development of a wide range of dual-excitation ratiometric probes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

Excited-state intramolecular proton transfer in 2-(2'-arylsulfonamidophenyl)benzimidazole derivatives: the effect of donor and acceptor substituents

A series of water-soluble 2-(2'-arylsulfonamidophenyl)benzimidazole derivatives containing electron-donating and accepting groups attached to various positions of the fluorophore pi-system has been synthesized and characterized in aqueous solution at 0.1 M ionic strength. The measured pK(a)'s for deprotonation of the sulfonamide group of monosubstituted derivatives range between 6.75 and 9.33 and follow closely Hammett's free energy relationship. In neutral aqueous buffer, all compounds undergo efficient excited-state intramolecular proton transfer (ESIPT) to yield a strongly Stokes-shifted fluorescence emission from the phototautomer. Upon deprotonation of the sulfonamide nitrogen at high pH, ESIPT is interrupted to yield a new, blue-shifted emission band. The peak absorption and emission energies were strongly influenced by the nature of the substituents and their attachment positions on the fluorophore pi-system. The fluorescence quantum yield of the ESIPT tautomers revealed a significant correlation with the observed Stokes shifts. The study provides valuable information regarding substituent effects on the photophysical properties of this class of ESIPT fluorophores in an aqueous environment and may offer guidelines for designing emission ratiometric pH or metal-cation sensors for biological applications.     

Optical Ratiometric Sensor for Alcohol Measurements

Abstract

The need for low‐cost, robust alcohol sensors has increased with the renewed interest in alternative fuels as well as high‐throughput screening of biological processes involving the production of ethanol. The goal of this research was to develop a miniaturized optical ratiometric ethanol sensor to be used for in situ measurements. The sensor is based on the fluorescent dye Nile Blue Chloride. When in solution, the dye exhibits a single fluorescence peak. However, a dual emission peak is observed upon physical immobilization of the dye in the hydrogel poly(ethylene glycol) dimethacrylate. The dual emission allows for ratiometric measurements, thus circumventing drawbacks associated with fluorescence intensity measurements such as signal variations due to dye bleaching, source intensity fluctuations, etc. In developing this sensor we investigated ethanol sensitivity; alcohol selectivity; response time; and cross‐sensitivity with pH, polarity, and ionic strength. We found that the sensor is sensitive to a broad range of ethanol concentrations, namely 5% to 90% v/v. Due to the hydrogel's restrictive pore size, the sensor is sensitive to short‐chain alcohols such as methanol, ethanol, and propanol, but lacks sensitivity to larger alcohols such as butanol and hexanol. We also found the sensor maintains full functionality after autoclaving. Sensor sensitivity to alcohol in solutions of varying ionic strength is negligible, whereas the solvent's polarity must be controlled to maintain meaningful results. The sensor is most sensitive in acidic and neutral environments, indicating promising use for yeast‐ based alcohol fermentations.     

Ratiometric fluorescent detection of acidic pH in lysosome with carbon nanodots

It is significant for cell physiology to keep the homeostasis of p H, and it is highly demanded to develop ratiometric fluorescent sensors toward p H. In this work, under mild condition, through the electrostatic interaction between carbon nanodots(CDs) and organic molecules, two novel ratiometric fluorescence hybrid nanosensors were fabricated for sensing acidic p H. These nanohybrid systems possess dual emission peaks at 455 and 527 nm under a single excitation wavelength of 380 nm in acidic p H condition.With the increasing of p H, the fluorescence of the 1,8-naphthalimide derivative completely quenches,while the blue fluorescence of CDs keeps constant. Furthermore, the CDsàorganic molecular nanohybrids exhibit excellent anti-disturbance ability, reversible p H sensing ability, and a linear response range in wide p H range respectively. Besides the ability to target lysosome, with one of the nanosensor, stimulated p H change has been successfully tracked in a ratiometric manner via fluorescence imaging.     

Ultrasensitive detection of aliphatic nitro-organics based on “turn-on” fluorescent sensor array

The broad class of explosives includes nitro aromatics as well as challenging aliphatic nitro-organics whose detection is important from counter-terrorism and national security perspectives. Here we report a turn-on fluorescent sensor array based on aggregation-induced emission (AIE) fluorophores as receptors. To achieve a good sensing system with fast response, good sensitivity and low detection limit, three receptors with abundant chemical diversities for target analytes were synthesized. The turn-on response of the individual receptor showed highly variable and cross-reactive analyte-dependent changes in fluorescence. The excellent ability to identify a variety of explosives, especially the challenging aliphatic nitro-organics (2,3-dimethyl-2,3-dinitrobutane (DMNB), 1,3,5-trinitro-1,3,5-triazinane (RDX), cyclotetramethylene tetranitramine (HMX) and entaerythritol tetranitrate (PETN)), was demonstrated in qualitative and quantitative analyses with 100% accuracy. The fluorescence signal amplification in the presence of explosives allows for application of these receptors in a sensor microarray suitable for high-throughput screening. These results suggested that the cross-reactive sensor array based on AIE fluorophores could find a wide range of applications for sensing various analytes or complex mixtures.     

氨基苯基类中氮茚化合物的合成及作为质子探针的研究

设计合成了3个氨基苯基类中氮茚化合物, 研究了其在不同pH值的缓冲溶液中的荧光强度变化. 结果表明， 在pH=2.1～4.2之间， 该类化合物具有荧光敏感性. 化合物3a和3b可以作为良好的质子控制的荧光开关器件信号分子.     

Novel fluorescent pH sensor based on 5-(4-carboxy-phenyl)-10,15,20-tris(phenyl)-porphyrin

A novel fluorescent pH sensor based on 5-(4-carboxy-phenyl)-10,15,20-tris(phenyl)-porphyrin, as sensing agent, has been developed. The carboxyl functionalized asymmetric porphyrin has been synthesized and characterized by high performance liquid chromatography (HPLC), thin layer chromatography (TLC), proton nuclear magnetic resonance (¹H NMR), Fourier transform infrared spectroscopy (FT-IR), ultraviolet–visible (UV–vis), emission, excitation and mass spectrometry (MS) spectra. Optical and fluorescence behaviors were investigated in relationship with variation of acid concentration in the range of acid pH from 1.5 to 5.5. Upon increasing the acidity of the solution, the decrease of the fluorescence intensity was noticed, as a linear function of pH. Different metal ions were tested to put into evidence the changes regarding the fluorescence intensity, but the fluorescence obtained results revealed no significant interference on pH determination. The conclusion is that the proposed fluorescent sensor can measure pH in acid range in the presence of different metal ions making this sensor a proper one for pH determinations in leaching solutions of the recyclable processes of valuable metals.     

A facile combinatorial approach to construct a ratiometric fluorescent sensor: application for the real-time sensing of cellular pH changes

Realtime monitoring of the cellular environment, such as the intracellular pH, in a defined cellular space provides a comprehensive understanding of the dynamics processes in a living cell. Considering the limitation of spatial resolution in conventional microscopy measurements, multiple types of fluorophores assembled within that space would behave as a single fluorescent probe molecule. Such a character of microscopic measurements enables a much more flexible combinatorial design strategy in developing fluorescent probes for given targets. Nanomaterials with sizes smaller than the microscopy spatial resolution provide a scaffold to assemble several types of fluorophores with a variety of optical characteristics, therefore providing a convenient strategy for designing fluorescent pH sensors. In this study, fluorescein (CF) and tetramethylrhodamine (CR) were assembled on a DNA nanostructure with controlling the number of each type of fluorophore. By taking advantage of the different responses of CF and CR emissions to the pH environment, an appropriate assembly of both CF and CR on DNA origami enabled a controlled intensity of fluorescence emission and ratiometric pH monitoring within the space defined by DNA origami. The CF and CR-assembled DNA origami was successfully applied for monitoring the intracellular pH changes.

A combinatorial assembly of two types of intensity-based fluorophores on a DNA nanostructure provided a ratiometric pH probe with high emission intensity for monitoring intracellular pH changes.     

Development of ratiometric fluorescent pH sensors based on chromenoquinoline derivatives with tunable pKa values for bioimaging

In this work, we first studied the pH-dependent characteristic of chromenoquinoline. Based on this, we then designed and synthesized two novel chromenoquinoline derivatives that can act as fluorescent pH sensors. The pK_a values of two novel chromenoquinoline derivatives can be modulated from 2.32 to 4.38 and 6.27 by introducing EDG on the backbone of chromenoquinoline. Furthermore, we demonstrate that the sensor 4 can be used as a ratiometric fluorescent pH sensor for fluorescence imaging in living cells.     

Developing a novel ratiometric fluorescent probe based on ESIPT for the detection of pH changes in living cells

As an important parameter of intracellular metabolism, pH plays important roles in maintaining normal physiological processes. The abnormal pH could cause disorder of cell function which may cause neurological diseases. Herein, we present two novel ratiometric fluorescent probes to detect pH changes. The probes employed 2-(2′-hydroxyphenyl)benzothiazole as fluorescent platform, and displayed desirable fluorescence response to pH on the basis of excited state intramolecular proton transfer (ESIPT) process. The probe BtyC-1 showed green fluorescence at 546 nm under acidic conditions, while it displayed strong blue fluorescence at 473 nm and weak green fluorescence at 546 nm under alkaline conditions. Biological experiments demonstrated that the probe BtyC-1 could be successfully applied for the ratiometric imaging of cellular pH and the NH₄Cl-induced pH changes in living cells.     

Harnessing a ratiometric fluorescence output from a sensor array

Ratiometric fluorescence-based sensors are widely sought after because they can effectively convert even relatively small changes in optical output into a strong and easy-to-read signal. However, ratiometric sensor molecules are usually difficult to make. We present a proof-of-principle experiment that shows that efficient ratiometric sensing may be achieved by an array of two chromophores, one providing an on-to-off response and the second yielding an off-to-on response in a complementary fashion. In the case that both chromophores emit light of different color, the result is a switching of colors that may be utilized in the same way as from a true ratiometric probe. The chromophore array comprises two sensor elements: i) a polyurethane membrane with embedded N-anthracen-9-yl-methyl-N-7-nitrobenzoxa-[1,2,5]diazo-4-yl-N',N'-dimethylethylenediamine hydrochloride and ii) a membrane with N,N-dimethyl-N'-(9-methylanthracenyl)ethylenediamine. A combination of photoinduced electron transfer (PET) and fluorescence resonance energy transfer (FRET) allows for green-to-blue emission switching in the presence of Zn(II) ions. The sensing experiments carried out with different Zn(II) salts at controlled pH revealed that the degree of color switching in the individual sensor elements depends on both the presence of Zn(II) ions and the counter anion. These results suggest that sensing of both cations and anions may perhaps be extended to different cation-anion pairs.