Ratiometric Fluorescent pH Sensor Research Articles

Boron Complexes with Propiolamidinato Ligands: Synthesis, Structure, and Photophysical Properties.

Two series of boron derivatives with propiolamidinato ligands, [BPh2{C(C≡CAr)(NR)2}] (Ar = Ph, p-MeOPh, p-FPh, p-Me2NPh, or phen; R = iPr or p-tolyl), were synthesized and structurally characterized. The corresponding propiolamidine (or propargylamidine) proligands have been obtained through sustainable methods. One is the catalytic hydroalkynylation of diisopropylcarbodiimide with different terminal alkynes, using simple ZnEt2 as a precatalyst. Alternatively, to obtain propiolamidines with aromatic groups on the nitrogen atoms, the formation of lithiated derivatives of terminal alkynes by reaction with n-BuLi in air and at room temperature, and subsequent addition to the di-p-tolylcarbodiimide, under the same conditions and using 2-MeTHF as a sustainable solvent, has been used for the first time. After reaction with BPh3, the corresponding boron amidinates were obtained, which are emissive in the solution state. The influence of the different substituents introduced into the ligands on the photophysical properties of the boron compounds has been studied. One of the obtained compounds can be used as a ratiometric fluorescent pH sensor in the acidic range.

Ratiometric fiber optic fluorescent pH sensor for hydroxide diffusion measurements in concrete

This article delineates the development of an advanced ratiometric pH optode designed for protracted monitoring of pH dynamics in cementitious materials—a crucial aspect of structural management. The optode features a dual-layer Hydromed™ D4 polymer pellet incorporating a pH-sensitive Naphth-AlkyneOMe fluorescent probe (pKa ≈ 12) and CdSe quantum dots as an internal reference. The polymer matrix, optimized with diamond powder and carbon black, enhances the signal while minimizing light artifacts. Demonstrating a robust response to pH fluctuations, the optode, utilizing ratiometric measurements, enables steadfast monitoring for up to 75 days.The experimental setup includes a Delrin® sample compartment housing a glass slide with the optode connected to an optical fiber via an SMA connector. HydroMed™ D4 films, forming the pH-sensitive layer, are deposited on the glass slide and secured. The optode configuration is fine-tuned to optimize signal intensity by reducing its length. For optode-detector coupling, a 1000 µm core diameter optical fiber links the optode, and a 405 nm LED illuminates the sensing film. The emission signal is collected through the optical fiber, and signals are processed using computer-controlled lock-in amplifiers.In the investigation of hydroxide ion diffusion through a concrete sample, the optode's efficacy was validated, aligning with pH electrode measurements. Addressing long-term stability, a degradation model uncovered leisurely kinetics with commendable stability. Slow degradation kinetics, with a reaction rate coefficient of 1.6 × 10⁻⁷ s⁻¹ and a half-life of 1180 h, exceeded expectations.This pH optode, showcasing an accurate and resilient response to pH variations within the 10 to 12.5 range, stands as a promising tool for monitoring pH dynamics in low-pH cementitious materials. The incorporation of a degradation model underscores fidelity over prolonged durations and offers a blueprint for enduring surveillance of concrete structures.

Preparation of a Novel Ratiometric Fluorescent Capillary-based pH Sensor

Preparation of a Novel Ratiometric Fluorescent Capillary-based pH Sensor

Carbon dots-based fluorescent films to act as a potential antioxidant agent and pH ratiometric sensor for skin applications

The wound healing process is accompanied by changes in pH values. Monitoring this physicochemical parameter can indicate the effectiveness of the applied treatment and act as early identification of wound infection. This study focuses on the development of a fluorescent film-based polyvinyl alcohol (PVA) and carbon dots (CDs) derived from lemon bagasse (CD-L) and ortho-phenylenediamine (CD-oPD) named to act as antioxidants and potential ratiometric fluorescent pH sensors, in wound applications. The I460/I550 intensity ratio, as a function of pH value for the dual-system CDs prepared from the mixture of CD-L and CD-oPD, named CD-L/oPD, was investigated. The fit corresponded to a sigmoidal function in the pH range of 5–10, with a relationship having a r2 = 0.992. The variation in the values of the I460/I550 ratio allows for the visualization of the color change from yellowish-green to green with increasing pH. Through a simulated ex vivo pig skin model, it was possible to note that the films prepared from mixed of the CD-L and CD-oPD carbon dots incorporated in a matrix PVA named CD-L/oPD-F was more efficient at visually discriminating color in relation to changes in pH than the films prepared from both individual CD-L (CD-L-F) and CD-oPD (CD-oPD-F) carbon dots. CD-L and CD-oPD demonstrated antioxidant capacity against reactive oxygen species (ROS). The IC50 values for CD-L and CD-oPD were 56.7 and 39.5 μg mL−1 in the DPPH● inhibition assay, and 25.1 and 63.4 μg mL−1 in the HOCl scavenging one, respectively. MTT viability assays using human non-tumoral skin fibroblast (HFF-1) cell showed a cell survival rate of over 80% for both CDs up to a concentration of 1,000 μg mL−1. Finally, the developed films can act in a bifunctional way, by monitoring healing through pH changes and by acting as an antioxidant agent in the treatment of wounds.

Photoluminescent gold nanoclusters as two-photon excited ratiometric pH sensor and photoactivated peroxidase.

Atwo-photon excited ratiometric fluorescent pH sensor is reportedby combining L-cysteine-protected AuNCs (Cys@AuNCs) with fluorescein isothiocyanate (FITC). Cys@AuNCs were synthesized through a one-step self-reduction route and showed pH-responsive photoluminescence at 650 nm. Benefiting from the opposite pH response of Cys@AuNCs and FITC, the fluorescence ratio (F515 nm/F650 nm) of FITC&Cys@AuNCs provided a large dynamic range of 200-fold for pH measurement in the response interval of pH 5.0-8.0. Based on the excellent two-photon absorption coefficient of Cys@AuNCs, the sensor was expected to achieve sensitive quantitation of pH in living cells under two-photon excitation. In addition, colorimetric biosensing based on enzyme-like metal nanoclusters has attracted wide attention due to their low-cost, simplicity, and practicality. It is crucial to develop high catalytic activity nanozyme from the viewpoint of practical application. The synthesized Cys@AuNCs exhibited excellent photoactivated peroxidase-like activity with high substrate affinity and catalytic reaction rate, promising for rapid colorimetric biosensing of field analysis and the control of catalytic reactions by photostimulation.

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes.

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pKa , off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.

Facile preparation of FITC-modified silicon nanodots for ratiometric pH sensing and imaging.

A ratiometric fluorescent pH sensor was facilely constructed by covalent modification of amino-terminated silicon nanodots (SiND) with pH-sensitive fluorescein isothiocyanate (FITC). After optimization, the SiND-FITC(40:1) material with a SiND:FITC initial mass ratio of 40:1 was selected for the sensing of hydrogen ions. It was observed that the material inherits the unique features of SiND and FITC, and there is significant improvement of SiND acid-base stability, which is a favorable factor in terms of providing fluorescence reference signal. The SiND-FITC(40:1) material displays not only high pH sensitivity, but also good stability and anti-interference ability, and the response process is highly reversible. Deploying the SiND-FITC(40:1) material, we have made available a simple, sensitive, and precise approach for pH sensing. In aqueous solutions, the I517/I466 fluorescence intensity ratio of SiND-FITC(40:1) increases linearly in the pH range of 5.40-7.76. This dual emission nanosensor was successfully applied for pH sensing and cellular fluorescence imaging.

Development of novel optical pH sensors based on coumarin 6 and nile blue A encapsulated in resin particles and specific support materials.

Inspired by traditional pH papers, two types of ratiometric fluorescent pH sensors for neutral and alkaline pH ranges were developed in this study by using two fluorescent dyes, coumarin 6 (C6) and nile blue A (NB). These dyes were encapsulated into melamine-formaldehyde (MF) resin particles, which were then incorporated with polymer Nafion (Nf) or polyurethane hydrogel (PU) to prepare pH-sensing membranes. MF-C6 particles immobilized into polymer Nafion (i.e., MF-C6-Nf membrane) showed a dynamic ratiometric pH detection range of 4.5-7.5 through shift in fluorescence emission spectra at acidic and neutral pH solutions, as well as distinct color transition under normal visual sense from pink to yellow color. By contrast, MF-NB particles immobilized into polyurethane hydrogel (i.e., MF-NB-PU membrane) displayed a dynamic ratiometric fluorescence detection range of pH 9 - pH 12 via change in ratiometric fluorescence intensity at two emission band edges. The membrane also showed a clear change from blue to purple color under sunlight at high pH values. These pH-sensing membranes also exhibited high sensitivity, good reversibility, and stability. They were then applied to measure pH values in real urine samples and fermentation media.

Bimetal-organic framework nanocomposite based point-of-care visual ratiometric fluorescence pH microsensor for strong acidity

pH is of great importance in understanding physiological and pathologic processes. Abnormal gastric juice pH is an indicator of many gastric diseases such as peptic ulcer and gastric cancer. In this work, a nanocomposite 1-hydroxypyrene@ Co/Tb- dipicolinic acid metal-organic framework (1-OHP@Co/Tb-DPA MOF) was constructed as a ratiometric fluorescent pH sensor for strong acidity. The nanocomposite-based pH sensor was response-rapid (30 s), linearly responsive in the broad pH range of 0.3–7.8 (r > 0.95) and ultra-sensitive in the pH range of 0.3–5.0. Importantly, the nanocomposite-loaded fiber paper was prepared as a visual microsensor for point-of-care pH detection via ratiometric chromaticity, coupled with a homemade portable determination device. The paper-based visual microsensor is response-rapid and sensitive, long-term stable (at least 30 days), and suitable for pH assay of microvolume (as little as 5 μL) samples at pH 0.3–5.0. Both the nanocomposite-based ratiometric fluorescent sensor and the paper-based visual microsensor can accurately determine the pH of artificial gastric juices (relative error <5.4%). To our knowledge, this is the first time to report a MOF-based ratiometric fluorescent pH sensor for strong acidity (pH<1) and a paper-based pH microsensor for point-of-care visual assay via ratiometric chromaticity.

Core-shell polymeric nanoparticles comprising BODIPY and fluorescein as ultra-bright ratiometric fluorescent pH sensors.

A new ratiometric fluorescent pH nanosensor is presented. It is based on ultrabright nanoparticles containing two spatially separated fluorophores: BODIPY covalently linked to the polystyrene core and fluorescein grafted to the nanoparticle shell. The nanoparticles comprise a large number (≥2500) of both fluorescent moieties. Their spectroscopic characteristics were studied at different pH and ionic strength. They could successfully be used to determine the solution pH between 5.5 and 7.5 by measuring the fluorescence intensity ratio of the sensor molecule (fluorescein) relative to the reference dye (BODIPY).

Dual colorimetric and ratiometric fluorescent pH sensors for neutral and alkaline pH ranges

Dual colorimetric and ratiometric fluorescent pH sensors for neutral and alkaline pH ranges

Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry.

The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a pKa of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.

One-step synthesis of fluorescent organic nanoparticles: The application to label-free ratiometric fluorescent pH sensor

In this work, we develop a facile one-step synthesis method of poly(3,4-dihydroxyphenylalanine) (poly(DOPA)) fluorescent organic nanoparticles (FONPs) by polymerization of DOPA in alkaline alcohol solution under ultrasonic radiation. The morphology and size of poly(DOPA)-FONPs are dependent on the synthesis conditions, such as solvent and alkali concentration. Under the optimized condition, the prepared poly(DOPA)-FONPs possess not only uniform size and morphology, but also strong green fluorescence, good solubility in water and organic solvents. Under excitation of single wavelength, two emission peaks can be clearly observed, which are pH-sensitive. Moreover, the intensity ratio of two emission peaks is linear against pH values from 4.0 to 8.0 in buffer solution. In addition, the pH-sensitive response of the poly(DOPA)-FONPs be free from the influence of various metal ions and biomolecules, indicating the excellent specificity to pH. Further, due to the excellent biocompatibility, the poly(DOPA)-FONPs might also be applied on biological imaging for monitoring intracellular pH. The as-prepared poly(DOPA)-FONPs would be promising to be applied on the low-cost, low-toxic and label-free ratiometric pH sensor.

Carbon-dot-based ratiometric fluorescent pH sensor for the detections of very weak acids assisted by auxiliary reagents that contribute to the release of protons

In this work, a carbon-dot-based ratiometric fluorescent pH sensor was fabricated by conjugating a pH-sensitive fluorescein, fluoresceinisothiocyanate (FITC), onto the surface of carbon dots (CD-FITCs). The fabrication condition was optimized so as to make the pH sensor possess a typical character of ratiometic probe that the two emission intensities ratio is independent on its concentration. As a result, the CD-FITCs exhibited high sensitivity and selectivity to pH, and the as-proposed method for pH sensing is of considerable repeatability. However, the CD-FITCs sensor is still not an effective tool for the direct detections of very weak acids based on pH sensing because of their extremely low dissociated constants. Interestingly, with the help of the auxiliary reagents that contribute to the release of protons, the sensitivities of the CD-FITCs sensor to the very weak acids can be considerably enhanced. For example, the very weak acids, boric acid and hydroxylamine hydrochloride, which can be transformed into relatively strong acids assisted by D-fructose and acetone, was successfully measured using the carbon-dot-based ratiometric fluorescent pH sensor in the real water samples (tap water and lake water). Moreover, the proposed method can be extended to sensing any analytes in chemical reactions associated with proton release.

Fluorescent pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging.

A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting spirolactam Rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pKa of 5.35, which indicated that L-SRhB has the potential for pH detection of acidic organelle. In addition, the two probes were internalized successfully by living cells through the endocytosis pathway and could distinguish normal cells from cancer cells by different cell staining rates. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, whereas it was nontoxic to normal cells in the same condition. L-SRhB might have potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH sensor and bioimaging dye for the recognition of cancer cells. The results also provided a new perspective to the high-value utilization of lignin.

A ratiometric fluorescent sensor for pH fluctuation and its application in living cells with low dark toxicity

It is highly demanded and challenging to develop small-molecular fluorescent sensors for the ratiometric detection of pH fluctuation. A ratiometric fluorescent pH sensor was constructed by integrating a pH-sensitive fluorophore with a pH-insensitive hemicyanine group. Its linear and reversible ratiometric fluorescent response from pH 7.0 to 8.8 and 6.0 to 9.0 respectively makes this sensor suitable for the practical tracking of pH fluctuation in live cells. With this sensor, stimulated pH fluctuation has been successfully tracked in a ratiometric manner via fluorescence imaging with low dark toxicity.

A quinoline based pH sensitive ratiometric fluorescent sensor: Structure and spectroscopy

A new quinoline based hydrazone was synthesized via a condensation reaction and characterized by NMR, mass and single crystal X-ray diffraction studies. It was investigated for suitability as a reversible ratiometric fluorescent pH sensor in acidic pH region. The sensor exhibits intramolecular charge transfer (ICT) type photophysical changes upon protonation of the quinoline ring. No significant interference on emission behavior was observed in the presence of various metal ions. A new quinoline based hydrazone was established as a reversible ratiometric fluorescent pH sensor in the acidic pH region and structurally characterized by single crystal X-ray diffraction studies. It exhibits intramolecular charge transfer (ICT) type photophysical changes upon protonation of quinoline ring.

2D ratiometric fluorescent pH sensor for tracking of cells proliferation and metabolism.

Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved.

A highly dispersible silica pH nanosensor with expanded measurement ranges

Two kinds of silica based colloidal ratiometric fluorescent pH sensors were successfully developed. The measurement range was expanded to almost 4 pH units.

Styrylcyanine-based ratiometric and tunable fluorescent pH sensors

Styrylcyanine derivatives based on indolenine and salicylaldehydes act as excellent fluorescent sensors with ratiometric changes in absorption and emission profiles in response to pH variation.