Ratiometric pH Probe Research Articles

A new insight into the luminescence mechanism of a FRET-ICT-based ratiometric and colorimetric pH probe

Recently, a ratio fluorescent probe molecule BP was designed and synthesized to detect pH in living cells. However, few have conducted in-depth theoretical exploration of the detailed transfer process. In this paper, the luminescence mechanisms of BP and BPH were studied with the results of geometric optimization structures and molecular orbitals at the CAM-B3LYP/TZVP computational level. It was found that a fluorescence resonance energy transfer (FRET) process occurs in the excited state of BP molecules, and the final fluorescence emission is attributed to the intramolecular charge transfer (ICT) of the acceptor styryl pyridine ring. Meanwhile, the fluorescence emission of BPH molecules is due to the ICT process caused by the local excitation process of the acceptor group protonated styryl pyridine ring itself. Therefore, this luminescence is a FRET “ON-OFF” combined ICT process, not like the FRET “OFF-ON” mechanism which the experiment suspected. Our work illustrates a specific FRET combined ICT process of probes BP and BPH from a theoretical perspective which could provide theoretical guidance for the future design of similar fluorescent probes.

Intrinsic dual emissive insulin capped Au/Ag nanoclusters as single ratiometric nanoprobe for reversible detection of pH and temperature and cell imaging

pH and temperature are two important characteristics in cells and the environment. These, not only in the well-done regulation of cell functions but also in diagnosis and treatment, have a key role. Protein-protected bimetallic nanoclusters are abundantly used in the building of biosensors. However, insulin-stabilized Au–Ag nanoclusters with dual intrinsic emission have not been investigated yet. In this work, Dual emissive insulin templated Au–Ag nanocluster (Ins(Au/Ag)NCs) were first synthesized in a simple and green one-put manner. The two emission wavelengths of, as-prepared NCs centered at 410 and 630 nm, excited in one excitation wavelength (330 nm). These two emission peaks were assigned to the di-Tyrosine cross-linked formation and bimetallic nanoclusters respectively. Further analysis displayed that each emission band of Ins(Au/Ag)NCs responded to one variable whilst another peak remained constant; For blue and red emission wavelengths, pH dependency and thermo-responsibility were observed respectively. As-prepared nanoprobe with the intrinsic dual emissive feature was used for ratiometric determination of these parameters, each with a discrete response from another. The linear range of 6.0–9.0 for pH and 1 to 71 °C for temperature was obtained, which comprises the physiological range of pH and temperature and afforded intracellular sensing and imaging capability. As-prepared NCs probe show excellent biocompatibility and cell membrane permeability, and so were successfully applied as direct ratiometric pH and temperature probes in HeLa and HFF cells. More interestingly, this dual emissive nanoprobe is capable of distinguishing cancer cells from normal ones.

Albumin Hydrogel-Coated Mesoporous Silica Nanoparticle as a Carrier of Cationic Porphyrin and Ratiometric Fluorescence pH Sensor.

Here, we report that a mesoporous silica nanoparticle (MSN) coated with a fluoresceine-labeled bovine serum albumin (F-BSA) hydrogel layer works as a temperature-responsive nanocarrier for tetrakis-N-methylpyridyl porphyrin (TMPyP) and as a fluorescence ratiometric pH probe. F-BSA hydrogel-coated MSN containing TMPyP (F-BSA/MSN/TMPyP) was synthesized by thermal gelation of denatured F-BSA on the external surface of MSN. The F-BSA hydrogel layer was composed of an inner hard corona layer and an outer soft layer and was stable under physiological conditions. F-BSA/MSN/TMPyP exhibited temperature-dependent exponential release of TMPyP. In this release profile, the MSN was found to be a suitable host for stable encapsulation of tetracationic TMPyP by electrostatic interactions, and the F-BSA hydrogel layer mediated the diffusion of TMPyP from the MSN pore interior into the solution phase. Increasing temperature promoted partitioning of TMPyP from the pore interior to the F-BSA hydrogel layer, from where it was spontaneously released into the bulk solution phase by cation exchange. F-BSA/MSN/TMPyP also gave a linear ratiometric fluorescence response (1.3 per pH unit) in the pH range from 6.1 to 8.9.

A phenyl-substituted hemicyanine with maintained pKa as ratiometric near-infrared fluorescent probe for in vitro and in vivo pH determinations

In this study a new ratiometric pH probe, PHE-C, was designed and synthesized with the aim of enlarging the wavelength difference (Δλfl) between the two peaks of the typical hemicyanine fluorophore without significantly affecting its pKa value. The two emission maxima at 682 and 730 nm, associated with the respective protonation states of the hydroxyl group in PHE-C, have a larger Δλfl (48 nm) than that of unsubstituted hemicyanine. Importantly, the pKa of PHE-C (6.55) is the same as that of the unsubstituted counterpart. Consequently, the ratio of the intensities of the 682 and 730 nm emission bands is linearly related to pH in the range of 5.5–7.5, which is appropriate for monitoring small changes in pH of weakly acidic physiological environments. Moreover, PHE-C possesses good photostability, sensitivity and reversibility, and it displays the same response as pH meter when determining the pH in bovine serum solutions, indicating that it can be utilized in complex biological environments. Finally, the results of studies aimed at assessing the applicability to monitoring pH changes in living cells and mice demonstrate that the new probe is a highly sensitive ratiometric probe for in vivo pH determinations.

Ratiometric, pH-Sensitive Probe for Monitoring siRNA Delivery.

Many RNA delivery strategies require efficient endosomal uptake and release. To monitor this process, we developed a 2'-OMe RNA-based ratiometric pH probe with a pH-invariant 3'-Cy5 and 5'-FAM whose pH sensitivity is enhanced by proximal guanines. The probe, in duplex with a DNA complement, exhibits a 48.9-fold FAM fluorescence enhancement going from pH 4.5 to pH 8.0 and reports on both endosomal entrapment and release when delivered to HeLa cells. In complex with an antisense RNA complement, the probe constitutes an siRNA mimic capable of protein knockdown in HEK293T cells. This illustrates a general approach for measuring the localization and pH microenvironment of any oligonucleotide.

Ratiometric fluorescent probes for pH mapping in cellular organelles.

The intracellular pH (pHi) in organelles, including mitochondria, endoplasmic reticulum, lysosomes, and nuclei, differs from the cytoplasmic pH, and thus maintaining the pH of these organelles is crucial for cellular homeostasis. Alterations in the intracellular pH (ΔpHi) in organelles lead to the disruption of cell proliferation, ion transportation, cellular homeostasis, and even cell death. Hence, accurately mapping the pH of organelles is crucial. Accordingly, the development of fluorescence imaging probes for targeting specific organelles and monitoring their dynamics at the molecular level has become the forefront of research in the last three decades. Among them, ratiometric fluorescent probes minimize the interference from the excitation wavelength of light, auto-fluorescence from probe concentration, environmental fluctuations, and instrument sensitivity through self-correction compared to monochromatic fluorescent probes, which are known as turn-on/off fluorescent probes. Small-molecular ratiometric fluorescent probes for detecting ΔpHi are challenging yet demanding. To date, sixty-two ratiometric pH probes have been reported for monitoring internal pH alterations in cellular organelles. However, a critical review on organelle-specific ratiometric probes for pH mapping is still lacking. Thus, in the present review, we report the most recent advances in ratiometric pH probes and the previous data on the role of mapping the ΔpHi of cellular organelles. The development strategy, including ratiometric fluorescence with one reference signal (RFRS) and ratiometric fluorescence with two reversible signals (RFRvS), is systematically illustrated. Finally, we emphasize the major challenges in developing ratiometric probes that merit further research in the future.

Imaging and Measuring Vesicular Acidification with a Plasma Membrane-Targeted Ratiometric pH Probe.

Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

A novel two-channel ratio fluorescent probe for monitoring intracellular pH fluctuations

Accurate measurement of pH is of great significance in the research of biology and medicine sciences. Cell is the basic unit of living organism and the proper pH of cytoplasm and organelles such as lysosome is the precondition to maintain its function. Alteration of pH may impair the normal process of cells and lead to occurrence of diseases. Sensitively sensing the pH of cytoplasm and organelles is of great value for biomedical research. Due to the complexity of biological microenvironment, it is still challenging to simultaneously detect the pH changes of cytoplasm and lysosome. Here, we reported a ratiometric pH probe with NIR fluorescence termed as PCHC, which realized sensitive cytoplasmic and lysosome pH detection. The probe was prepared by connecting coumarin with dicyanmethylene-4H-pyran via amidation reaction, and displays large Stoke-shift, good biosafety and pH selectivity. After entering the cell, the probe shows blue in cytoplasm and NIR fluorescence in lysosome due to respective pH. The ratio of blue and NIR fluorescence signal can be served as indicator to monitor pH change of cytoplasm and lysosome simultaneously, which has potential application for investigation of pH alteration related diseases.

A ratiometric pH probe for acidification tracking in dysfunctional mitochondria and tumour tissue in vivo.

Cellular dysregulated pH and mitochondrial metabolism are commonly two central factors for solid tumour progression. pH regulation and long-term mitochondrial tracking provide a great opportunity for tumour treatment. pH probes with suitable pKa and satisfactory mitochondria-immobilizing character are demanded for tumour recognition and therapy. Here, we report a ratiometric fluorescent probe, CouDa, for pH imaging in mitochondria and tumour tissue. CouDa displays an obvious blue-shifted emission (about 160 nm) in alkaline environment, with a pKa around 7.4 suitable for monitoring mitochondrial pH change. NMR and MS data confirmed an addition reaction mechanism of OH- upon the positively charged conjugated structure of hemicyanine fluorophore. Mitochondrial immobilization and acidification monitoring were realized by CouDa in cells treated with a mitochondrial uncoupler. Moreover, CouDa could distinguish acidified tumour tissue in living mice. Comparing with its analogue, the pH-sensitivity and mitochondria-immobilizing property are attributed to a hydrophobic long alkyl chain on indolium N atom. This work provides an effective strategy to track nucleophilic substances in dysfunctional mitochondria.

A Ratiometric pH Responsive Fluorescence Probe for Monitoring Wide pH Range

AbstractA novel ratiometric pH probe has been designed and synthesized on a Rhodamine B platform bearing one anthraquinone moiety and the photochemical properties of this system are evaluated. Changing the pH from acidic to basic condition, this probe exhibited 92 nm red‐shift of the emission maximum. This change is sufficiently large to permit its use as a ratiometric pH probe. With fast response time this chemosensor is fully reversible which is important and advantageous for practical applications. Furthermore, the pKa values of this probe can be predicted from the fluorescence titration. This design strategy is very simple and being modular, can be applicable to develop ratiometric pH probes for various applications.

Aggregation induced emission in 1,8-naphthalimide embedded nanomicellar architecture as a platform for fluorescent ratiometric pH-probe with biomedical applications

pH sensitive nanomicelles core embedded with fluorescent 1,8-naphthalimide aggregates are prepared. Due to the aggregation induced emission of the core embedded fluorophore in alkaline and neutral media, the novel micelles show green fluorescence which is switched in blue monomeric 1,8-naphthalimide emission in acid media after destabilization of the micellar architecture. Based on this change, the obtained nanomicelles are successfully applied as a fluorescent ratiometric probe for determination of pH of the media. The prepared nanomicelles show high cell-permeability and low cytotoxicity, which indicate high potential for future biomedical applications.

Genetic engineering of circularly permuted yellow fluorescent protein reveals intracellular acidification in response to nitric oxide stimuli

Intracellular pH (pHi) is a crucial parameter in cell biology; thus, a series of pH probes have been developed to determine pHi changes in living cells. However, more sensitive and non-perturbing ratiometric pH probes are needed for accurate pHi measurements. While the fluorescence of circular permutated YFP (cpYFP) is hypersensitive to pH changes due to its intrinsic properties, the single excitation peak of this protein restricts its capacity of becoming a rational type of pH sensor. Herein, we collected several cpYFP-based probes with dual excitation peaks and constructed their corresponding loss-of-function mutants to screen for a potential competent pH probe. The most sensitive probe was named NocPer. NocPer consists of cpYFP inserted into inactive-mutated GAF and AAA+, which are two regulatory domains of E. coli NorR, a nitric oxide (NO)-specific transcription factor. Fluorescence emission of NocPer peaks at 517 nm while exhibiting dual excitation peaks at 420 and 495 nm, which can be used for ratiometric imaging. This new pH sensor has a large ratio response dynamic (pH range of 7.0–11.0), which covers the physiological pH range (pH 7.0–8.0), and exhibits an approximately 3-fold higher fluorescent signal in response to a pH increase from 7.0 to 8.0 than that of pHluorin. Using NocPer, we discovered a new biological phenomenon in which NO exposure decreases the E. coli pHi, which led to the hypothesis that pathogens decrease their own pHi during infection. Further, we elucidated that the NO-induced inhibition of cytochrome c oxidase in the respiratory chain is responsible for the decline in pHi, which might represent a protective strategy of E. coli under NO stress conditions. Our results demonstrated that NocPer is a ratiometric pH probe with high sensitivity for the physiological pH range.

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 dual-channel Hill-type small-molecule pH probe.

By combining a Hill-type pH probe and a pH-insensitive naphthalimide fluorophore, we synthesized a FRET-based ratiometric pH probe (PHHF), exhibiting a reduced pH transition width, representing a unique approach for development of sensitive probes for detection of biorelevant pH changes.

A general strategy to increase emission shift of two-photon ratiometric pH probes using a reversible intramolecular reaction of spiro-oxazolidine.

Fluorescent pH probes have been served as powerful tools in biological and pathological studies in recent years due to the important roles of pH values in various physiological processes. Although plenty of pH probes have been delivered, development of two-photon ratiometric pH probes with large emission shift for detecting the variation of intracellular pH values is still a greatly challenging task. To address this concern, in this work, we have discovered a general strategy designing pH probes by means of a pH-dependent reversible intramolecular reaction of spiro-oxazolidine which can efficiently change their conjugation length and the electronic effect concurrently. To display the generality of the strategy, we have synthesized six pH probes, and all these probes exhibit short emission in basic conditions and dramatically red-shifted emission in acid environments. The emission shift of the six probes is more than 150nm and even up to 210nm, much larger than shift of all commercial and reported pH probes. The chemical sensing mechanism of intramolecular ring opening/closing reaction of spiro-oxazolidine has been confirmed with 1H NMR spectra and density functional theory (DFT) calculations. Finally, we have used one of six with one- and two-photon properties to successfully image lysosomal pH changes under confocal and two-photon microscopes in a ratiometric manner. We believed that this spiro-oxazolidine strategy can serve as a general and powerful platform for the design of ideal pH probes.

Green and high-yield synthesis of carbon dots for ratiometric fluorescent determination of pH and enzyme reactions.

In this work, the carbon dots (CDs) were prepared by one-pot hydrothermal method with chitosan as a carbon source and hydrogen peroxide as an oxidant, this route is a novel, simple and green. The prepared CDs has a relatively high photoluminescence (PL) quantum yield (QY) (18.9%) and good water solubility. CdTe quantum dots (CdTe QDs) possess the characteristic of responding to pH rapidly. So a ratiometric fluorescent probe for measuring pH was constructed using the prepared CDs and CdTe QDs. Under the excitation of 330nm, the probe shows dual (blue and red) emission with peaks at 430 and 600nm, respectively. When the pH of the solution changes, the blue fluorescence intensity of the CDs remains stable as a reference signal, while the red fluorescence intensity of CdTe QDs changes accordingly. This method has obvious pH-sensitive feature, stable fluorescence properties, and a great linear relationship with pH values in the range of pH3.0-11.0. What is more, the method can monitor proton-producing enzyme-catalyzed reactions by acting as a ratiometric pH probe.

Single-wavelength Excited Ratiometric Fluorescence pH Probe to Image Intracellular Trafficking of Tobacco Mosaic Virus

As a typical plant virus which has biocompatibility and high transfection efficiency, tobacco mosaic virus (TMV) has shown broad application potential in drug or gene delivery field. Elucidating its intracellular trafficking is of great importance in investigation of its cytotoxicity, targeting site, and delivery efficiency, and is advantageous to designing new TMV-based drug delivery systems with different targets. By taking advantage of the regulated pH value of different organelles in a mammalian cell, we exploit a pH detection strategy to investigate the intracellular trafficking pathway of TMV. Here, we report a single-wavelength excited ratiometric fluorescent pH probe. This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate (FITC) and pH-insensitive rhodamine B isothiocyanate (RBIRC) onto the inner surface of TMV. The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents. By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy, it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes. This work may provide important guidance on construction of TMV-based nano carriers.

An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications

Fluorescent ratiometric probes based on monomer/excimer of pyrene group are often used to detect cations. However, only a few pyrene-containing ratiometric pH probes have been reported so far, and almost all of them produce intramolecular pyrene excimer. In this work, three new ratiometric fluorescent pH probes based on intermolecular pyrene excimer, pyrene-1-carboxamide (1), N-(2-hydroxyethyl)pyrene -1-carboxamide (2) and N,N-bis(2-hydroxyethyl) pyrene-1-carboxamide (3), were synthesized. These probes, 1, 2, and 3, have pKa value of 1.93, 1.85 and 2.07, respectively, and can fluorescently determine the pH value of the extremely acidic aqueous solution with a detection range of about 1.0–2.5. When the pH value was decreased from 2.5 to 1.0, these probes showed a ratiometric fluorescent response signal due to the conversion of monomer/excimer of pyrene group. Therefore, intermolecular excimer of the pyrene group from these probes can be produced in extremely acidic environment. Further tests show that these probes have potential as excellent pH fluorescent probes, with high sensitivity, fast response time, good photostability, strong anti-ion interference ability and high repeatability, etc. The applicability of these probes was demonstrated with environment water samples. In addition, fluorescent pH test paper was successfully developed, which can monitor pH values in the solution through pH-related change of fluorescent color of the probes.

A through-bond energy transfer-based ratiometric fluorescent pH probe: For extreme acidity and extreme alkaline detection with large emission shifts

A ratiometric fluorescence pH probe 1 based on through-bond energy transfer (TBET) with a 2-(2-hydroxyphenyl)benzoxazole (HBO) as donor and a Rhodamine derivative as acceptor is developed through simple condensation reaction. The probe exhibits a ratiometric fluorescence emission (I593/I422) characteristics and linear response to extreme acidity range of 5.00–2.88, and a ratiometric fluorescence emission (I555/I422) characteristics and linear response to extreme alkaline range of 10.00–13.78, respectively. Moreover, 1 possesses highly selective response to pH over metal ions, good reversibility and excellent photostability. Probe 1 is cellpermeable and can distinguish near pH 5.55 fluctuations in Hela cells. Furthermore, 1 can be immobilized on a test paper, which shows a rapid and reversible colorimetric response to HCl/NH3 vapor by the naked-eye.

A near-IR luminescent ratiometric ytterbium pH probe.

The hypersensitive 2F5/2 to 2F7/2 transition of Yb3+ can be used to monitor perturbations of the coordination sphere in ytterbium(iii) complexes. An envelope of Stark components gives rise to a relatively broad and asymmetric emission band, whilst changes in their relative intensity and energy enable a ratiometric response. We report a new ytterbium complex with a sulphonamide arm that binds reversibly to Yb3+ as a function of pH, giving rise to significant pH dependent changes in the Yb emission spectrum.