pH荧光探针的研究进展

本文评述了近二十年来荧光素类、半萘酚罗丹荧类、金属配合物类、8-羟基芘-1,3,6-三磺酸盐等pH荧光探针的发展。按种类分别讨论了其荧光随pH变化的性质、适用的pH测定范围以及分析应用。引用文献56篇。     

19F NMR based pH probes: lanthanide(III) complexes with pH-sensitive chemical shifts

Experimental measurements and theoretical analysis of magnetic properties, structural dynamics and acid-base equilibria for several lanthanide(III) complexes with tetraazacyclododecane derivatives as 19F NMR chemical shift pH probes are presented; pKa values vary between 6.9 and 7.7, with 18 to 40 ppm chemical shift differences between the acidic and basic forms for Ho(III) complexes possessing T1 values of 10 to 30 ms (4.7-9.4 T, 295 K).     

Fluorescent pH probes for alkaline pH range based on perylene tetra-(alkoxycarbonyl) derivatives

The pH detection in the alkaline range is particularly important in many fields such as leather processing, waste water treatment, paper industry, and metal mining and finishing. Compared with traditional analysis methods such as colorimetric sensors and electrochemical sensors, the fluorescence and colorimetric probes for pH measurements have attracted much more attention due to their advantages of high sensitivity, excellent selectivity, noninvasiveness, low cost, fast response time, the possibility of continuously measurement, etc. However, there are few fluorescent probes fiting for alkaline pH monitoring. Acturally, the design and synthesis of them were more significant for new probes producing. In this study, the design, synthesis, and practical application of two novel fluorescent pH probes for alkaline pH assay were discussed. Both of the two probes were derived from perylene tetra-(alkoxycarbonyl). The red or blue shift of the absorption/fluorescence spectrum was caused by the introduction of electron donor amino or oxygen ring in the bay region. Due to electronic separation of the OH group from the electron-withdrawing core, the probes have high pKa values and cover the pH range from 8 to 12. They exist in either fluorescent acid form or non-fluorescent basic form. It was investigated that the amino substituent at bay region had a higher pKa value than O-heterocyclic annulated perylene, which showed that the adjustable pKa value could be achieved by the modification of electron withdrawing groups. The probes would have a wide use for testing strips measurements and monitoring pH changes in concrete.     

Synthesis and luminescence studies of aryl substituted tetraamide complexes of europium(III): a new approach to pH responsive luminescent europium probes

DOTA-tetraamide ligands having extended phenol or pyridine substituents have been synthesized. The (5)D(0) --> (7)F(J) deltaJ = 1 and 2 emission bands in the corresponding europium(III) complexes differ in their sensitivity to solution pH. This offers the potential for developing pH responsive probes for in vivo imaging that are independent of probe concentration.     

A Theoretical Investigation of Two Typical Two‐Photon pH Fluorescent Probes

Intracellular pH plays an important role in many cellular events, such as cell growth, endocytosis, cell adhesion and so on. Some pH fluorescent probes have been reported, but most of them are one‐photon fluorescent probes, studies about two‐photon fluorescent probes are very rare. In this work, the geometrical structure, electronic structure and one‐photon properties of a series of two‐photon pH fluorescent probes have been theoretically studied by using density functional theory (DFT) method. Their two‐photon absorption (TPA) properties are calculated using the method of ZINDO/sum‐over‐states method. Two types of two‐photon pH fluorescent probes have been investigated by theoretical methods. The mechanisms of the Photoinduced Charge Transfer (PCT) probes and the Photoinduced Electron Transfer (PET) probes are verified specifically. Some designed strategies of good two‐photon pH fluorescent probes are suggested on the basis of the investigated results of two mechanisms. For the PCT probes, substituting a stronger electron‐donating group for the terminal methoxyl group is an advisable choice to increase the TPA cross section. For the PET probes, the TPA cross sections increase upon protonation.     

Fiber-optic pH detection in small volumes of biosamples

Determining the pH values of microscopic plant samples may help to explain complex processes in plants, so it is an area of interest to botanists. Fiber-optic probes with small dimensions can be used for this purpose. This paper deals with the fiber-optic detection of the pH values of droplets of plant xylem exudate based on ratiometric fluorescence intensity measurements with an internal reference. For this purpose, novel V-taper sensing probes with a minimum diameter of around 8 μm were prepared that enable the delivery of fluorescence signal from the detection site on the taper tip to the detector. The taper tips were coated with pH-sensitive transducer (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt; HPTS) and a reference [dichlorotris-(1,10-phenanthroline) ruthenium (II) hydrate (Ru-phen dichloride)] immobilized in a xerogel layer of propyltriethoxysilane and (3-glycidoxy)propyl trimethoxysilane. The prepared probes were sensitive to pH values mainly in the range from 6.0 to 9.0. In the pH range 6–9, the results were limited by measurement errors of about 0.2 pH units, and in the pH range 5–6 by measurement errors of about 0.5 pH units. Using the developed V-taper sensing probes, the pH values of in vivo and in vitro samples of small volumes (∼6 μl) of exudate were measured. The results were validated by comparison with conventional electrochemical pH measurements.     

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes

Tracking pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade, different small probes and supramolecular systems have emerged for in cellulo or in vivo pH tracking. However, pH reporting still presents critical limitations, such as background reduction, improved sensor stability, cell targeting, endosomal escape, near- and far-infrared ratiometric pH tracking and adaption to new imaging techniques (i.e., super-resolution). These challenges will require the combined efforts of synthetic and supramolecular chemistry working together to develop the next generation of smart materials that will resolve current limitations. Herein, recent advances in the synthesis of small fluorescent probes, together with new supramolecular functional systems employed for pH tracking, are described with an emphasis on ratiometric probes. The combination of organic synthesis and stimuli-responsive supramolecular functional materials will be essential to solve future challenges of pH tracking, such as improved signal to noise ratio, on target activation and microenvironment reporting.     

Lysosomal pH Rise during Heat Shock Monitored by a Lysosome‐Targeting Near‐Infrared Ratiometric Fluorescent Probe

Heat stroke is a life‐threatening condition, featuring a high body temperature and malfunction of many organ systems. The relationship between heat shock and lysosomes is poorly understood, mainly because of the lack of a suitable research approach. Herein, by incorporating morpholine into a stable hemicyanine skeleton, we develop a new lysosome‐targeting near‐infrared ratiometric pH probe. In combination with fluorescence imaging, we show for the first time that the lysosomal pH value increases but never decreases during heat shock, which might result from lysosomal membrane permeabilization. We also demonstrate that this lysosomal pH rise is irreversible in living cells. Moreover, the probe is easy to synthesize, and shows superior overall analytical performance as compared to the existing commercial ones. This enhanced performance may enable it to be widely used in more lysosomal models of living cells and in further revealing the mechanisms underlying heat‐related pathology.     

Carboxymethyl poly(L‐histidine) as a new pH‐sensitive polypeptide at endosomal/lysosomal pH

A carboxymethyl poly(L ‐histidine) has been synthesized as a new pH‐sensitive polypeptide at endosomal/lysosomal pH. Because of its poor water solubility at physiological pH, an application of poly(L ‐histidine) with a pK_a around 6.0 has been limited in spite of the native possession of the pH‐dependent property change at endosomal pH. Although the unmodified poly(L ‐histidine) suddenly precipitates out of the aqueous medium above pH 6.0 as the result of the deprotonation of the imidazole groups, the water solubility of the resulting carboxymethyl poly(L ‐histidine) has been improved at physiological pH. A solution turbidity measurement proved that no significant effect on a rapid aggregate formation or phase separation of serum proteins is induced by carboxymethyl poly(L ‐histidine). Hemolysis assay showed that the carboxymethyl poly(L ‐histidine) enhances membrane disruptive ability at endosomal/lysosomal pH. The cellular uptake of luciferase in the presence of the carboxymethyl poly(L ‐histidine) increases intracellular luciferase activity, which suggests that the carboxymethyl poly(L ‐histidine) makes the luciferase escape from lysosomal degradation. The carboxymethyl poly(L ‐histidine) would be the fundamental compound for designing various drug carriers with the pH sensitivity at endosomal/lysosomal pH. Copyright © 2007 John Wiley & Sons, Ltd.     

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.     

Colorimetric and Fluorescent Bimodal Ratiometric Probes for pH Sensing of Living Cells

pH measurement is widely used in many fields. Ratiometric pH sensing is an important way to improve the detection accuracy. Herein, five water‐soluble cationic porphyrin derivatives were synthesized and their optical property changes with pH value were investigated. Their pH‐dependent assembly/disassembly behaviors caused significant changes in both absorption and fluorescence spectra, thus making them promising bimodal ratiometric probes for both colorimetric and fluorescent pH sensing. Different substituent identity and position confer these probes with different sensitive pH‐sensing ranges, and the substituent position gives a larger effect. By selecting different porphyrins, different signal intensity ratios and different fluorescence excitation wavelengths, sensitive pH sensing can be achieved in the range of 2.1–8.0. Having demonstrated the excellent reversibility, good accuracy and low cytotoxicity of the probes, they were successfully applied in pH sensing inside living cells.     

Syntheses of three terbium complexes as fluorescent probes and their application on the pH detection of routine urine test

By modifying the salicylic-acid moiety with electron-withdrawing or –donating groups, three new terbium complexes(L~Ⅰ·Tb, L~(Ⅱ)·Tb, L·~(Ⅲ)Tb) based on tripodal carboxylate ligands were synthesized. Due to different pull-push electronic effects of ligands, the fluorescence intensities of these terbium complexes significantly varied, that is: L~(Ⅱ)·Tb L~(Ⅲ)·Tb L~Ⅰ·Tb. Meanwhile, the characteristic peaks at 492 nm(~5D_4→~7F_6) and 547 nm(~5 D_4→~7F_5) showed "Off–On–Off" fluorescence response to various p H conditions,which indicated that all of them can be used as the highly sensitive pH fluorescent probes. Notably, using L~(Ⅱ)·Tb with the best fluorescence performance as a probe, some patients' urine samples can be easily monitored through the response triggered by pH value. Therefore, L~(Ⅱ)·Tb has the potential to auxiliarily diagnose some diseases in clinical practice through p H detection of routine urine test.     

Two 1,8-naphthalimide-based proton-receptor fluorescent probes for pH determination

Two newly synthesised 1,8-naphthalimide-based proton-receptor fluorescent probes N-allyl-4-(4’-N,N-diethylpropionamide-acetamido-piperazinyl)-1,8-naphthalimide I and N-(N,N-dibenzylpro- pionamide-acetamido)-4-allyl-1-piperazinyl-1,8-naphthalimide II were synthesised to monitor the change in pH in such a way that the presence of protons can increase the fluorescence intensity of these compounds. Unlike most of the other pH-sensitive probes reported, the probes possess the obvious advantage of being able to detect stronger acids at pH ≈ 2 and a combination of the two probes could detect a wider pH scale from 1.98 to 6.59; this should be very useful for monitoring the pH of the environment.     

Two near-infrared highly sensitive cyanine fluorescent probes for pH monitoring

Two near-infrared(NIR) p H-activated heptamethine indocyanine probes with quaternary ammonium unit were designed and synthesized. The absorption and emission titrations indicate that cationic structure improves the cyanine dye's aqueous solubility and these two probes exhibit highly sensitive response to p H in acid condition. Their fluorescence intensities both gradually increase about 25-fold from p H 7.60 to 3.00 with p Ka values of 4.72 and 4.45 respectively, which are suitable for studying acidic organelles in living cells. Moreover, their fluorescence intensities are linearly proportional to p H values in the range of 5.50–4.00. These results are probably attributed to the protonation of the indole nitrogen atoms, which are verified by 1H NMR spectra. Furthermore, these two probes can achieve real-time imaging of cellular p H and detection of p H in situ in living He La cells due to their excellent properties,including good reversibility, desirable photostability, high selectivity, low cytotoxicity and remarkable membrane permeability.     

Rational design of ratiometric near-infrared fluorescent pH probes with various pKa values, based on aminocyanine

Novel ratiometric, near-infrared fluorescent pH probes with various pK(a) values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pK(a) values of these probes can be predicted from the calculated pK(a) values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications.     

A new rhodamine B-based lysosomal pH fluorescent indicator

We designed and synthesized a new pH fluorescent probe, RCE, based on structural changes of rhodamine dye at different pH values. The probe exhibits high selectivity, high sensitivity and quick response to acidic pH, as well as low cytotoxicity, excellent photostability, reversibility and cell membrane permeability. Fluorescence intensity at 584 nm was increased more than 150-fold within pH range 7.51–3.53. This probe has pK_a value 4.71, which is valuable for studying acidic organelles. Because of its long absorption and emission wavelengths, RCE can avoid associated cell damage. The probe can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

苯乙烯类菁染料pH探针的合成与活细胞成像

合成了一个以苯乙烯类菁染料为母体的含胺类基团的pH荧光探针, 合成方法简单, 产物收率高, 提纯方便. 在乙醇-水体系中的性能测试结果表明, 随着pH值的降低, 其非质子化形态的吸收峰强度逐渐降低, 而质子化形式的长波长吸收峰强度逐渐升高, 长、短波长相差(Δλ)130 nm, 颜色由黄色变成粉红色, 利于可视观察; 探针在氨基呈非质子化形式时没有荧光, 氨基质子化后探针的荧光强度显著增强. 活细胞实验表明, 探针可以穿透细胞膜, 在细胞质内pH值偏低区域显示红色荧光, 可以用来定性探测酸性细胞器.     

Wavelength-ratiometric near-physiological pH sensors based on 6-aminoquinolinium boronic acid probes

We describe the pH response of a set of isomeric water-soluble fluorescent probes based on both the 6-aminoquinolinium and boronic acid moieties. These probes show spectral shifts and intensity changes with pH, in a wavelength-ratiometric and colorimetric manner. Subsequently, changes in pH can readily be determined around the physiological level.Although boronic acid containing probes are known to exhibit pH sensitivity along with an ability for saccharide binding/chelating, the new probes reported here are considered to be unique and show an unperturbed pH response, even in the presence of high concentrations of background saccharide, such as with glucose and fructose, allowing for the predominant pH sensitivity. The response of the probes is based on the ability of the boronic acid group to interact with strong bases like OH⁻, changing from the neutral form of the boronic acid group, R-B(OH)₂, to the anionic ester, R-B⁻(OH)₃, form, which is an electron donating group. The presence of an electron deficient quaternary heterocyclic nitrogen center and a strong electron donating amino group in the 6-position of the quinolinium backbone, provides for the spectral changes observed upon OH⁻ complexation. In addition, by comparing the results obtained with systems separately incorporating 6-methoxy or 6-methyl substituents, the suppressed response towards monosaccharides, such as with glucose and fructose, can clearly be observed for these systems. Finally we compare our results to those of a control compound, BAQ, which does not contain the boronic acid group, allowing a rationale of the spectral changes to be made.     

Monitoring the pH fluctuation of lysosome under cell stress using a near-infrared ratiometric fluorescent probe

Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organelle-specific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe (BLT) with lysosomes localization capability was developed. The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable pK_a value has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced (APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.     

Ratiometric Fluorescent pH Probes Based on Glycopolymers

Effectively detecting pH changes plays a critical role in exploring cellular functions and determining physiological and pathological processes. A novel ratiometric pH probe based on a glycopolymer, armored with properties of serum‐stability, tumor‐targeting, and pH monitoring, is designed. Random copolymers of 2‐(methacrylamido) glucopyranose and fluorescein O‐methacrylate are first synthesized by reversible addition fragmentation chain transfer polymerization. Acryloxyethyl thiocarbamoyl rhodamine B is then attached to the polymer chain to prepare ratiometric fluorescent pH probes via a thiol‐ene reaction. The synthesized polymeric probes are characterized by NMR, gel permeation chromatography, UV–vis spectroscopy, and transmission electron microscopy, and the fluorescence responses are examined in phosphate buffer at different pHs. The cytotoxicity and confocal imaging experiments of the probes are detected using HeLa cells, demonstrating a low toxicity and superior biocompatibility for detecting pH changes in bioapplications.