Carbazole as an excited state proton transfer fluorescent probe for lipid bilayers in alkaline medium

In our effort to look for novel excited state proton transfer (ESPT) fluorescent probes in alkaline pH range, we have examined carbazole as a possible candidate because of its high extinction coefficient, high quantum yield and a larger difference in ionization constant between the ground and excited state (pKa - pKa*). The photodissociation of carbazole was studied in liposome membrane by steady state fluorescence measurements at alkaline pH ranges. The neutral form and the anionic form of carbazole emit at 362 and 417 nm, respectively. This large shift in emission makes it convenient to monitor the physical properties of liposomes. The neutral form fluorescence intensity of carbazole is sensitive to phase changes in the membrane and also shows a maximum at phase transition temperature. This variation of intensity can be explained in terms of redistribution of probe between the surface and interior of the liposomes. Cholesterol induced phase changes of liposomes were also sensed by the ESPT of carbazole.     

Visual pH Sensors: From a Chemical Perspective to New Bioengineered Materials

Many human activities and cellular functions depend upon precise pH values, and pH monitoring is considered a fundamental task. Colorimetric and fluorescence sensors for pH measurements are chemical and biochemical tools able to sense protons and produce a visible signal. These pH sensors are gaining widespread attention as non-destructive tools, visible to the human eye, that are capable of a real-time and in-situ response. Optical “visual” sensors are expanding researchers’ interests in many chemical contexts and are routinely used for biological, environmental, and medical applications. In this review we provide an overview of trending colorimetric, fluorescent, or dual-mode responsive visual pH sensors. These sensors include molecular synthetic organic sensors, metal organic frameworks (MOF), engineered sensing nanomaterials, and bioengineered sensors. We review different typological chemical entities of visual pH sensors, three-dimensional structures, and signaling mechanisms for pH sensing and applications; developed in the past five years. The progression of this review from simple organic molecules to biological macromolecules seeks to benefit beginners and scientists embarking on a project of pH sensing development, who needs background information and a quick update on advances in the field. Lessons learned from these tools will aid pH determination projects and provide new ways of thinking for cell bioimaging or other cutting-edge in vivo applications.     

Development of selective,visible light-excitable,fluorescent magnesium ion probes with a novel fluorescence switching mechanism

Novel fluorescent Mg2+ probes, 2'-carboxyfluorescein (2'-CF) and its derivatives, that are excitable by visible light, were designed, synthesized and characterized. After complexation with Mg2+, the absorption and emission maxima of these fluorescent probes were red-shifted and the fluorescence intensity was increased 11-fold at neutral pH. The apparent dissociation constant (Kd) of the Mg2+-2'-CF complex was 15.8 mM at neutral pH. The Kd of the Ca2+-2'-CF complex was about 10 times larger than that of the Mg2+ complex and the other derivatives showed similar characteristics. In contrast, all the commercially available fluorescent Mg2+ probes have a higher affinity for Ca2+ than Mg2+. 2'-CF fluoresced in alkaline solution (pH > 8) and the pK, value was 8.8. The pKa value of the Mg2+-2'-CF complex was 6.8, and the fluorescence intensity was increased in the neutral conditions. Thus, the addition of Mg2+ resulted in a lowering of the pKa, and also an increase of the fluorescence intensity.     

Towards highly fluorescent and water-soluble perylene dyes

A systematic approach towards highly fluorescent, water-soluble perylene-3,4:9,10-tetracarboxylic acid diimide chromophores is presented. Water solubility was introduced first through the attachment of four hydrophilic substituents onto the bay region of the perylene dye. Positively and negatively charged groups were then applied to the chromophore, and their number and their distance from the aromatic scaffold were systematically varied. To suppress aggregation, the chromophore was further isolated within a dendritic shell. Such variation of structural features and a thorough investigation of the resulting optical properties facilitated the first synthesis of perylene-3,4:9,10-tetracarboxylic acid diimides combining the properties of water solubility and fluorescence quantum yields (FQYs) close to unity, which makes them attractive as high-performance fluorescence probes in aqueous media.     

响应范围可调的pH荧光传感器的构建及其性能研究

合成并表征了化合物N,N'-二谷氨酸铵盐-3,4,9,10-苝四羧酸二酰亚胺(PTCDG).此化合物水溶性好,荧光量子产率高.基于此,设计了一组新颖的响应范围可调的pH荧光传感器.PTCDG与顺磁性的Fe3+可按1∶1络合,络合常数为6.5×105,考察了其它金属离子的影响,表明PTCDG对Fe3+有良好的选择性.PTCDG与Fe3+结合后,由于PET效应,荧光猝灭,伴随pH值增大,Fe3+与OH-形成Fe(OH)3,从体系中沉淀出来,荧光团被释放,从而荧光恢复.在不同的Fe3+的良好配体存在条件下,形成Fe(OH)3的pH范围向碱性方向移动.PTCDG,PTCDG/Fe3+,PTCDG/Fe3+/三乙胺,PTCDG/Fe3+/吡啶、PTCDG/Fe3+/邻菲罗啉的响应范围分别为4.0～6.0,5.0～10,5.0～7.0,6.0～8.0和7.0～9.0,涵盖了pH值4.0～10的区间,实现了响应范围的可调.考察了内在的干扰物对该传感器的效能的影响,探讨了此体系的荧光传感机理.此传感器具有很多优异的性能,响应的范围可调且近中性,荧光和颜色变化明显,响应快,具有较高的灵敏性.     

Highly selective 4-amino-1,8-naphthalimide based fluorescent photoinduced electron transfer (PET) chemosensors for Zn(II) under physiological pH conditions

The design and synthesis of two novel fluorescent sensors based on the photoinduced electron transfer (PET) concept, and , for the detection of zinc under competitive media is described. These sensors are based on the 4-amino-1,8-naphthalimide fluorophore, which has an absorption band centred at 450 nm and emits in the green with lambda(max) approximately 550 nm. By functionalizing the chromophore with a simple benzyl or ethyl-aryl based iminodiacetate receptor at the 4-position, both high selectivity and sensitivity were achieved for the sensing of Zn(II) over other competitive transition and Group I and II metal ions. These sensors were also shown to be pH independent, with a pKa of 2.3 being determined for , which allows these to be used in highly competitive pH media. Upon sensing of Zn(II) the fluorescence emission spectrum is 'switched on' demonstrating the suppression of PET from the receptor to the fluorophore. For , the sensing of Zn(II) was achieved with Kd = 4 nM when measured in pH 7.4 buffered solution, in the presence of 1.1 mM of EGTA.     

A new water-soluble near-neutral ratiometric fluorescent pH indicator

Donor-pi-acceptor fluorene derivative 1c is a near-neutral pH indicator whose pKa of approximately 7.0 was determined by both absorption and fluorescence methods. 1c satisfies important criteria for a sensitive ratiomeric fluorescent pH indicator with a distinctive isoemissive point, good dispersion in cell cytosol, and low cytotoxicity. Furthermore, its 2PA cross section of 100 GM in its neutral form suggests its potential in two-photon fluorescence imaging applications.     

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.     

Change of pH indicator's pKa value via molecular imprinting

The pKa value of the indicator is significantly changed up to several orders of magnitude by the imprinting synthesis; this fine-tuning of the reactivity for pH indicators via the imprinting synthesis may find novel applications in fabricating pH sensors with tailored pKa values.     

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.     

BODIPY-based hydroxyaryl derivatives as fluorescent pH probes

[structures: see text] Seven new 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes with phenolic or naphtholic subunits on position 8 and with substituents having different electron driving forces on positions 3 and 5 were synthesized. Their absorption and steady-state fluorescence properties were investigated as a function of solvent. The novel compounds, with the exception of 4,4-difluoro-8-(4-hydroxyphenyl)-3,5-bis-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene, are characterized by absorption maxima in the range 493-515 nm and small (400-600 cm(-1)) Stokes shifts. The exceptional dye has absorption maxima in the 570-580 nm region and fluorescence emission maxima around 610-620 nm, depending on the solvent. In aqueous solution, the dyes show a large fluorescent enhancement upon increasing the acidity of the solution. They can be used in aqueous solution as fluorescent pH probes excitable with visible light, with pKa values ranging from 7.5 to 9.3, depending on the substitution pattern on positions 3, 5, and 8.     

A series of simple curcumin-derived colorimetric and fluorescent probes for ratiometric-pH sensing and cell imaging

Colorimetric and fluorescent probes have emerged as a potent tool for pH sensing due to easy operation and high sensitivity. However, most of the existing bimodal probes require complicated synthesis,which greatly limits their wide applications. Herein, a simple fluorescent dye（called BFCUR） featuring a D-π-A-π-D conjugated system was developed from the natural polyphenol curcumin（CUR）. BFCUR exhibited significant red-shift in UV absorption and fluorescence emission as pH increased because of th...     

Fiber optic pH sensing with long wavelength excitable Schiff bases in the pH range of 7.0-12.0

Most of the fluorescent pH probes work near neutral or acidic regions of the pH scale. In this work, two different fluorescent Schiff bases, chloro phenyl imino propenyl aniline (CPIPA) and nitro phenyl imino propenyl aniline (NPIPA), have been investigated for pH sensing in the alkaline region. Absorption and emission based spectral data, quantum yield, fluorescence lifetime, photostability and acidity constant (pK(a)) of the Schiff bases were determined in conventional solvents and in PVC. The long wavelength excitable immobilized Schiff bases CPIPA (lambda(ex)=556 nm) and NPIPA (lambda(ex)=570 nm) exhibited absorption and emission based optical response to proton in the pH range of 8.0-12.0 and 7.0-12.0, respectively. Response of the CPIPA was fully reversible within the dynamic working range. The response times were between 3-13 min. A relative signal change of 95% and 96% have been achieved for sensor dyes of CPIPA and NPIPA, respectively. The CPIPA displayed better fluorescence quantum yield (varphi(F)=3.7 x 10(-1)) and higher matrix compatibility compared to NPIPA (varphi(F)=1.6 x 10(-1)) in immobilized PVC. The CPIPA and NPIPA exhibited a slight cross sensitivity to the ions of Hg(+) and Fe(3+), respectively.     

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.     

氟硼二吡咯类pH荧光探针PET光谱特性的理论计算

氟硼二吡咯(BODIPY)类pH荧光探针分子是基于光诱导电子转移(PET)的荧光探针分子, 识别基团氮原子上引入不同取代基可呈现不同的光学灵敏度. 本文应用密度泛函理论(DFT)及含时密度泛函理论(TD-DFT)方法对六种含不同取代基的探针分子进行了几何构型优化及激发态计算, 探讨了不同取代基对PET效应影响. 计算结果表明: 基态时这些探针分子的最高占有分子轨道(HOMO)和最低未占有分子轨道(LUMO)都在荧光母体BODIPY的π, π^*轨道, 而识别基团上氮原子孤对电子所在的轨道为HOMO-1轨道. 但是在激发态, 当氮原子上有两个取代基时, HOMO-1→LUMO跃迁的激发能都小于荧光团的HOMO→LUMO跃迁, 这将有可能产生PET效应并导致荧光熄灭, 而当氮原子上有一个取代基时不会出现这种现象. 通过激发态结构优化可以发现, 无论识别基团氮原子上有一个还是两个取代基, N原子的轨道对称性都发生变化, 由sp³→sp², 孤对电子占据在p轨道上, 其轨道能级升高至荧光团的HOMO和LUMO轨道之间, 将导致不同程度的PET效应, 与实验结果一致.     

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.     

New fluorescent perylene bisimide indicators—a platform for broadband pH optodes

Asymmetric perylene bisimide (PBI) dyes are prepared and are shown to be suitable for the preparation of fluorescence chemosensors for pH. They carry one amino-functional substituent which introduces pH sensitivity via photoinduced electron transfer (PET) while the other one increases solubility. The luminescence quantum yields for the new indicators exceed 75% in the protonated form. The new indicators are non-covalently entrapped in polyurethane hydrogel D4 and poly(hydroxyalkylmethacrylates). Several PET functions including aliphatic and aromatic amino groups were successfully used to tune the dynamic range of the sensor. Because of their virtually identical spectral properties, various PBIs with selected PET functions can easily be integrated into a single sensor with enlarged dynamic range (over 4 pH units). PBIs with two different substitution patterns in the bay position are investigated and possess variable spectral properties. Compared with their tetrachloro analogues, tetra-tert-butyl-substituted PBIs yield more long-wave excitable sensors which feature excellent photostability. Cross-sensitivity to ionic strength was found to be negligible. The practical applicability of the sensors may be compromised by the long response times (especially in case of tetra-tert-butyl-substituted PBIs).     

Spectroscopic studies of a near-infrared absorbing pH sensitive aminodienone-carbocyanine dye system

Synthetic red and near-infrared absorbing dyes may be used as probe molecules in a large number of applications. Dyes exhibiting spectral changes with hydrogen ion concentration are useful as pH probes. Those dyes which have their absorption and fluorescence maxima in the long wavelength region of the visible spectral region are specially valuable because of decreased interference and semiconductor laser applications. In this paper we have evaluated an aminodienone dyes 1 which demostrates pH dependent absorption and fluorescence spectra as well as solvent polarity dependence. In organic solvents the long wavelength absorption band of the dye is in the reduced interference region. The absorption maximum is at 535 nm in neutral or alkaline solutions in methanol. The absorption spectra undergo a strong bathochromic shift in the presence of acids (lambda(max) = 709 nm) with a concomitant change in the fluorescence spectra. This pH sensitive dye was found to be specially especially useful for organic solvents. The analytical utility of this and similar near-infrared absorbing dyes is discussed.     

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.     

Membrane-permeable and -impermeable sensors of the Zinpyr family and their application to imaging of hippocampal zinc in vivo

Esterification of fluorescent biosensors is a common strategy used to trap probes within the cell. Zinpyr-1 (ZP1) is a fluorescein-based bright fluorescent sensor for divalent zinc that is cell permeable without prior modification. We describe here the synthesis and characterization of ZP1 sensors containing a carboxylic acid or ethyl ester functionality at the 5 or 6 position of the fluorescein. The presence of an electronegative carboxylate decreases the proton-induced background fluorescence of the probe by lowering the pKa of the benzylic amines responsible for fluorescence quenching. The charged species ZP1(6-CO2-) is membrane-impermeant, whereas the permeability of the neutral ZP1(5/6-CO2Et) is similar to that of the parent sensor. Intracranial microinfusion of ZP1(6-CO2Et) into rat hippocampus produces reduced staining of vesicular zinc in neuropil and very clear delineation of zinc-positive injured neuronal somata and dendrites as compared with ZP1.