Fluorescent pH Sensor Research Articles

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.

Magnetic microdrill as a modulated fluorescent pH sensor

Remotely exploring biological tissues using magnetically driven micromachines could one day allow for medical diagnostics and treatment. We developed a magnetically driven drill capable of sensing pH changes. The drill is coated on one side with a fluorescent indicator dye that blinks when the drill rotates; extracting the blinking signal reduces background interference by over a factor of 100.

A fluorescent chemosensor for wide-range pH detection

Simple polyamines, L1-L3, bearing anthracene and benzophenone units at the respective ends, behave as a fluorescent pH sensor applicable to wide-range pH detection.

Novel Fluorescent pH Sensors Based on Intramolecular Hydrogen Bonding Ability of Naphthalimide.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Novel fluorescent pH sensors based on intramolecular hydrogen bonding ability of naphthalimide.

4-Piperidine-naphthalimide derivatives containing potential acceptors for hydrogen bonding were synthesized, and their fluorescence properties were examined. Some of the compounds with a 2-imino-oxalidin (thiazolidin) side chain at the imide moiety exhibit a strong fluorescence quench and some red shift in weakly acidic conditions, caused by the formation of an intramolecular hydrogen bond. Their pK(a) values were estimated to be about 6.4-7.5. The results showed that these compounds could serve as novel fluorescent pH sensors for further application. [structure: see text]

Fluorescent pH sensors with negligible sensitivity to ionic strength.

Optical pH determination has the fundamental disadvantage of measuring a signal that depends on the ionic strength of the sample. The problem originates from the complex relationship between the proton activity and the concentration of the pH-sensitive dye. The effect of ionic strength on the signal depends on the charge of the indicator and its environment, e.g. the immobilisation matrix. We present novel lipophilic fluorescein esters carrying one negative charge. They are embedded in an uncharged, highly proton-permeable hydrogel to give optical pH sensors that show a negligible cross-sensitivity towards ionic strength. The fluorescent dyes differ in their substituents. This variation of substituents results in dissociation constants between 5.5 and 8.5. The indicators were made lipophilic by esterification of the carboxy group with a C(18) alkyl chain. Since their spectral properties are quite similar, two indicators may be used in one sensor. This results in an optical pH sensor with a dynamic range that extends from pH 4.5 to 8.

Anthrylmethylamines and anthrylmethylazamacrocycles as fluorescent pH sensors—a systematic study of their static and dynamic properties

The pH dependent fluorescence of various 9-mono- and 9,10-disubstituded anthracenes is reported. They have a compartmental structure: fluorophore (anthracene)–spacer (methylene)–and ionophore (mono-, polyamines, azamacrocycles). In these n-basic anthrylmethylamines the fluorescence of anthracene is intramolecularly quenched by unprotonated amine groups. The pKA values have been evaluated by titration in order to calculate the amount (mole fraction) of each of the n + 1 differently charged species and their dependence on the pH. The fluorescence–pH profiles (intensities and lifetime versus pH) have been determined, and interpreted as the formation of differently protonated species with different fluorescence intensities and hence different fluorescence lifetimes. The experimentally determined fluorescence lifetime–pH profiles can be described by three lifetimes. This is corroborated by global analysis. The measurement of the fluorescence quantum yields of the completely protonated species yielded the radiative lifetimes. The rate constants and quantum yields of the photoinduced electron transfer (PET) are calculated for the species with shorter lifetimes and hence smaller fluorescence intensities.

Modeling of mixing in 96-well microplates observed with fluorescence indicators.

Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.

Electrostatic Core Shielding in Dendritic Polyglutamic Porphyrins

Polyglutamic dendritic porphyrins of the general formula H2PophGluNOR (H2Porph=free-base meso-tetra-4-carboxyphenylporphyrin (H2TCPP), Glu=dendrimer layer composed of L-glutamates, N=1-3: dendrimer generation number, R=terminal group (All, H)) were synthesized and characterized with NMR and MALDI-TOF mass spectroscopy. The free-acid terminated compounds were found to be highly soluble in water, with both their absorption and fluorescence spectra dependent on pH. The value of the porphyrin mono-protonation constant, measured by fluorescence rationing, increased monotonously in the studied series of dendrimers (pK3=6.31, 6.70, and 6.98, for N=1, 2, 3, respectively). For the largest dendrimer, H2PorphGlu3OH, pK3 was found shifted by almost two pH units relative to the non-modified H2Porph. The second protonation constant (K4) was much less affected by the dendritic substituents. At pH values less than 3.5 there were noticeable changes in fluorescence intensity and quantum yield even for the highly soluble H2PorphGlu3OH. This suggests that interactions between individual dendritic molecules in solution are favored by full protonation of the peripheral glutamic carboxyls. The “dendrimer-protected” porphyrins are convenient fluorescent pH sensors in the biological pH range.

Electrostatic core shielding in dendritic polyglutamic porphyrins.

Polyglutamic dendritic porphyrins of the general formula H2PophGlu(N)OR (H2Porph = free-base meso-tetra-4-carboxyphenylporphyrin (H2TCPP), Glu=dendrimer layer composed of L-glutamates, N= 1-3: dendrimer generation number, R = terminal group (All, H)) were synthesized and characterized with NMR and MALDI-TOF mass spectroscopy. The free-acid terminated compounds were found to be highly soluble in water, with both their absorption and fluorescence spectra dependent on pH. The value of the porphyrin mono-protonation constant, measured by fluorescence rationing, increased monotonously in the studied series of dendrimers (pK3=6.31. 6.70, and 6.98, for N=1, 2, 3, respectively). For the largest dendrimer, H2PorphGlu(3)OH, pK3 was found shifted by almost two pH units relative to the non-modified H2Porph. The second protonation constant (K4) was much less affected by the dendritic substituents. At pH values less than 3.5 there were noticeable changes in fluorescence intensity and quantum yield even for the highly soluble H2PorphGlu(3)OH. This suggests that interactions between individual dendritic molecules in solution are favored by full protonation of the peripheral glutamic carboxyls. The "dendrimer-protected" porphyrins are convenient fluorescent pH sensors in the biological pH range.

Arenedicarboximide Building Blocks for Fluorescent Photoinduced Electron Transfer pH Sensors Applicable with Different Media and Communication Wavelengths

A whole family of modular photoinduced electron transfer (PET) sensors has been constructed from a single simple reaction type with commercially available materials. This family, one member of which is shown here, possesses amine receptors for protons and arenedicarboximide fluorophores addressable at different wavelengths. Its pH-sensing behaviour is examined in solution and in polymer membrane matrices, which are crucial for the use of fluorescent PET sensors as optode pH monitors.

Pyridyl-, bipyridyl-, and terpyridyl-functionalised azamacrocycles

Abstract A series of azamacrocycles which have been N-functionalised with pendent pyridylmethyl-(pyCH2-), bipyridylmethyl-(bipyCH2-) and terpyridylmethyl- (terpyCH2-) arms have been synthesised and characterised, and some of their coordination chemistry with transition metal ions is reported. By attaching the pendent-arms at the 5- rather than the 6-position of the py, bipy and terpy, new ligands are generated which can be used to form polynuclear metal complexes in a controlled and systematic fashion. Fluorescent pH and transition metal ion sensors have been developed by reacting the azamacrocyclic N-pendent bipyCH2 arm(s) with cis-[Ru(bipy)2Cl2], to give macrocycles with up to four attached [Ru(bipy)3]2+ groups. That based on 1,4,7-triazacyclononane (9N3), with three attached [Ru(bipy)3]2+ groups, has a first photo excited state pKa of 7.1, and is a useful fluorescent sensor for physiological pH at below micromolar concentrations. The analogous derivative of cyclam (1,4,8,11-tetraazacyclotetradecane) ca...

Dependence of the fluorescence of immobilized 1-hydroxypyrene-3,6,8-trisulfonate on solution pH: Extension of the range of applicability of a pH fluorosensor

1-Hydroxy-3,6,8-pyrenetrisulfonate (HPTS), covalently immobilized on cellulose attached to a plastic strip, has been used as a fluorescent optical pH sensor for the neutral pH region. At near neutral pH, HPTS fluoresces green, i.e., from its conjugate base form, exclusively and independent of whether it was excited in its conjugate base form or acidic (phenol) form, or both. However, the fluorescence of immobilized HPTS also varies in the low pH region from about pH 3 to below pH 1, in that not only the green emission almost completely disappears, but also a second (blue) band appears which can be attributed to the fluorescence of the conjugate acid (the phenol). The proton transfer in the lowest excited singlet state which accounts for the variation of the fluorescence in the low pH region is here evaluated kinetically. The application of this sensor to measure pH over a wide range of pH values is suggested.

Luminescence and charge transfer. Part 4. ‘On–off’ fluorescent PET (photoinduced electron transfer) sensors with pyridine receptors: 1,3-diaryl-5-pyridyl-4,5-dihydropyrazoles

1,3-Diaryl-5-pyridyl-4,5-dihydropyrazoles, some of which have been previously synthesized for use as optical brighteners, satisfy the design criteria for fluorescent PET pH sensors by a proton-induced ‘switching off’ of fluorescence. In practice, they are the first family of ‘on–off’ fluorescent PET pH sensors whose signals cover most of the visible spectrum. They also show significant absorptiometric pH sensing action due to the coupling of the protonatable pyridine with the ICT (internal charge transfer) excited state of the 1,3-diaryl-4,5-dihydropyrazole. Experimentally identical pKa values arise from the fluorimetric and absorptiometric pH dependences. The examination of 2-, 3- and 4-pyridyl regioisomers shows understandable differences in pKa and maximum fluorescene quantum yield [φF(base)] values, but show no significant variations in fluorescence switching factors (FE).

Miniaturization of organically doped sol-gel materials: a microns-size fluorescent pH sensor

Miniaturization of a reactive doped sol-gel material is described. A reversible fluorescent pH sensor was constructed by placing a pyranine doped SiO 2 sol-gel matrix at a 10 μm tip of a metal coated micropipette. Subsecond response time was achieved by further downscaling to ≈ 1 μm. Using optical near-field microscopy configuration, these observations demonstrate the potential of constructing a variety of miniaturized sensors and chemical manipulators, by doping the sol-gel matrix with a suitable reagent.

Luminescence and charge transfer. Part 3. The use of chromophores with ICT (internal charge transfer) excited states in the construction of fluorescent PET (photoinduced electron transfer) pH sensors and related absorption pH sensors with aminoalkyl side chains

7-Methoxycoumarin, 3-aminobenzimidazo[2,1-a]benz[de]isoquinolin-7-one and 4-nitroaniline derivatives, useful as chromatographic derivatizing agents, dyes and solvent polarity indicators respectively, are easily transformed into optical pH sensors 3a–c, 4a–c and 5a–c respectively by attachment of a proximal, non-adjacent amine moiety. The availability of ICT (internal charge transfer) excited singlet states in each of these sensor families leads to protonation-induced wavelength shifts of their electronic absorption spectra. A similar phenomenon is observed in those fluorescence emission spectra where the proton-free form of the sensor is weakly emissive due to the sluggishness of the designed PET (photoinduced electron transfer) process. Except for these differences, sensors 3a–c and 4a–c show the expected pH dependence of fluorescence quantum yields, thus extending the scope of the fluorescent PET pH sensor design logic to electronic push-pull fluorophores besides aromatic hydrocarbons.