Fluorescent pH Indicator Research Articles

Synthesis and photophysics of a series of lipophilic phosphazene‐based fluorescent indicators

AbstractThis work explores the 7‐amino‐4‐(trifluoromethyl)coumarin backbone for designing lipophilic fluorescent pH indicators. Four novel fluorescent phosphazene bases with an advantageous combination of properties—high lipophilicity of both neutral and charged forms, lack of localized charges in the cations, and prominent spectral changes upon protonation/deprotonation—were synthesized using the Staudinger reaction. The molecular structures of the synthesized compounds were confirmed using attenuated total reflectance Fourier‐transform infrared spectroscopy (ATR‐FT‐IR), nuclear magnetic resonance (NMR), and Fourier‐transform ion‐cyclotron resonance (FT‐ICR) high‐resolution mass spectrometry. The compounds were further characterized by ultraviolet‐visible (UV‐Vis) absorption and fluorescence emission spectra, lipophilicities (logP values) and pKa values in water and acetonitrile, as well as “biphasic” pKa values in the water/1‐octanol solvent system. The bases could potentially be useful as working agents in optical pH sensors and acid‐base indicators for lipophilic membranes.

On-line sample preconcentration by polarity switching in floating electrode-integrated microchannel.

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly-dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)-integrated straight-channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530-fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high-pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode-embedded channel, the mixture of AF488-labeled proteins was also well concentrated and separated within 100 s.

NIR optical carbon dioxide gas sensor based on simple azaBODIPY pH indicators.

Two simple boron-dipyrromethene-type fluorophore (azaBODIPYs) dyes are synthesized and tested for the determination of CO2 gas by an inner filter process. The indicators are noncovalently entrapped in suitable polymers according to their polarity, featuring absorption maxima at 620 nm and fluorescent emission maxima in the range 675-720 nm. Molar absorptivity and fluorescence quantum yield data were determined for these two synthesized azaBODIPYs. These indicators have high molar absorption coefficients of 7.1 × 104 and 2.1 × 104 M-1 cm-1 and quantum yields of 21 and 9%. The pKa values of the indicators are determined from absorbance and fluorescence measurements with values of 7.9 and 8.5, depending on the positioning of the substitution pattern of the electron-donating functionalities. The two azaBODIPYs present excellent photostability, making them suitable for long duration measurements. These azaBODIPY dyes act as fluorescent pH indicators in a polymeric sensing membrane along with microcrystalline powder of chromium-doped gadolinium aluminium borate as the luminophore, a transfer phase agent (tetraoctyl or tetramethyl ammonium hydroxide) and a plasticizer or surfactant to improve membrane permeability to gaseous CO2. The response time ranges from 42 to 60 s and recovery time from 103 to 120 s, with a detection limit of 0.04 and 0.57% CO2. The store time of the sensing membranes is longer than 570 days in the best case, and it does not need to be kept in any special atmosphere other than darkness.

V-ATPase (Vacuolar ATPase) Activity Required for ABCA1 (ATP-Binding Cassette Protein A1)-Mediated Cholesterol Efflux.

Objective- We have shown that ABCA1 (ATP-binding cassette protein A1) mediates unfolding of the apoA1 (apolipoprotein A1) N-terminal helical hairpin during apoA1 lipidation. Others have shown that an acidic pH exposes the hydrophobic surface of apoA1. We postulated that the V-ATPase (vacuolar ATPase) proton pump facilitates apoA1 unfolding and promotes ABCA1-mediated cholesterol efflux. Approach and Results- We found that V-ATPase inhibitors dose-dependently decreased ABCA1-mediated cholesterol efflux to apoA1 in baby hamster kidney cells and RAW264.7 cells; and similarly, siRNA knockdown of ATP6V0C inhibited ABCA1-mediated cholesterol efflux to apoA1 in RAW264.7 cells. Although ABCA1 expression did not alter total cellular levels of V-ATPase, ABCA1 increased the cell surface levels of the V0A1 and V1E1 subunits of V-ATPase. We generated a fluorescein isothiocyanate/Alexa647 double-labeled fluorescent ratiometric apoA1 pH indicator whose fluorescein isothiocyanate/Alexa647 emission ratio decreased as the pH drops. We found that ABCA1 induction in baby hamster kidney cells led to acidification of the cell-associated apoA1 pH indicator, compared with control cells without ABCA1 expression. The V-ATPase inhibitor bafilomycin A1 dose-dependently inhibited the apoA1 pH shift in ABCA1-expressing cells, without affecting the levels of cell-associated apoA1. However, we were not able to detect ABCA1-mediated extracellular proton release. We showed that acidic pH facilitated apoA1 unfolding, apoA1 solubilization of phosphatidycholine:phosphatidyserine liposomes, and increased lipid fluidity of these liposomes. Conclusions- Our results support a model that ABCA1 recruits V-ATPase to the plasma membrane where V-ATPase mediates apoA1 acidification and membrane remodeling that promote apoA1 unfolding and ABCA1-mediated HDL (high-density lipoprotein) biogenesis and lipid efflux.

Polyvinyl alcohol magnetic microspheres containing 2-aminopyrimidin-bis(ethene)bis(dialkylphenylamine) compounds as fluorescent pH indicators

The fluorescent dyes 4,4′-(1E,1′Е)-2,2′-(2-aminopyrimidin-4,6-diyl) bis(ethene-2,l-diyl) bis(N,N-dimethylphenylamine) (АРМ) and 4′-(1E,1′Е)-2,2′-(2-aminopyrimidin-4,6-diyl) bis(ethene-2,l-diyl) bis(N,N-diethylphenylamine) (APE) were incorporated into glutaraldehyde-crosslinked polyvinyl alcohol microspheres containing magnetite. Fluorescence spectra of the obtained microspheres depend on pH of the medium. A technique for pH measurement within the range of 4–9 based on calculation of the green/red ratio of the fragments of color microimages of the microspheres was developed. only negligible loss of the fluorochromes from the microspheres occured during the storage time of 180 days. Thus the microspheres may be used as local pH indicators, e. g. for phagocytosis studies.

Local pH and Effective pK of a Polyelectrolyte Chain: Two Names for One Quantity?

In recent experiments, the "local pH" near polyelectrolyte chains was determined from the shift in the effective acidity constant of fluorescent pH indicators attached to the macromolecules. This indirect determination raises the question if the analyzed quantity was indeed the "local pH" and what this term actually means. In this study, we combined experiments and simulations to demonstrate that the shift in ionization constant is slightly lower than the difference between the pH and the "local pH". This offset is caused by correlations between fluctuations in chain conformation, small-ion distribution, and fluorophore ionization.

Surface pH buffering to promote degradation of mesoporous silica nanoparticles under a physiological condition

Despite significant advancement of mesoporous silica nanoparticle (MSN)-based biomedical research, studies have not been done enough to understand biodegradability of functional MSNs for better clinical efficacy. Polyethyleneimine (PEI) is one of the mostly used surface functionalities of MSNs, owing to the amine-rich chemical composition and the well-known proton sponge effect. In this paper, we study degradation behaviors of PEI-coated MSNs (PEI-MSNs) under a neutral or acidic physiological condition in comparison to those of surface-uncoated or nonionic F-127-encapsulated MSNs. The results showed that the surface coating by PEI could promote particle degradation in both neutral and acidic phosphate buffered saline (PBS) solution (i.e., pH 7.4 and 5.0). Importantly, we demonstrated that the local pH buffering by the surface PEI could lead to a greater total degradation quantity of particles even in the acidic PBS solution. The PEI-induced pH buffering phenomenon was confirmed by using a fluorescent pH indicator dye, fluorescein, which was attached to the surface of PEI-MSNs (F-PEI-MSNs). The observed pH-insensitive fluorescing behavior of fluorescein attained by surface coating with PEI corroborates the buffering effect that minimizes the surface pH change regardless of the external pH. The presented results may offer a useful insight into the degradability of silica nanomaterials with PEI or related surface functionalities, especially in the acidic subcellular microenvironment.

Determination of intracellular pH in phytoplankton using the fluorescent probe, SNARF, with detection by fluorescence spectroscopy

The maintenance of pH homeostasis is critical for a variety of cellular metabolic processes. Although ocean acidification is likely to influence cellular metabolism and energy balance, the degree to which intracellular pH in phytoplankton differs from the external environment under varying environmental pH levels is not well characterized. While there are numerous existing methods for the determination of intracellular pH in the form of single peak emission (e.g., BCECF) and radioisotopic (e.g., 14C-DMO) indicators for use with phytoplankton, the fluorescent pH indicator seminaphtharhodafluor (SNARF) has not been established as a robust method for measuring in vivo pH in phytoplankton. SNARF has superior accuracy and sensitivity since it exhibits dual emission peaks from a single excitation wavelength and the ratio of the two are related to pH. The use of a ratio limiting variations in fluorescence due to dye loading, photobleaching, and instrument variation; moreover, like other fluorescence-based assays, it does not require the specialized equipment and permits that radioisotopic methods do. As a first step, we tested the performance of SNARF for measuring intracellular pH in vivo in a number of phytoplankton taxa. SNARF detection was accomplished using fluorescence spectroscopy (FS) and laser scanning microscopy (LSM). Since SNARF fluorescence is activated by cleavage of an ester group from the core fluorophore by non-specific esterases, we measured esterase activity using fluorescein diacetate (FDA) to characterize variability in esterase activity among phytoplankton taxa, with a view towards its influence on assay performance. Esterase activity cell volume; however, there was no indication that enzyme specificity and differences in individual esterase profiles adversely affected SNARF performance in phytoplankton. Assays of intracellular pH using SNARF were comparable to those made with 14C-labeled DMO, an accepted standard method. Thus, SNARF provides robust measurements of intracellular pH in phytoplankton, constituting a useful tool in investigations of the effects of ocean acidification and fluctuations in environmental pH on cellular physiology.

A fluorescent AND logic gate based on a ferrocene-naphthalimide-piperazine format responsive to acidity and oxidizability

A novel fluorescent molecular logic gate 1 is demonstrated as a two-input AND logic gate for the detection of acidity and oxidizability. Designed according to an electron-donor–spacer–fluorophore–spacer–receptor’ format, the modules are represented by ferrocene as the electron donor, 1,8-naphthalimide as the fluorophore and piperazine as the proton receptor. In the presence of elevated concentrations of H+ and Fe3+, the molecule switches ‘on’ emitting at λmax = 530 nm with Φf = 0.060 at 10−4 M H+ and 50 μM Fe3+. The H+ binding and apparent Fe3+ binding constants determined by fluorimetric titrations in 1:1 (v/v) methanol/water are log βH+ = 8.1 and log βFe3+ = 4.7. Modulation of the fluorescence output results from controlling competing photoinduced electron transfer (PET) at the ferrocene end and internal charge transfer (ICT) at the piperazine end. Time-resolved fluorescence spectroscopy reveals a single fluorescent lifetime of 5.8 ns, while from absorption transient spectroscopy a remarkable fast decay signal <2 ps is observed. Comparison is made with methylpiperazine 2 and piperazine fluorescent pH indicator 3 as model compounds.

A Fluorescence Indicator at Extreme Low pH Region Based on Dissolution of Zn Tetra-2,3-Pyridoporphyradine (TPP) Nanocrystal Suspension.

Conventional organic pH indicators are widely used for chemical synthesis and analysis based on change of π conjugation length depending on pH values. However, there are a few organic pH indicators for strong acid conditions, because organic compounds are usually decomposed at extreme low pH. Tetra-2,3-pyridoporphyradine (TPP) has high chemical stability and crystallinity, and its nano-crystal is widely used as stable pigments. ZnTPP exhibits red fluorescence, but ZnTPP crystals don't exhibit fluorescence due to concentration quenching. In this paper, we show a UV-assisted synthesis of ZnTPP nano-crystals and application for fluorescence pH indicator at extreme low pH condition by quaternization of ZnTPP nano-crystals. ZnTPP crystals were successfully synthesized and then dispersed into 1 to 10 M HCl aq. UV-Vis and fluorescence spectroscopy revealed that absorption and fluorescence change were observed depending on HCl concentrations at extreme low pH conditions due to quarternization of ZnTPP and dissolution of ZnTPP nano-crystals. These results indicate that ZnTPP nano-crystals can be used for fluorescence pH indicators at extreme low pH.

Fabrication of microstructured binary polymer brush "corrals" with integral pH sensing for studies of proton transport in model membrane systems.

Binary brush structures consisting of poly(cysteine methacrylate) (PCysMA) "corrals" enclosed within poly(oligoethylene glycol methyl ether methacrylate) (POEGMA) "walls" are fabricated simply and efficiently using a two-step photochemical process. First, the C-Cl bonds of 4-(chloromethyl)phenylsilane monolayers are selectively converted into carboxylic acid groups by patterned exposure to UV light through a mask and POEGMA is grown from unmodified chlorinated regions by surface-initiated atom-transfer radical polymerisation (ATRP). Incorporation of a ratiometric fluorescent pH indicator, Nile Blue 2-(methacryloyloxy)ethyl carbamate (NBC), into the polymer brushes facilitates assessment of local changes in pH using a confocal laser scanning microscope with spectral resolution capability. Moreover, the dye label acts as a radical spin trap, enabling removal of halogen end-groups from the brushes via in situ dye addition during the polymerisation process. Second, an initiator is attached to the carboxylic acid-functionalised regions formed by UV photolysis in the patterning step, enabling growth of PCysMA brushes by ATRP. Transfer of the system to THF, a poor solvent for PCysMA, causes collapse of the PCysMA brushes. At the interface between the collapsed brush and solvent, selective derivatisation of amine groups is achieved by reaction with excess glutaraldehyde, facilitating attachment of aminobutyl(nitrile triacetic acid) (NTA). The PCysMA brush collapse is reversed on transfer to water, leaving it fully expanded but only functionalized at the brush-water interface. Following complexation of NTA with Ni2+, attachment of histidine-tagged proteorhodopsin and lipid deposition, light-activated transport of protons into the brush structure is demonstrated by measuring the ratiometric response of NBC in the POEGMA walls.

Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana

Plant cells are embedded within cell walls, which provide structural integrity, but also spatially constrain cells, and must therefore be modified to allow cellular expansion. The long-standing acid growth theory postulates that auxin triggers apoplast acidification, thereby activating cell wall-loosening enzymes that enable cell expansion in shoots. Interestingly, this model remains heavily debated in roots, because of both the complex role of auxin in plant development as well as technical limitations in investigating apoplastic pH at cellular resolution. Here, we introduce 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a suitable fluorescent pH indicator for assessing apoplastic pH, and thus acid growth, at a cellular resolution in Arabidopsis thaliana roots. Using HPTS, we demonstrate that cell wall acidification triggers cellular expansion, which is correlated with a preceding increase of auxin signaling. Reduction in auxin levels, perception, or signaling abolishes both the extracellular acidification and cellular expansion. These findings jointly suggest that endogenous auxin controls apoplastic acidification and the onset of cellular elongation in roots. In contrast, an endogenous or exogenous increase in auxin levels induces a transient alkalinization of the extracellular matrix, reducing cellular elongation. The receptor-like kinase FERONIA is required for this physiological process, which affects cellular root expansion during the gravitropic response. These findings pinpoint a complex, presumably concentration-dependent role for auxin in apoplastic pH regulation, steering the rate of root cell expansion and gravitropic response.

‘Pourbaix sensors’: fluorescent molecular logic gates for pE and pH

From the cross-fertilisation of fluorescent pH indicators and fluorescent redox switches, our group has established a new class of molecular sensor that operates as two-input molecular logic gates. These molecular sensors, known as ‘Pourbaix sensors’, are named in honour of Marcel Pourbaix, who developed the pH–potential diagrams for the various states of metal ion species in aqueous solution. This review highlights the evolution of ‘Pourbaix sensors’ based on anthracene and naphthalimide fluorophores. Potential applications of this class of molecule in fields such as corrosion science, cell biology and biomedical diagnostics are highlighted.

A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs.

Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P.falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P.falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by PfGR in CQ-resistant versus CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.

Integrating chemical imaging of cationic trace metal solutes and pH into a single hydrogel layer

Gel-based, two-dimensional (2D) chemical imaging techniques are versatile methods for investigating biogeochemically active environments at high spatial resolution (sub-mm). State-of-the-art solute imaging techniques, such as diffusive gradients in thin films (DGT) and planar optodes (PO), employ passive solute sampling or sensing. Combining these methods will provide powerful tools for studying the biogeochemistry of biological niches in soils and sediments. In this study we aimed at developing a combined single-layer gel for direct pH imaging using PO and sampling of anionic and cationic solutes by DGT, with subsequent analysis of the bound solutes by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). We tested three ultra-thin (<100 μm) polyurethane-based gels, incorporating anion and cation binding materials and the fluorescent pH indicator DCIFODA (2′,7′-dichloro-5(6)-N-octadecyl-carboxamidofluorescein).Results showed that PO-based pH sensing using DCIFODA was impossible in the presence of the anion binding materials due to interferences with DCIFODA protonation. One gel, containing only a cation binding material and DCIFODA, was fully characterized and showed similar performance characteristics as comparable DGT-only gels (applicable pH range: pH 5–8, applicable ionic strength range: 1–20 mmol L-1, cation binding capacity ∼24 μg cm−2). The dynamic range for PO-based pH mapping was between pH 5.5 and 7.5 with t90 response time of ∼60 min.In a case study we demonstrated the gel's suitability for multi-analyte solute imaging and mapped pH gradients and concurrent metal solubility patterns in the rhizosphere of Salix smithiana. pH decreases in the rooted soil were co-localized with elevated solute fluxes of Al3+, Co2+, Cu2+, Fe, Mn2+, Ni2+ and Pb2+, indicating pH-induced metal solubilisation.

Simultaneous detection of pH value and glucose concentrations for wound monitoring applications

Aging population and longer life expectancy are the main reasons for an increasing number of patients with wound problems. Although the interest in wound care increases continuously, wound management still remains a challenge mainly due to the higher occurrence of chronic wounds, which require intensive care and constant monitoring. Here, we demonstrate a fluorescent sensing system to monitor the wound status and to distinguish between an autonomously healing and a chronic wound at an early stage. The system allows monitoring two of the most relevant fluctuating wound parameters during the healing process which are pH and glucose concentration. A fluorescent pH indicator dye, carboxynaphthofluorescein, and a metabolite-sensing enzymatic system, based on glucose oxidase and horseradish peroxidase, were immobilized on a biocompatible polysaccharide matrix to develop a functional hydrogel coating for wound monitoring. The changes in metabolite and enzyme concentration in artificial wound extract were converted into a fluorescent signal.

Defective small intestinal anion secretion, dipeptide absorption, and intestinal failure in suckling NBCe1-deficient mice.

The electrogenic Na+HCO3− cotransporter NBCe1 (Slc4a4) is strongly expressed in the basolateral enterocyte membrane in a villous/surface predominant fashion. In order to better understand its physiological function in the intestine, isolated mucosae in miniaturized Ussing chambers and microdissected intestinal villi or crypts loaded with the fluorescent pH-indicator BCECF were studied from the duodenum, jejunum, and colon of 14- to 17-days-old slc4a4-deficient (KO) and WT mice. NBCe1 was active in the basal state in all intestinal segments under study, most likely to compensate for acid loads imposed upon the enterocytes. Upregulation of other basolateral base uptake mechanism occurs, but in a segment-specific fashion. Loss of NBCe1 resulted in severely impaired Cl− and fluid secretory response, but not HCO3− secretory response to agonist stimulation. In addition, NBCe1 was found to be active during transport processes that load the surface enterocytes with acid, such as Slc26a3 (DRA)-mediated luminal Cl−/HCO3− exchange or PEPT1-mediated H+/dipeptide uptake. Possibly because of the high energy demand for hyperventilation in conjunction with the fluid secretory and nutrient absorptive defects and the relative scarcity of compensatory mechanisms, NBCe1-deficient mice developed progressive jejunal failure, worsening of metabolic acidosis, and death in the third week of life. Our data suggest that the electrogenic influx of base via NBCe1 maintains enterocyte anion homeostasis and pHi control. Its loss impairs small intestinal Cl− and fluid secretion as well as the neutralization of acid loads imposed on the enterocytes during nutrient and electrolyte absorption.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-016-1836-3) contains supplementary material, which is available to authorized users.

Resting state of respiratory Complex I from Escherichia coli.

Respiratory Complex I from Escherichia coli may exist in two states, resting (R) and active (A). The conversion from the R- to A-forms occurs spontaneously upon turnover. The fast resting-to-active (R/A) transition of membrane-bound and purified Complex I was studied with the stopped-flow technique by following NADH oxidation either by absorption decay at 340 nm or using the fluorescent pH indicator, trisodium 8-hydroxypyrene-1,3,6-trisulfonate (pyranine). The R/A transition of Complex I from E. coli occurs upon its turnover in a time interval of ~ 1.5 s. Comparisons between the bacterial Complex I R/A transition and the active/deactive transition of mitochondrial Complex I are discussed.

Abstract 549: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that acidic pH exposes apoAI’s hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at acidic pH (EC 50 for unfolding at 0.94 and 0.75 M guanidine at pH 7.5 and 5, respectively), and confirmed that acidic conditions promoted apoAI solubilization of and binding to egg PC:PS (8:2 mole ratio) liposomes. We generated a FITC/Alexa647-labeled fluorescent ratiometric apoAI pH indicator whose FITC/Alexa647 emission ratio decreased as pH decreased. Using flow cytometry in BHK and RAW264.7 cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to substantial acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, eliminated the pH shift of apoAI on plasma membrane, and dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI. Although ABCA1 did not alter total cellular levels of V-ATPase, we showed that ABCA1 increased the cell surface levels of the V 0 A1 and V 1 E1 subunits of V-ATPase by cell surface biotinylation and immunofluorescence. Moreover, inhibition of apoAI acidification by bafilomycin decreased apoAI unfolding on the cell surface. Conclusions: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which promotes apoAI unfolding and lipidation.

BODIPY-derived piperazidine fluorescent near-neutral pH indicator and its bioimaging

Two new 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes (1 and 2) with piperazine subunits on position 3 or 5 were synthesized. Their absorption and steady-state fluorescence properties were investigated as a function of solvent. In water/MeCN (9:1, V/V) solution, the dyes show fluorescent enhancement upon increasing the acidity of the solution. They can be used in water/MeCN (9:1, V/V) solution as fluorescent pH probes excitable with visible light, with pKa values near-neutral (around 6.5 and 6.8). Dyes 1 and 2 are readily taken up by biological cells in the cytosol where it has no obvious toxic effects.