Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values

The stability at room temperature (25°C) of recombinant green fluorescent protein (GFPuv), expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction column, was studied. The GFPuv was diluted in buffered (each 10 mM: Tris-HCl, pH 8.0; phosphate, pH 6.0 and 7.0 and acetate, pH 5.0) and in unbuffered (water for injection [WFI]; pH 6.70 ± 0.40) glucose solutions (from 1.5 to 50%). By assaying the loss of fluorescence intensity as a measure of denaturation, the stability of GFPuv in these solutions was evaluated relative to glucose concentration, pH, osmolarity, density, conductivity, and viscosity. The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time required to reduce 90% of the initial fluorescence intensity of GFPuv. The D-value between 56 and 83 h of GFPuv at 1.5–15% glucose in WFI was equivalent to 20–30% glucose in a phosphate. The stability of GFPuv in 50% glucose was similar for all buffers studied and four times higher than in WFI. By the convenient measure of fluorescence intensity, GFPuv can be used as an indicator to report the extent of denaturation rates of other proteins in glucose solutions.     

A Cu protein cavity mimicking fluorescent chemosensor for selective Cu recognition: tuning of fluorescence quenching to enhancement through spatial placement of anthracene unit

Four new fluoroionophores possessing four ligating sites (2S+2N) and an essential hydrophobic environment, as prevailing in the plastocyanin and rusticyanin proteins, have been synthesized. In these PET fluoroionophores, the position of fluorophore anthracene moiety effectively modulates the Cu²⁺ induced emission properties (quenching vs enhancement) of the fluorophore. The addition of Cu²⁺ to solution of receptor with anthracene moiety in its center caused quenching in emission intensity through photoinduced fluorophore-to-metal electron transfer mechanism and in cases where anthracene is present at terminus nitrogen, the emission intensities increased by nearly 1000% due to inhibition of the photoinduced electron transfer from receptor-to-fluorophore in the presence of Cu²⁺ ions. The hydrophobic environment created by various aromatic rings clearly manifested the stability of fluorescence of these molecules above pH 2.0 and their Cu²⁺ complexes above pH 4. The application of such fluoroionophores has been elaborated for building OR and AND logic gates.     

Design and synthesis of a Cu(II)-complex-based carbazole-hemicyanine hybrid for fluorescent sensing of H2S in SDS micellar solution

A novel fluorescent chemosensor HACBA with carbazole-hemicyanine fluorophore as signal reporter and N,N,N'-tri(2-pyridylmethyl)ethylenediamine (TPEA) as binding sites was designed and synthesized. Its assemblies with anionic surfactant sodium dodecyl sulfate (SDS) show improved fluorescence emission stability and enhanced fluorescence intensity. HACBA/SDS system can selectively recognize Cu²⁺, which led to a dramatic fluorescence quenching. The in situ resultant HACBA-Cu(II)/SDS ensemble functioned as a highly selective and sensitive sensor for H₂S with a turn-on fluorescent response. Our results show that the “on-off-on” molecular switch occured through the reversible formation-dissociation reaction between HACBA-Cu(II) complex and HACBA/CuS in the SDS micellar solution, and at least 3 cycles of on-off-on switches were observed.     

Studies on the photophysical characteristics of poly(carboxylic acid)s bound protoporphyrin IX and metal complexes of protoporphyrin IX

The copolymers of methacrylic acid with protoporphyrin IX (PPIX) and the metal complexes, zinc protoporphyrin IX and magnesium protoporphyrin IX were synthesised and characterised. Corresponding acrylic acid copolymers were also synthesised. The steady state absorption and fluorescence spectral properties of the macromolecular bound fluorophores PPIX, Zn-PPIX and Mg-PPIX were investigated. Poly(methacrylic acid) bound protoporphyrin IX, zinc protoporphyrin IX and magnesium protoporphyrin IX show an increase in the fluorescence intensity and lifetime with increase in the pH in the range 2-8 with a marked transition around pH 6.0-7.0. The fluorophore concentration in the dilute solution of the copolymers is micromolar and the fluorophore to the carboxylic acid monomer ratios in the copolymer is around 10⁻³. The molecular weight of the copolymers is 100 ± 10 kD. The fluorescence decay curves of all the fluorophore bound polymers follow biexponential decay fit independent of pH. Poly(MAA-co-PPIX) and poly(MAA-co-MgPPIX) undergo well marked pH induced structural transitions in the pH range of 6.0-7.0 whereas poly(MAA-co-ZnPPIX) undergoes pH induced structural transitions in the pH range of 4.0. In the case of polyacrylic acid copolymers the changes observed in the steady state and time resolved fluorescence studies are less marked. The distinct hydrophobic and hydrophilic environments experienced by the fluorophore bound to PMMA are attributed to the dynamics of the macromolecules in dilute aqueous solutions manifested by the α-methyl group present in the copolymer. The studies carried out using the fluorophores in the time windows from 2 ns to 12 ns indicate evolving trends in the dynamic coiling and reverse coiling of poly methacrylic acid chain.     

Proton "off-on" behaviour of methylpiperazinyl derivative of naphthalimide: a pH sensor based on fluorescence enhancement

In a search for new type pH sensing fluorophores, the possibility of using the proton "off-on" switch behaviour of naphthalimide derivatives for optical pH sensor preparation has been explored. A new compound, N-allyl-4-(4[prime or minute]-methyl-piperazinyl)-1,8-naphthalimide (AMPN), was synthesized. The enhancement of fluorescence of AMPN with the increase of hydrogen ion concentration is based on arresting photo-induced electron transfer to the naphthalimide fluorophore from aliphatic amine group after its protonation. The Stokes Shift of the proposed type of pH sensing fluorophore is significantly larger than that of the fluorescein counterparts. To avoid the leakage of the fluorophore, AMPN was photo-copolymerized with 2-hydroxyethyl methacrylate and acrylamide on the glass surface. The fluorescence intensity of membrane contacted with a pH 3.50 buffer is 4.7 times of that for pH 12.00 buffer solutions. The proposed pH sensor is not susceptible to ionic strength and shows good selectivity, repeatability and short response time. The membrane shows a good stability with a lifetime over two months. The sensor can be used for the determination of pH in the range of pH 4.5-9.0, without interference of most commonly co-existing inorganic ions and some organic species. The sensor has been applied to the analysis of urine samples.     

Fluorescence investigation of isochlorotetracycline: ground-state and excited-state acid-base equilibria

The fluorescence emission from isochlorotetracycline is shown to be associated with the hydroxyphthalide portion of the molecule. Neutral, anionic, zwitterionic and cationic forms of the fluorophore are proposed to account for the fluorescent behaviour in aqueous and organic media of different acidities. In strongly acidic aqueous solution intermolecular photoautomerism is observed, while in chloroform intramolecular phototautomerism occurs. In aqueous solution an excited-state pK_a of 3.3 was observed for the fluorophore. From a study of the pH vs. fluorescence profile with excitation at 350 nm, the ground-state microscopic dissociation constants for isochlorotetracycline hydrochloride were calculated. The optimum conditions for the fluorimetric determination of isochlorotetracycline are an alkaline medium (pH > 10) with κ_ex = 350 nm and κ_em = 415 nm.     

A dual role of phenylboronic acid as a receptor for carbohydrates as well as a quencher for neighboring pyrene fluorophore

A simple amino acid based compound (1) containing a phenyl boronic group and pyrene fluorophore showed an enhanced fluorescence in aqueous solutions at physiological pH through suppression of the photoinduced electron transfer from pyrene to boronic acid on carbohydrate binding. The compound exhibited an interesting fluorescence change depending on pH with decreased emission intensity at acidic pH but enhanced emission intensity at basic pH unlike the fluorescent carbohydrate chemosensors using a PET process with amine and aryl-boronic acid. We have characterized a dual role of phenylboronic acid as a receptor for carbohydrates as well as a quencher for the fluorescence of pyrene fluorophore.     

A New Near-Infrared Neutral pH Fluorescent Probe for Monitoring Minor pH Changes and its Application in Imaging of HepG2 Cells

A new near-neutral pH near-infrared (NIR) fluorescent probe utilizing a fluorophore–receptor molecular framework that can modulate the fluorescence emission intensity through a fast photoinduced electron transfer process was developed. Our strategy was to choose tricarbocyanine (Cy), a NIR fluorescent dye with high extinction coefficients, as a fluorophore, and N-methylpiperazine (MP) as a receptor. The pH titration indicated that MP-Cy can monitor the minor physiological pH fluctuations with a pKa of ～7.10 near physiological pH, which is valuable for intracellular pH researches. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.05–7.10 and exhibits strong dependence on pH changes. As expected, the real-time imaging of cellular pH and the detection of pH in situ was achieved successfully in living HepG2 cells by this probe. It is shown that the probe effectively avoids the influence of autofluorescence and native cellular species in biological systems and meanwhile exhibits high sensitivity, good photostability, and excellent cell membrane permeability.     

Studies on the dynamics of poly(carboxylic acids) with covalently bound thionine and phenosafranine in dilute aqueous solutions

The poly(carboxylic acid) bound phenosafranine and thionine dyes show that, the fluorescence intensity and lifetime increases first and starts to decrease after reaching a maximum at pH 4.0. The fluorescence decay curve of the fluorophore bound polymers follow the biexponential decay fit independent of pH, while poly(MAA-Th) follows single exponential function above pH 4.0. At low pH, a more compact environment of the fluorophore exerts a more hydrophobic environment. In the subnanosecond time domain the solvation process is found to be incomplete while in the nanosecond time scale the solvation of the macromolecular chains is found to be over. The time resolved fluorescence spectra of the polymer bound fluorophores at different pH indicate distinct hydrophobic and hydrophilic environments due to the dynamics of the macromolecules in dilute aqueous solutions. For the first time structural transitions involving solvent are observed in the nanosecond and picosecond time domains for the same macromolecule.     

Synthesis and fluorescence properties of naphthalimide-containing Tröger’s bases

Tröger’s bases based on the naphthalimide fluorophore have been prepared from N-alkyl-4-amino-1,8-naphthalimides. The fluorescence emission intensity of these dyes is highly medium dependent. In cyclohexane, these dyes emit near 440 nm with high quantum yields; addition of cosolvents reduces the fluorescence intensity near 440 nm and leads to increased fluorescence intensity around 480 nm.     

Modular fluorescence sensors for saccharides

Modular and modular polymer supported fluorescence photoinduced electron transfer (PET) sensors 2 and 3 with two boronic acid receptor units, a pyren-1-yl fluorophore, and hexamethylene linker show selective saccharide binding in aqueous methanolic solution at pH 8.21.     

Enhancement of the fluorescence of the zinc-morin complex by a non-ionic surfactant

A method is described for the fluorimetric determination of zinc, based on formation of a zinc-morin complex in the presence of a non-ionic surfactant. The complex has practically no fluorescence in the absence of surfactant, but the addition of Genapol PF-20 (non-ionic surfactant, ethylene oxide-propylene oxide condensate) makes possible the fluorimetric determination of low concentrations of zinc as it enhances the fluorescence of the complex about 75-fold. Maximum fluorescence is produced at pH 4.7 +/- 0.2 (acetic acid-acetate buffer), with 1.5% surfactant and 0.009% morin. The fluorescence is excited at 433 nm and measured at 503 nm. The calibration graph is linear up to 150 ng/ml zinc concentration and the detection limit is 3 ng/ml. The relative standard deviation (11 replicates) is 2.4% for zinc at 20 ng/ml concentration and 1.7% for 100 ng/ml. Of 29 ions studied, Al(3+), Be(2+), Zr(4+) and Cd(2+) strongly increase the fluorescence of the system, and Fe(3+), Ni(2+), Cu(2+), Ti(IV) and Co(2+) decrease the fluorescence signal.     

Photometric and fluorimetric assay of alkaline phosphatase with new coumarin-derived substrates

Summary Two new coumarin-derived synthetic substrates for use in the direct and continuous kinetic assay of alkaline phosphatase are presented. They have been studied with respect to optimum pH (9.5) and rate of enzymatic hydrolysis (1.5–1.8 nmol/min at pH 9.5) by alkaline phosphatase from calf intestine. Detection limits were 0.0005 units/ml for the photometric assay, and 0.00001 units/ml for the fluorimetric one. The relatively longwave shifted absorption and emission maxima of the new substrates in addition to the large Stoke's shifts allow the determination of enzyme activities in a spectral range distinctly outside the intrinsic fluorescence of biological matter such as serum.     

Acid–base chemistry at the single ion limit

We present the results of acid–base experiments performed at the single ion (H⁺ or OH⁻) limit in ∼6 aL volume nanopores incorporating electrochemical zero-mode waveguides (E-ZMWs). At pH 3 each E-ZMW nanopore contains ca. 3600H⁺ ions, and application of a negative electrochemical potential to the gold working electrode/optical cladding layer reduces H⁺ to H₂, thereby depleting H⁺ and increasing the local pH within the nanopore. The change in pH was quantified by tracking the intensity of fluorescein, a pH-responsive fluorophore whose intensity increases with pH. This behavior was translated to the single ion limit by changing the initial pH of the electrolyte solution to pH 6, at which the average pore occupancy 〈n〉_pore ∼3.6H⁺/nanopore. Application of an electrochemical potential sufficiently negative to change the local pH to pH 7 reduces the proton nanopore occupancy to 〈n〉_pore ∼0.36H⁺/nanopore, demonstrating that the approach is sensitive to single H⁺ manipulations, as evidenced by clear potential-dependent changes in fluorescein emission intensity. In addition, at high overpotential, the observed fluorescence intensity exceeded the value predicted from the fluorescence intensity-pH calibration, an observation attributed to the nucleation of H₂ nanobubbles as confirmed both by calculations and the behavior of non-pH responsive Alexa 488 fluorophore. Apart from enhancing fundamental understanding, the approach described here opens the door to applications requiring ultrasensitive ion sensing, based on the optical detection of H⁺ population at the single ion limit.

Visualizing dynamic change in the number of protons during electroreduction of protons in attoliter volume zero-mode waveguides.     

A novel colorimetric and “turn-on” fluorescent sensor for selective detection of Cu2+

In this work, a new highly selective and sensitive fluorescent sensor for detecting Cu²⁺ was developed based on rhodamine fluorophore. It displayed strong fluorescence “turn-on” effect upon addition of Cu²⁺, and possessed the function of naked eye recognition. The fluorescence enhancement also enabled the sensor to quantitatively analyze Cu²⁺ due to the formation of a stable 1:1 metal–ligand complex in a short time, and the complex possesses relatively good pH stability. In addition, the density functional theory calculations were adopted to investigate the molecular orbitals as well as the spatial structure. Simultaneously, the cell imaging and zebra fish experiments provided a broader application prospect in biological system.     

Fluorimetric determination of amino acids and photochemical stability of their reaction products with ortho-phthalic aldehyde under irradiation by high-power pulsed laser

The opportunity of the highly sensitive determination of amino acids using the fluorescence of their derivatives that form in the reaction with aqueous solutions of ortho-phthalic aldehyde and nucleophilic agents, potassium cyanide, sodium sulfite, and N-acetyl-L-cysteine was studied as an example. The chemical stability of forming fluorescent compounds (substituted isoindoles) and their photochemical stability under the action of power pulsed laser radiation is higher, and the analytic characteristics of reagents are highly competive with a standard applied reagent for determining amino acids and a mixture ortho-phthalic aldehyde with 2-mercaptoethanol.     

Tracking bacterial infection of macrophages using a novel red-emission pH sensor

The relationship between bacteria and host phagocytic cells is key to the induction of immunity. To visualize and monitor bacterial infection, we developed a novel bacterial membrane permeable pH sensor for the noninvasive monitoring of bacterial entry into murine macrophages. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. A piperazine moiety was used as the pH-sensitive group. Because of the strong electron-donating and -withdrawing units conjugated in the sensing moiety M, the fluorophore emitted in the red spectral window, away from the autofluorescence regions of the bacteria. Following the engulfment of sensor-labeled bacteria by macrophages and their subsequent merger with host lysosomes, the resulting low-pH environment enhances the fluorescence intensity of the pH sensors inside the bacteria. Time-lapse analysis of the fluorescent intensity suggested significant heterogeneity of bacterial uptake among macrophages. In addition, qRT-PCR analysis of the bacterial 16 S rRNA gene expression within single macrophage cells suggested that the 16 S rRNA of the bacteria was still intact 120 min after they had been engulfed by macrophages. A toxicity assay showed that the pH sensor has no cytotoxicity towards either E. coli or murine macrophages. The sensor shows good repeatability, a long lifetime, and a fast response to pH changes, and can be used for a variety of bacteria.     

Influence of micellar media on the fluorescence of various benzo- and methyl-quinolizinium salts

The bebaviour of several different micelar systems (anionic, cationic and non-ionic) on the fluorescence of quinolizinium salts is studied. Important factors, such as pH and ionic strength that influence fluorescence parameters, are discussed. Fourteen quinolizinium salts (benzo and methyl derivatives) are examined as fluorescent probes in micellar media. All of them showed a marked increase of fluorescence intensity when sodium dodecyl sulfate solutions of critical micelle concentration (CMC) are added. The presence of non-ionic surfactants did not change the fluorescent emission of the probes. The emission intensity is much decreased when N-cetyl-N,N,N- trimethylammonium bromide concentrations are above the CMC. Changes in pH ido not significantly affect the fluorescence intensity of the benzo derivatives. Increasing the ionic strength decreases the fluorescence. For 9-cyanobenzo[a]phenanthro [9,10-g] quinolizinium chloride, the spectrum changes when the surfactant concentration is high than the CMC thereforre this compound is considered to be a good fluorescent probe in icell     

Fluorescence spectroscopy of fulvic acids’ interaction with surfactants

Fluorescence spectra of two fulvic acid (FA) samples, FA0 from underground water and FA1 from forest soil, were recorded in various surfactant solutions. Alkyltrimethylammonium ions with different alkyl chain lengths induced a decrease in the fluorescence intensity for both FAs at concentrations below the critical micelle concentration (cmc) and an enhancement above the cmc. The intensity minimum thus obtained at the cmc was deeper for surfactants with longer alkyl chains. This effect was attributable to the formation of insoluble FA–surfactant complexes below the cmc and to the solubilization of the complex into micelles above the cmc. Dodecylpyridinium chloride caused a monotonic decrease in the FA fluorescence even far above the cmc. This was attributable to the quenching of FA fluorescence by the positioning of the pyridinium head group near the FA fluorophore. Anionic and nonionic surfactants showed little to no effect on the FA fluorescence.