Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity

Live-cell pH measurements: An environment-sensitive fluorophore (green) was site-specifically introduced on HdeA, an acid-resistant chaperone showing pH-mediated conformational changes under low pH conditions. A survey of the attachment sites led to the discovery of one position on HdeA at which the attached fluorophore showed a strong fluorescence increase upon acidification.     

Fine tuning of a solvatochromic fluorophore for selective determination of Fe: a new type of benzimidazole-based anthracene-coupled receptor

We synthesized a novel benzimidazole-based, anthracene-coupled fluorescent receptor capable of recognizing and estimating the concentrations of Fe³⁺ in semi-aqueous solution by ratiometric estimation. Our sensor can be made highly selective for Fe³⁺ over other metal ions by changing the solvent composition.     

Strong solvatochromic fluorescence from the intramolecular charge-transfer state created by excited-state intramolecular proton transfer

In this work, we report a peculiar positive solvatochromism in the keto emission of the acceptor-substituted 2-(2'-hydroxyphenyl)benzoxazoles (HBO), which originates from the excited-state intramolecular proton transfer (ESIPT) followed by the intramolecular charge transfer (ICT) and subsequent solvent relaxation. This transient evolution of enhanced ICT characteristic triggered by ESIPT, which is first observed in this work, is responsible for the novel concept of a fast hyperpolarizability modulator as well as the unique solvatochromic behavior.     

A new equation of state for polar and nonpolar pure fluids

Chung, T.H., Khan, M.M., Lee, L.L. and Starling, K.E., 1984. A new equation of state for polar and nonpolar pure fluids. Fluid Phase Equilibria, 17: 351–372A new equation of state based on the concept of perturbation theory and the hard-convex-body equation of state has been developed successfully for nonpolar compounds. The equation can predict the thermodynamic properties (density, enthalpy departure and vapor pressure) of a wide range of pure fluids from small, spherical (argon-like) molecules to large, structurally complex molecules. For nonpolar compounds, the equation employs three parameters: the shape, size and energy parameters. For normal paraffins, the size parameter (hard-core volume) is related to the measurable van der Waals volume given by Bondi. For most other compounds, it is related to the critical volume. The shape-parameter values reflect the structure and degree of acentricity of the compound of interest. The equation has been extended to polar and associating compounds by using the mean-potential model. For polar compounds, a fourth parameter is required. The equation has been tested extensively for polar (dipolar and quadrupolar) and hydrogen-bonding compounds. The applicability of this equation for such a wide variety of substances provides an important first step in the development of a composition-dependent equation of state for mixtures.     

Analysis of titanium nanoparticles created by laser irradiation under liquid environments

The results of studies of the formation of various titanium-based nanoparticles by laser ablation of a titanium rod in liquid environments comprised of water, ethanol, 2-propanol, and n-hexane are reported. The effect of fluence on nanoparticle characteristics was studied by ablation with a 532 nm Nd:YAG operating at 10 Hz, showing that mean particle size and the size distribution increase with increasing laser intensity. The solvent plays a crucial role in the nature of the nanoparticles, as solvent components are incorporated into the nanoparticles during formation. Titanium nanoparticles formed in oxygen-rich solvents incorporate oxygen, while those formed in a carbon-rich environment are found to contain carbon. The nanoparticles created in their respective liquid environments are very stable, maintain their character over time, and remain in solution for months after creation.     

Detection of the local structural changes in the dimer interface of BamHI initiated by DNA binding and dissociation using a solvatochromic fluorophore

To detect the local structural change in an interface between proteins induced by the substrate binding and dissociation, a solvatochromic fluorescent N(beta)-L-alanyl-5-(N,N-dimethylamino)-naphthalene-1-sulfonamide (DanAla) was introduced into 132 position of the dimer interface in BamHI. Before addition of the substrate, the fluorescence from the normal planer excited state of DanAla moiety was observed as a main emission, and thereby the DanAla in the dimer interface is located in the hydrophobic microenvironment. The incubation with the substrate for 20 min induced the gradual increase in fluorescence intensity around 430 nm. The fact reflects that the polarity is reduced by the slight structural change initiated by the formation of the complex with the substrate. Furthermore, the incubation for more than 20 min caused the slight decrease in fluorescence around 430 nm and an appearance of fluorescence (560 nm) due to twisted intramolecular charge transfer (TICT) excited state. Therefore, the DanAla is exposed to comparative polar environment after the dissociation of the substrate. The fluorescence lifetime as a minor component, which is attributed to the TICT excited state, was reduced by addition of the substrate. The results provide that the hydrophobicity in the dimer interface is increased by the substrate binding. Interestingly, we found that the structure of an initial form is different from that of a refolded form after the dissociation of the substrate using a spectral subtraction technique. We have achieved detection of the changing structure induced by the substrate binding and dissociation using a steady-state and time-resolved fluorescence.     

A versatile amino acid analogue of the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide: a powerful tool for the study of dynamic protein interactions

We have developed a new unnatural amino acid based on the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) for application in the study of protein-protein interactions. The fluorescence quantum yield of this chromophore is highly sensitive to changes in the local solvent environment, demonstrating "switch-like" emission properties characteristic of the dimethylaminophthalimide family of fluorophores. In particular, this new species possesses a number of significant advantages over related fluorophores, including greater chemical stability under a wide range of conditions, a longer wavelength of excitation (408 nm), and improved synthetic accessibility. This amino acid has been prepared as an Fmoc-protected building block and may readily be incorporated into peptides via standard solid-phase peptide synthesis. A series of comparative studies are presented to demonstrate the advantageous properties of the 4-DMN amino acid relative to those of the previously reported 4-N,N-dimethylaminophthalimidoalanine and 6-N,N-dimethylamino-2,3-naphthalimidoalanine amino acids. Other commercially available solvatochromic fluorophores are also include in these studies. The potential of this new probe as a tool for the study of protein-protein interactions is demonstrated by introducing it into a peptide that is recognized by calcium-activated calmodulin. The binding interaction between these two components yields an increase in fluorescence emission greater than 900-fold.     

A dual fluorophore system for simultaneous bioassays

A detection scheme for the simultaneous evaluation of two bioassays based on fluorescence spectroscopy is presented. For the determination of hydrogen peroxide-generating enzymes or peroxidases, the non-fluorescent 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) is converted to the strongly fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Phosphatases are detected based on the cleavage of the non-fluorescent 5-fluorosalicyl phosphate (5-FSAP) under formation of the fluorescent 5-fluorosalicylic acid (5-FSA). While excitation of the fluorophores may be carried out at the same wavelength, their emission spectra differ significantly. This allows the read-out of both assays using commercially available microplate readers without additional chemometric tools. Compared with individual assays, limits of detection are similar, and linearity of the calibration functions for both enzymes is observed over 2-3 concentration decades starting at the limit of quantification. The simultaneous determination of glucose oxidase and acid phosphatase in honey is presented as example for the application of the detection scheme.     

A2-rhodopsin: a new fluorophore isolated from photoreceptor outer segments

A2E and iso-A2E are fluorescent amphiphilic pyridinium bisretinoids involved in age-related macular degeneration (AMD). It is now shown that the presence of high exogenous concentrations of all-trans-retinal in photoreceptor outer segments leads to the formation of A2-rhodopsin (A2-Rh), an unprecedented fluorescent rhodopsin adduct which consists of bisretinoids (A2) linked to each of three lysine residues in rhodopsin (Rh) and which exhibits an emission spectrum similar to A2E. The fluorophore to protein ratio was determined by MALDI-TOF-MS and UV-VIS spectroscopy. Enzymatic degradation with thermolysin and cathepsin D showed that two of the A2 moieties were located in the region of the third cytoplasmic loop and 8th helix of Rh. Examination of A2-Rh and A2-PE (the precursor of A2E) fluorescence in relation to all-trans-retinal concentration indicated that whereas A2-PE formation is favored over that of A2-Rh, for a single rhodopsin molecule only one phosphatidylethanolamine molecule is available to react with all-trans-retinal; this phosphatidylethanolamine is probably tightly associated with the protein.     

A new fluorescent fluoride chemosensor based on conformational restriction of a biaryl fluorophore

A new fluorescent anion sensor 1, based on a biaryl–thiourea system, exhibits a fluorescence emission enhancement via conformational restriction upon a hydrogen bond-mediated complexation of fluoride anions.     

A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid-based imaging technology

In the development of orthogonal extra base pairs for expanding the genetic alphabet, we created novel, unnatural base pairs between fluorophore and quencher nucleobase analogues. We found that the nucleobase analogue, 2-nitropyrrole (denoted by Pn), and its 4-substitutions, such as 2-nitro-4-propynylpyrrole (Px) and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (NH(2)-hx-Px), act as fluorescence quenchers. The Pn and Px bases specifically pair with their pairing partner, 7-(2,2'-bithien-5-yl)imidazo[4,5-b]pyridine (Dss), which is strongly fluorescent. Thus, these unnatural Dss-Pn and Dss-Px base pairs function as reporter-quencher base pairs, and are complementarily incorporated into DNA by polymerase reactions as a third base pair in combination with the natural A-T and G-C pairs. Due to the static contact quenching, the Pn and Px quencher bases significantly decreased the fluorescence intensity of Dss by the unnatural base pairings in DNA duplexes. In addition, the Dss-Px pair exhibited high efficiency and selectivity in PCR amplification. Thus, this new unnatural base pair system would be suitable for detection methods of target nucleic acid sequences, and here we demonstrated the applications of the Dss-Pn and Dss-Px pairs as molecular beacons and in real-time PCR. The genetic alphabet expansion system with the replicable, unnatural fluorophore-quencher base pair will be a useful tool for sensing and diagnostic applications, as well as an imaging tool for basic research.     

Oligothiophene isothiocyanates as a new class of fluorescent markers for biopolymers.

The regioselective synthesis of fluorescent oligothiophene isothiocyanates is described. The isothiocyanates were reacted with bovine serum albumin (BSA) following standard procedures and the optical properties of the oligothiophene-BSA conjugates were analyzed as a function of oligomer concentration, time, and irradiation power. The oligothiophene-BSA conjugates were chemically very stable and their photoluminescence characteristics persisted unaltered for several months. Photoluminescence data relative to the conjugate of an oligothiophene-S,S-dioxide isothiocyanate with monoclonal anti-CD8 antibody are reported. No fluorescence quenching was observed following the binding of the isothiocyanate to the antibody and the conjugate displayed high chemical stability and photostability.     

A photoactivatable push-pull fluorophore for single-molecule imaging in live cells

We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.     

A new continuous fluorometric assay for acetylcholinesterase activity and inhibitor screening with emissive core-shell silica particles containing tetraphenylethylene fluorophore

Emissive core-shell silica particles with tetraphenylethylene moieties were prepared and characterized. Fluorescence quenching was observed for the silica particles upon addition of compound 2 (Dabcyl-ACh). This was attributed to the electrostatic interaction between the silica particles and 2 and the resulting photoinduced energy transfer between them. After incubation with AChE, the fluorescence intensity started to increase. The fluorescence enhancement became more significant when the concentration of AChE was higher. The reaction kinetic parameters for AChE were successfully estimated with the silica particles and 2. These results reveal that the ensemble of the silica particles and 2 can be utilized for AChE assay. Moreover, the fluorescence spectra of the ensemble of the silica particles and 2 containing AChE were also measured after further addition of either neostigmine or tacrine which are typical inhibitors of AChE. The results manifest that the ensemble of the emissive silica particles and 2 is also useful for screening the inhibitors of AChE.     

A dsDNA-lighted fluorophore for monitoring protein-ligand interaction through binding-mediated DNA protection

Because of their important roles in cellular functions, life activities, drug screening, and disease treatment, the development of efficient methods for monitoring protein-ligand interaction is essential. In this study, inspired by our previous studies on DNA conformation-selective fluorescent indicators, we developed a new sensing platform for monitoring protein-ligand interaction and detecting protein activity based on binding-mediated DNA protection and the dsDNA-lighted fluorophore, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9 H-carbazol-9-yl)] butanoate(EBCB). The ligand was purposefully linked to the 3?-terminal of a hairpin DNA probe to selectively bind with the target protein and protect the DNA from cleavage by exonuclease III. By virtue of EBCB's outstanding capacity to discriminate DNA conformation, the protein-ligand interaction could be effectively monitored through a fluorescence change in EBCB. A high fluorescence signal was detected when the hairpin DNA was protected in the presence of the target protein, whereas a much lower signal was observed in the presence of nontarget proteins.Our results demonstrated that the proposed strategy had high potential, such as high selectivity and relatively high sensitivity, for monitoring protein-ligand interaction and detecting protein activity. We believe these results will pave the way for applying dsDNA-lighted fluorophore EBCB as an effective signal transducer for DNA conformation transformation-mediated biochemical sensing.     

A new strategy of exploring metabolomics data using Monte Carlo tree

Large amounts of data from high-throughput metabolomics experiments have become commonly more and more complex, which brings a number of challenges to existing statistical modeling. Thus there is a need to develop a statistically efficient approach for mining the underlying metabolite information contained by metabolomics data under investigation. In this work, we provide a new strategy based on Monte Carlo cross validation coupled with the classification tree algorithm, which is termed as the MCTree approach. The MCTree approach inherently provides a feasible way to uncover the predictive structure of metabolomics data by the establishment of many cross-predictive models. With the help of the sample proximity matrix such obtained, it seems to be able to give some interesting insights into metabolomics data. Simultaneously, informative metabolites or potential biomarkers can be successfully discovered by means of variable importance ranking in the MCTree approach. Two real metabolomics datasets are finally used to demonstrate the performance of the proposed approach.     

A ratiometric approach for pH optosensing with a single fluorophore indicator

A new fiber-optic prototype of luminometer has been designed in order to perform ratiometric-based measurements for optical sensing purposes. The coupling of a pH-selective sensing phase to the fiber-optic prototype has been evaluated for robust pH optosensing in drinking water. The pH-sensitive material has been synthesized by entrapping a pH-sensitive luminescent indicator (mercurochrome) in a sol-gel inorganic matrix. The pH optosensing is based on the detection of pH-induced reversible changes in the mercurochrome fluorescent emission and in the light reflected by the sensing phase.The instrument has been constructed using low-cost and simple optoelectronic components. The active phase was excited by means of a visible 470 nm high intensity light emitting diode (LED). The radiant power of the LED was modulated using a sinusoidal function so that scattered light due to light sources of different frequency than the modulating signal (e.g. sunlight) can be easily removed by adequate electronic filtering of the emission signal. Both the fluorescence emission from the dye and the sensing phase reflected light were collected in a bifurcated fiber-optic to allow the ratiometric measurement.Two different ratiometric approaches have been evaluated. The analytical performance of the pH optrode using both measurement methods have been compared, between them and with simple fluorescence intensity measurements, in terms of sensitivity, measurement range, response time, repeatability and insensitivity to changes in excitation light intensity.The applicability of the developed pH optrode and methods has been tested for pH analysis in tap and bottled still mineral water samples. The results obtained showed good agreement with the corresponding pH values provided by a commercial glass electrode.In this work, pH was selected as a model analyte to evaluate the performance of the proposed methodology, although other optical sensors for different applications/analytes could benefit of this approach.     

A new zinc(II) fluorophore 2-(9-anthrylmethylamino)ethyl-appended 1,4,7,10-tetraazacyclododecane

A new 2-(9-anthrylmethylamino)ethyl-appended cyclen, L(3) (1-(2-(9-anthrylmethylamino)ethyl)-1,4,7,10-tetraazacyclododecane) (cyclen = 1,4,7,10-tetraazacyclododecane), was synthesized and characterized for a new Zn(2+) chelation-enhanced fluorophore, in comparison with previously reported 9-anthrylmethylcyclen L(1) (1-(9-anthrylmethyl)-1,4,7,10-tetraazacyclododecane) and dansylamide cyclen L(2). L(3) showed protonation constants log K(a)(i)() of 10.57 +/- 0.02, 9.10 +/- 0.02, 7.15 +/- 0.02, <2, and <2. The log K(a3) value of 7.15 was assigned to the pendant 2-(9-anthrylmethylamino)ethyl on the basis of the pH-dependent (1)H NMR and fluorescence spectroscopic measurements. The potentiometric pH titration study indicated extremely stable 1:1 Zn(2+)-L(3) complexation with a stability constant log K(s)(ZnL(3)) (where K(s)(ZnL(3)) = [ZnL(3)]/[Zn(2+)][L(3)] (M(-)(1))) of 17.6 at 25 degrees C with I = 0.1 (NaNO(3)), which is translated into the much smaller apparent dissociation constant K(d) (=[Zn(2+)](free)[L(3)](free)/[ZnL(3)]) of 2 x 10(-)(11) M with respect to 5 x 10(-)(8) M for L(1) at pH 7.4. The quantum yield (Phi = 0.14) in the fluorescent emission of L(3) increased to Phi = 0.44 upon complexation with zinc(II) ion at pH 7.4 (excitation at 368 nm). The fluorescence of 5 microM L(3) at pH 7.4 linearly increased with a 0.1-5 microM concentration of zinc(II). By comparison, the fluorescent emission of the free ligand L(1) decreased upon binding to Zn(2+) (from Phi = 0.27 to Phi = 0.19) at pH 7.4 (excitation at 368 nm). The Zn(2+) complexation with L(3) occurred more rapidly (the second-order rate constant k(2) is 4.6 x 10(2) M(-)(1) s(-)(1)) at pH 7.4 than that with L(1) (k(2) = 5.6 x 10 M(-)(1) s(-)(1)) and L(2) (k(2) = 1.4 x 10(2) M(-)(1) s(-)(1)). With an additionally inserted ethylamine in the pendant group, the macrocyclic ligand L(3) is a more effective and practical zinc(II) fluorophore than L(1).