Endo- and exo-levanases from Bacillus subtilis HM7: Catalytic components, synergistic cooperation, and application in fructooligosaccharide synthesis.

Levan-type fructooligosaccharides (LFOS) exhibit significant biological activities and selectively promote the growth of certain beneficial bacteria. Levanase is an important enzyme for LFOS production. In this study, two isoforms of levanases, exo- and endo-type depolymerizing enzymes, from Bacillus subtilis HM7 isolated from Dynastes hercules larvae excrement were cloned, expressed, and characterized. The synergistic effect on the levan hydrolysis and kinetic properties of both isoforms were evaluated, indicating their cooperation in levan metabolism, where the endo-levanase catalyzes a rate-limiting step. In addition, homology models and molecular dynamics simulations revealed the key amino residues of the enzymes for levan binding and catalysis. It was found that both isoforms possessed distinct binding residues in the active sites, suggesting the importance of the specificity of the enzymes. Finally, we demonstrated the potential of endo-type levanase in LFOS synthesis using a one-pot reaction with levansucrase. Overall, this study fills the knowledge gap in understanding levanase's mechanism, making an important contribution to the fields of food science and biotechnology.

A fluorescent electrophile for CLIPS: self indicating TrkB binders.

Combination of cysteine-containing peptides with electrophiles provides efficient access to cyclo-organopeptides. However, there are no routes to intrinsically fluorescent cyclo-organopeptides containing robust, brilliant fluorophores emitting at wavelengths longer than cellular autofluorescence. We show such fluorescent cyclo-organopeptides can be made via SNAr reactions of cysteine-containing peptides with a BODIPY system. Seven compounds of this type were prepared to test as probes; six contained peptide sequences corresponding to loop regions in brain-derived neurotrophic factor and neurotrophic factor 4 (BDNF and NT-4) which bind tropomyocin receptor kinase B (TrkB). Cellular assays in serum-free media indicated two of the six key compounds induced survival of HEK293 cells stably transfected with TrkB whereas a control did not. The two compounds inducing cell survival bound TrkB on those cells (Kd ∼40 and 47 nM), illustrating how intrinsically fluorescent cyclo-organopeptides can be assayed for quantifiable binding to surface receptors in cell membrane environments.

Detection of hazardous mercury ion using [5]helicene-based fluorescence probe with "TurnON" sensing response for practical applications.

A new fluorescence probe based on [5]helicene derivative (MT) was designed and synthesized. The chemical structure of the probe was fully characterized by NMR, mass spectrometry and X-ray crystallography. MT which is the combination of thioamide[5]helicene with Schiff base-thiophene moiety, exhibited a high selectivity to detect Hg2+ through irreversible desulfurization reaction with "TurnON" fluorescence response and large Stokes shift of 110 nm in aqueous methanol solution. The detection limit of MT was 1.2 ppb (6.0 × 10-3 µM), which is lower than the limit of Hg2+ level in drinking water, as specified by WHO (6.0 ppb) and U.S. EPA (2.0 ppb). The Hg2+ detection range of the probe was 0.07-1.6 µM with good linearity. Under UV irradiation, MT possessed the capability to detect Hg2+ in diverse context of real samples, including drinking and sea waters, vegetable tissue and brain tumor cell. In addition, MT could be used as a paper test strip for monitoring and screening of Hg2+ contamination in environment.

Rapid and visual detection of Cd2+ based on aza-BODIPY near infrared dye and its application in real and biological samples for environmental contamination screening.

Cadmium highly toxic and hazardous, and it can adversely affect human health leading to serious disorders. Herein, a water-soluble near-infrared sensor based on aza-BODIPY (1) was developed for dual determination of Cd2+ in environmental and biological media. This sensor exhibited color change from colorless to green along with a fluorescence enhancement in the near-infrared (NIR) region via photoinduced electron transfer (PET) after complexation with Cd2+. Sensor 1 can be employed in aqueous media at physiological pH for quantitative monitoring. It shows rapid response with high sensitivity (detection limit of 2.8 ppb; linear correlation over [Cd2+] 1.33 - 6.67 µM) and selectivity over potentially interfering ions. NIR sensor 1 can be used to determine [Cd2+] in living cells and environmental samples.

Cu2+-selective NIR fluorescence sensor based on heptamethine cyanine in aqueous media and its application.

A near-infrared (NIR) colorimetric fluorescence sensor, Cy7C3, based on heptamethine cyanine dye was synthesized for determining the presence of Cu2+ ions. The sensor showed highly sensitive fluorescence quenching toward Cu2+ ions in acetonitrile/buffer solution at physiological pH with long emission wavelength of 718 nm. Cy7C3 also provided an excellent selectivity to Cu2+ ions over other competing metal ions, with a low detection limit of 9 ppb, which was lower than the maximum concentration of Cu2+ ions in drinking water of U.S. EPA. Cy7C3 could achieve naked-eye detection of Cu2+ ions via the color change from blue to colorless, which allowed determination of Cu2+ ions in hydroponic fertilizers. Additionally, the sensor was developed to detect Cu2+ ions in HepG2 cancer cells via fluorescence imaging.

A Near-Infrared Fluorescence Chemosensor Based on Isothiocyanate-Aza-BODIPY for Cyanide Detection at the Parts per Billion Level: Applications in Buffer Media and Living Cell Imaging.

A near-infrared chemodosimeter based on an aza-BODIPY dye was designed and synthesized. The sensor contains isothiocyanate groups for cyanide ion sensing. The sensing function was illustrated via the fluorescence changes in near-infrared frequencies as well as chromogenic changes which could be easily visualized with a detection limit of 19 ppb. The sensor provides high selectivity to CN- and discriminates other anions such as CH3 COO- , HPO4- , HSO4- , ClO3- , CO32- , SO42- , NO3- , Cl- , F- , Br- , I- , and phenylalanine (Phe) in 50 % PBS buffer/acetonitrile at physiological pH. The potential of the sensor for CN- detection in both aqueous buffer solutions and living cells imaging was demonstrated.

Dual-Analyte Fluorescent Sensor Based on [5]Helicene Derivative with Super Large Stokes Shift for the Selective Determinations of Cu2+ or Zn2+ in Buffer Solutions and Its Application in a Living Cell.

A new fluorescent sensor, M201-DPA, based on [5]helicene derivative was utilized as dual-analyte sensor for determination of Cu2+ or Zn2+ in different media and different emission wavelengths. The sensor could provide selective and bifunctional determination of Cu2+ in HEPES buffer containing Triton-X100 and Zn2+ in Tris buffer/methanol without interference from each other and other ions. In HEPES buffer, M201-DPA demonstrated the selective ON-OFF fluorescence quenching at 524 nm toward Cu2+. On the other hand, in Tris buffer/methanol, M201-DPA showed the selective OFF-ON fluorescence enhancement upon the addition of Zn2+, which was specified by the hypsochromic shift at 448 nm. Additionally, M201-DPA showed extremely large Stokes shifts up to ∼150 nm. By controlling the concentration of Zn2+ and Cu2+ in a living cell, the imaging of a HepG2 cellular system was performed, in which the fluorescence of M201-DPA in the blue channel was decreased upon addition of Cu2+ and was enhanced in UV channel upon addition of Zn2+. The detection limits of M201-DPA for Cu2+ and Zn2+ in buffer solutions were 5.6 and 3.8 ppb, respectively. Importantly, the Cu2+ and Zn2+ detection limits of the developed sensors were significantly lower than permitted Cu2+ and Zn2+ concentrations in drinking water as established by the U.S. EPA and WHO.

Near-infrared aza-BODIPY fluorescent probe for selective Cu2+ detection and its potential in living cell imaging.

A near-infrared (NIR) fluorescent sensor 1 composed of an aza-boron-dipyrromethene (aza-BODIPY) core covalently bound to two di-2-picolylamine moieties was conceived for Cu2+ detection in aqueous solutions. Spectroscopic properties and binding abilities with several metal ions were investigated in phosphate buffered saline (pH 7.4): acetonitrile (95 : 5 v/v) with Triton X-100 via fluorometric titrations. The fluorescence of sensor 1 was quenched selectively by cupric ions in the presence of alkali- and transition-metal-ions. A detection limit of 13 ppb was measured for this system, and this is significantly lower than permissible levels of Cu2+ in drinking water according to the guidelines described by the U.S. Environmental Protection Agency (EPA) and by the World Health Organization (WHO). Application of the sensor in detecting Cu2+ in HepG2 cells was demonstrated.

Oligoethylene glycol-substituted aza-BODIPY dyes as red emitting ER-probes.

This study features aza-BODIPY (BF2-chelated azadipyrromethene) dyes with two aromatic substituents linked by oligoethylene glycol fragments to increase hydrophilicity of aza-BODIPY for applications in intracellular imaging. To prepare these, two chalcones were attached α,ω onto oligoethylene glycol fragments, then reacted with nitromethane anion. Conjugate addition products from this reaction were then subjected to typical conditions for synthesis of aza-BODIPY dyes (NH4OAc, (n)BuOH, 120 °C); formation of boracycles in this reaction was concomitant with creation of macrocycles containing the oligoethylene glycol fragments. Similar dyes with acyclic oligoelythene glycol substituents in the same position were used to compare the efficiencies of the intra- and inter-molecular aza-BODIPY forming reactions, and the characteristics of the products. All the fluors with oligoethylene glycol fragments, i.e. cyclic or acyclic, localized in the endoplasmic reticulum of a fibroblast cell line (WEHI-13VAR), the human pancreatic cancer cell line (PANC-1, rough ER predominates) and human liver cancer cell line (HepG2, smooth ER prevalent). These fluors are potentially useful for near IR (λmax emis at 730 nm) ER staining probes.

"Naked-eye" colorimetric and "turn-on" fluorometric chemosensors for reversible Hg2+ detection.

Two new Hg(2+)-colorimetric and fluorescent sensors based on 2-[3-(2-aminoethylsulfanyl) propylsulfanyl]ethanamine covalently bound to one and two units of rhodamine-6G moieties, 1 and 2, were synthesised, and their sensing behaviors toward metal ions were investigated by UV/Vis and fluorescence spectroscopy. Upon the addition of Hg(2+), the sensors exhibited highly sensitive "turn-on" fluorescence enhancement as well as a color change from colorless to pink, which was readily noticeable for naked eye detection. Especially, 1 exhibited the reversible behavior and revealed a very high selectivity in the presence of competitive ions, particularly Cu(2+), Ag(+), Pb(2+), Ca(2+), Cd(2+), Co(2+), Fe(2+), Mn(2+), Na(+), Ni(2+), K(+), Ba(2+), Li(+) and Zn(2+), with a low detection limit of 1.7 ppb toward Hg(2+).

Assessment of heavy metals in sediments of the Don Hoi Lot area in the Mae Klong estuary, Thailand.

The status and seasonal variation of heavy metals in surface sediment were investigated at Don Hoi Lot, located in the Mae Klong estuary, Thailand. Results revealed that all the measured heavy metals, except Zn, in the sediments had lower concentrations than in other nearby estuaries. Only Zn may be of concern for potential negative effects on estuarine biota in the study area. With the exception of Fe, all the studied heavy metals showed seasonal variation, but the patterns were diverse. Organic matter and the clay fraction in sediments were good sinks for heavy metals, excluding Zn, while Fe and Mn were good catchers. Principal component analysis suggested that Zn might have different origins and/or mechanisms of transport, accumulation and circulation, compared with the other heavy metals studied. A better understanding of sources and the behavior of Zn would enhance the efficiency of the estuary management plan in this study area.

Synthesis of a novel fluorescent sensor bearing dansyl fluorophores for the highly selective detection of mercury (II) ions.

A new macromolecule possessing two dansyl moieties and based on 2-[4-(2-aminoethylthio)butylthio]ethanamine was prepared as a fluorescent sensor and its mercury sensing properties toward various transition metal, alkali, and alkali earth ions were investigated. The designed compound exhibited pronounced Hg2+-selective ON-OFF type fluorescence switching upon binding. The new compound provided highly selective sensing to Hg2+ in acetonitrile-water solvent mixtures with a detection limit of 2.49 x 10(-7) M or 50 ppb. The molecular modeling results indicated that ions-recognition of the sensor originated from a self assembly process of the reagent and Hg2+ to form a helical wrapping structure with the favorable electrostatic interactions of Hg2+coordinated with sulfur, oxygen, nitrogen atoms and aromatic moieties.

Reversible photoswitchable wettability in noncovalently assembled multilayered films.

Reversible and irreversible photoinduced changes in surface wettability were observed in noncovalently assembled multilayered films. The multilayered films studied were fabricated from a self-assembled monolayer (SAM) consisting of 4-(10-mercaptodecyloxy)pyridine-2,6-dicarboxylic acid on gold, Cu(II) ions complexed to the pyridine head group of the SAM, and either cis- (film 1) or trans- (film 2) stilbene-4,4'-dicarboxylic acid complexed to the Cu(II) ions. Irradiation of film 1 at wavelengths corresponding to the absorption band of the cis-stilbene isomer resulted in an irreversible chemical change and an irreversible increase in wettability, as indicated by surface contact angle and grazing incidence IR measurements. However, no evidence for cis-/trans-photoisomerization was observed. Films 3 and 4, similar to films 1 and 2 in that they consist of an underlying SAM, an intermediate layer consisting of Cu(II) ions, and either cis- or trans-stilbene-4,4'-dicarboxylic acid as the capping ligand, were fabricated with a mixed SAM that contained both 4-(10-mercaptodecyloxy)pyridine-2,6-dicarboxylic acid and 4-tert-butylbenzenethiol. Irradiation of these films at wavelengths corresponding to stilbene isomer absorption bands resulted in reversible cis- to trans- (film 3) and trans- to cis- (film 4) photoisomerization and reversible switching of the surface wettability between a low wetting state (cis-stilbene) and a high wetting state (trans-stilbene). The difference in observed behavior between films 1 and 2 and films 3 and 4 is attributed to the greater surface spacing afforded by the mixed monolayer, which allows greater conformational flexibility and lowers the steric barriers to isomerization.

Surface-based lithium ion sensor: An electrode derivatized with a self-assembled monolayer.

Self-assembled monolayers (SAMs) of 21-(16-mercaptohexadecan-1-oyl)-4,7,13,16-tetraoxa-1,10,21-triazabicyclo[8.8.5]tricosane-19,23-dione were prepared on gold. Characterization of the SAMs was carried out by sessile drop contact angle, ellipsometry, grazing angle FT-IR spectroscopy, and electrochemical techniques. The cation recognition properties of the SAM were studied by cyclic voltammetry and impedance spectroscopy. The films show moderate selectivity for detection of Li+ ions in solution over K+ and Na+, with selectivity values calculated to be log K(Li+,Na+) approximately -1.30 and log K(Li+,K+) approximately -0.92. To the best of our knowledge, this is the first demonstration of a lithium sensor fabricated using self-assembled monolayer technology.

A highly selective bicyclic fluoroionophore for the detection of lithium ions.

The macrobicyclic molecule, 21-(9-anthrylmethyl)-4,17,13,16-tetraoxa-1,10,21-triazabicyclo [8.8.5]tricosane-19,23-dione, I, was designed, synthesized and characterized as a fluoroionophore for the selective, optical detection of lithium ions. Compound I is based on a bridged diazacrown structure, which provides a semirigid binding framework. Binding takes place by electrostatic interactions between the oxygen atoms of the crown and the cation and is transduced to fluorescence emission from an attached anthracene fluorophore. In a 75:25 dichloromethane/tetrahydrofuran solvent mixture, I acts as an intramolecular electron transfer "off-on" fluorescence switch, exhibiting a greater than 190-fold enhancement in fluorescence emission intensity in the presence of lithium ions. The relative selectivity of I for lithium ions over sodium, potassium and ammonium ions was found to be log K(Li+,Na+) approximately -3.36, log K(Li+,K+) approximately -1.77 and log K(Li+,NH4+) approximately -2.78.