A novel method for synthesis of carbon dots and their applications in reactive oxygen species (ROS) and glucose detections.

A simple and novel method is proposed for preparation of water-soluble fluorescent carbon dots (C-dots), which have potential to be applied in detecting reactive oxygen species (ROS). The C-dots with high fluorescence quantum yield were created by hydrothermal methods with lactose as the carbon source and tris(hydroxylmethyl)aminomethane (Tris) as the surface passivation reagent. The C-dots have some unique characteristics such as excellent biocompatibility with a broad pH working range of 5-11 and high fluorescence, which makes them especially useful in the bio-detection field. The optical properties, surface groups, and element components of the prepared C-dots have been systematically studied by fluorescence spectroscopy. This facile approach is efficient and environmentally friendly and allows large-scale production of the C-dots without any further post-treatment. The C-dots have been adopted as probes for fluorescence turn-off detection owing to their high sensitivity to the hydroxyl radical. The detection limit can reach ∼0.1 µM under optimized conditions when using hydrogen peroxide as the source for generating ROS. Moreover, when paired with glucose oxidase, these C-dots can track glucose concentrations in samples. This adaptability suggests their potential in detecting various metabolites, paving the way for practical uses in disease detection.

A selective colorimetric chemosensor for Fe3.

Two simple colorimetric receptors PS and PP (thiophene and pyridine appended derivative) were prepared and their cation sensing properties were investigated. Receptors PS and PP displayed a selective colorimetric change (from colorless to orange) upon binding to Fe3+ in MeOH solution. The association constants for receptors PS-Fe3+ and PP-Fe3+ in MeOH were determined to be 1.15 × 106 and 4.31 × 106  M-1 , respectively, using Hill plots. The detection limits of PS and PP were 490.7 ppm and 393.7 ppm, respectively.

A colorimetric sensor for the selective detection of fluoride ions.

A colorimetric receptor L was prepared. Receptor L can selectively sense F- based on distinct color changes among a series of ions. It can selectively sense F- through an intramolecular hydrogen bond interaction. A Job plot indicated a 1:1 complexation stoichiometry between receptor L and F- . The association constant for L-F- in CH3 CN was determined as 9.70 × 104  M-1 using a Stern-Volmer plot.

A Selective Colorimetric and Turn-on Fluorescent Chemosensor for Hg2+ in Aqueous Solution.

A simple colorimetric and turn-on fluorescence receptor FT (thiophene appended fluorescein-hydrazone derivative) was prepared and its cation-sensing properties were investigated. Receptor FT displayed a selective colorimetric change (from colorless to orange color) upon binding to Hg2+ in DMSO/H2O (1:9, v/v) solution. The association constant of FT-Hg2+ complex was calculated to be 3.03 × 109 M-1, and the detection limit for Hg2+ was found to be 0.24 ppm.

A Schiff-based sensor with turn-on fluorescence for selective detection of Hg2+.

A simple Schiff-base colorimetric receptor 1 was prepared. It exhibits an 'off­on-type' mode with high sensitivity in the presence of Hg2+. The change in color is very easily observed by the naked eye in the presence of Hg2+, whereas other metal cations do not induce such a change. A Job plot indicated a 1 : 1 complexation stoichiometry between receptor 1 and Hg2+. The association constant for 1­Hg2+ in Tetrahydrofuran (THF) was determined to be 1.3 × 10(9)M-1 using a Hill plot.

A turn-on indole-based sensor for hydrogen sulfate ion.

A simple indole-based receptor 1 was prepared by a simple Schiff-base reaction of 1H-indole-3-carbaldehyde with ethane 1,2-diamine and its fluoroionophoric properties toward anions were investigated. Indole-based receptor 1 acts as a selective turn-on fluorescent sensor for HSO4(-) in methanol among a series of tested anions. Fluorescence spectroscopy, ultraviolet and nuclear magnetic resonance imaging support that the HSO4(-) indeed interacted with imine nitrogen and the proton of nitrogen in indole ring.

A Schiff-based colorimetric fluorescent sensor with the potential for detection of fluoride ions.

A simple Schiff-based colorimetric fluorescent receptor 1 was prepared. It exhibits a "turn-on-type" mode with high sensitivity in the presence of F(-). The change in color is very easily observed by the naked eye in the presence of F(-), whereas other anions do not induce such a change. Job plot indicated a 1:2 complexation stoichiometry between receptor 1 and F(-). The association constant for 1-F(-) in CH3CN was determined as 1.32*10(5) M(-2) by a Hill plot.

A turn-on and reversible Schiff base fluorescence sensor for Al3+ ion.

A simple Schiff-base receptor 2 was prepared. It exhibits an "off-on-type" mode with high sensitivity in the presence of Al3+. The receptor 2 exhibited a high association constant with submicromolar detection for Al3+ in EtOH­H2O (95:5 v/v) solution. The addition of EDTA quenches the fluorescence of the receptor 2·Al3+ complex offering receptor 2 as a reversible chemosensor.

Euphorbia formosana root extract induces apoptosis by caspase-dependent cell death via Fas and mitochondrial pathway in THP-1 human leukemic cells.

Acute myeloid leukemia (AML), a very rare type of cancer, generally affects patients over 50 years old. While clinical drugs to treat advanced stages of AML exist, the disease becomes increasingly resistant to therapies. Euphorbia formosana Hayata (EF) is a native Taiwanese medicinal plant used to treat rheumatism, liver cirrhosis, herpes zoster, scabies, and photoaging, along with tumor suppression. However, the mechanisms by which it suppresses tumors have not been explored. Here, we provide molecular evidence that a hot-water extract of Euphorbia formosana (EFW) selectively inhibited the growth of human leukemic cancer cells more than other solid human cancer cell lines. Most importantly, the plant extract had limited toxicity toward healthy peripheral blood mononuclear cells (PBMCs). After THP-1 leukemic cells were treated with 50-100 µg/mL EFW for one day, the S phase DNA content of the cells increased, while treatment with 200-400 µg/mL caused the cells to accumulate in the G0/G1 phase. Notably, EFW did not affect A-549 lung cancer cells. The effectiveness of EFW against THP-1 cells may be through caspase-dependent apoptosis in leukemic cells, which is mediated through the Fas and mitochondrial pathways. The potent antileukemic activity of EFW in vitro warrants further investigation of this plant to treat leukemias and other malignancies.

A turn-on and reversible fluorescence sensor for zinc ion.

A simple Schiff base type fluorescent receptor was prepared and evaluated for its fluorescence response to heavy metal ions. Receptor 1 exhibits an "off-on-type" mode with high selectivity in the presence of Zn(2+) ion. The addition of EDTA quenches the fluorescence of receptor 1-Zn(2+) complex, making receptor 1 a reversible chemosensor. The selectivity of 1 for Zn(2+) is the consequence of combined effects of CHEF, C=N isomerization and inhibition of ESIPT.

Hydrolysis of cellulose in synergistic mixtures of ß-glucosidase and endo/exocellulase Cel9A from Thermobifida fusca.

The synergism between the endo/exocellulase, Cel9A, and ß-glucosidase (ßgl) of Thermobifida fusca was investigated. Wild type ßgl or S319C, a ßgl mutant with significantly improved cellobiase activity, were added to Cel9A. Both wild type and mutant ßgl enhanced the Cel9A hydrolysis of carboxymethyl cellulose (CMC) and filter paper by 50-100% compared to Cel9A alone. No enhancement occurred with addition of E388A, an inactive form of ßgl. HPLC analysis showed that, with Cel9A alone, the resulting hydrolysate of glucose and cellobiose contained about half glucose; after addition of equimolar amounts of either wild type ßgl or mutant S319C to Cel9A, the hydrolysate contained more than 85% glucose. ßgl thus acted synergistically with Cel9A by converting cello-oligomers to glucose; this reduced the soluble sugar accumulation during hydrolysis of cellulose.

Mutagenesis and mechanistic study of a glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Trichoderma koningii.

A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

Rapid and selective isolation of beta-xylosidase through an activity-based chemical approach.

beta-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of beta-xylosidase. Activity probe LCL-6X targeting beta-xylosidase was utilized in this study. It carries a beta-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the labeled proteins. LCL-6X could selectively label a model beta-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi-functional enzyme with alpha-L-arabinofuranosidase/beta-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective beta-xylosidase in the culture medium of Aspergillus fumigatus. The beta-xylosidase activities from numerous microbes were also screened using the LCL-6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chemical probe that has potential applications in xylan-related research.

Identification of the two essential groups in the family 3 beta-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis.

beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) catalyses the hydrolysis of beta-1,4-glucosidic bonds via a two-step double-displacement mechanism in which two amino acid residues act as nucleophile and acid/base catalyst. Definitive identification of these two residues is provided by the two active-site-directed inactivators, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-d-glucoside (2FDNPG) and N-bromoacetyl-beta-d-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively. Pseudo-first-order inactivation rate constants (k(i)) of 0.25+/-0.01 and 0.05+/-0.01 min(-1) and dissociation constants (K(i)) of 90+/-15 and 4.4+/-0.2 mM are determined for 2FDNPG and NBGN, respectively. Proteolytic digestion of the labelled proteins, followed by peptide mapping and tandem MS analysis identify Asp-247 and Glu-473 as the catalytic nucleophile and acid/base residues, respectively, of Fbgl. This study confirms that the catalytic nucleophile of family 3 glycohydrolase is conserved across sub-families. However, different sub-families may have unique general acid/base catalysts.