Oriented conjugation of antibodies against the epithelial cell adhesion molecule on fluorescently doped silica nanoparticles for flow-cytometric determination and <Emphasis Type="Italic">in vivo</Emphasis> imaging of EpCAM,a biomarker for colorectal cancer

The authors report on the conjugation of monoclonal antibodies against the biomarker epithelial cell adhesion molecule (EpCAM) to silica nanoparticles doped with the dye Cy5 (Cy5-SiNPs). Conjugation was performed on the Cy5-SiNPs that were previously coated with a layer of protein G which serves as a linker controlling the orientation of the antibody. The conjugation method takes advantage of site specific interactions between the protein G and constant domains (Fc) of the antibody. The method warrants the antibody binding sites (Fab) to be faced outwards such that the conjugates maintain their affinity for binding the analyte (EpCAM). In vitro analysis by confocal fluorescence imaging and flow cytometry using analytical wavelengths comparable with the excitation and emission wavelength of Cy5-SiNPs at 643 and 662 nm, respectively. The result demonstrated the oriented conjugate to specifically bind to target cells (HT-29) with a sensitivity that is 12 times higher than that of conjugates prepared by conventional EDC coupling. In vivo fluorescence imaging of mice bearing the HT-29 tumor highlighted time-dependent accumulation of the oriented conjugates at the tumor site. As indicated by biodistribution studies hepatic excretion of the oriented probes occurs, however tumor fluorescence still remains for up to 14 days post injection. This research demonstrates that the oriented conjugates derived herein can improve target cell detection sensitivity and can be successfully applied in tumor imaging, which should drive further development of new classes of effective fluorescence contrast agents for cancer diagnostics.

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Graphical abstract Cy5 dye-doped silica nanoparticles were conjugated to antibodies specific for the epithelial cell adhesion molecule. The nanoparticles were previously coated with protein G to control the orientation of the antibody. This warrants enhanced sensitivity for in vitro analysis and also enables in vivo imaging.

     

Mixed metal oxide catalysts for the selective oxidation of ethylbenzene to acetophenone

MgAl and MgMAl oxides(M = Co,Ni and Cu) with a Mg:M:Al molar ratio = 4:1:1 were synthesized from the calcination of their corresponding layered double hydroxide(LDHs) precursors.Their catalytic activities were examined for the oxidation of ethylbenzene using tert-butylhydroperoxide(TBHP) as an oxidant.The oxidized product was mainly acetophenone.The catalytic activities were in the order of MgCuAl>MgNiAl～NiAl～MgCoAl～CoAl>CuAl>MgAl oxides.Reusability studies show that the catalysts are stable under the re...     

Ion Chromatographic Analysis of Monosaccharides and Disaccharides in Raw Sugar

A simple and effective method using ion chromatography was developed for the simultaneous determination of five monosaccharides (arabinose, glucose, fructose, xylose, and ribose) and two disaccharides (sucrose and lactose) in raw sugar samples. The separation was performed on a CarboPac PA 10 column using the gradient elution of sodium hydroxide and water as the mobile phase. Monosaccharides and disaccharides were detected by an integrated pulsed amperometric detection (IPAD) using gold working electrode. Acid hydrolysis was used for sample preparation before the analysis of glucose and fructose. All the studied sugars showed good linear ranges within 0.5–100 µg mL⁻¹ with the correlation coefficients higher than 0.997. The limits of detection were all less than 0.5 µg mL⁻¹. The RSDs of the method were less than 10 %. The recoveries of the sugars that spiked in raw sugar samples ranged from 96.1 to 102.4 %. The method was successfully applied for the analysis of sugars in raw sugar samples. Sucrose is the major constituent found in the samples at 97 %.

     

Microemulsion electrokinetic chromatography for separation and analysis of curcuminoids in turmeric samples

Microemulsion EKC (MEEKC) was developed for quantitative analysis of curcuminoids, such as curcumin (C), demethoxycurcumin (D), and bis-demethoxycurcumin (B). MEEKC separation of curcuminoids was optimized, and a change in resolution was explained using a modified equation for resolution in MEEKC without electroosmosis. The suitable MEEKC conditions for separation of curcuminoids were obtained to be the microemulsion buffer containing 50 mM phosphate buffer at pH 2.5, 1.1% v/v n-octane as oil droplets, 180 mM SDS as surfactant, 890 mM 1-butanol as cosurfactant, and 25% v/v 2-propanol as organic cosolvent; applied voltage of -15 kV; and separation temperature 25 degrees C. Achieved baseline resolution of C:D and D:B was obtained with R(s) -2.4 and analysis time within 18 min. In addition, high accuracy and precision of the method were obtained. This MEEKC method was used for quantitative determination of individual curcuminoids in medicinal turmeric capsules and powdered turmeric used as coloring additive in food, with simple sample preparation such as solvent extraction, dilution, and filtration, and without cleaning up by SPE.     

Quantitative estimation of minor conductive hearing loss with distortion product otoacoustic emissions in the guinea pig

Subclinical conductive hearing losses (CHLs) can affect otoacoustic emissions and therefore limit their potential in the assessment of the cochlear function. Theoretical considerations to estimate a minor CHL from DPOAE measurements [Kummer et al. (2006). HNO 54, 457-467] are evaluated experimentally. They are based on the fact, that the level difference of the stimulus tones L(1) and L(2) for optimal excitation of the inner ear is given by L(1)=aL(2)+b. A CHL is presumed to attenuate both L(1) and L(2) to the same extent such that excitation of the inner ear is no longer optimal. From the change of L(1) that is necessary to restore optimal excitation of the inner ear and thus to produce maximal DPOAE levels, the CHL can be estimated. In 10 guinea pig ears an experimental CHL was produced, quantified by determination of compound action potential (CAP) thresholds at 8 kHz (CHL(CAP)) and estimated from DPOAE measurements at 8 kHz (CHL(DPOAE)). CHLs up to 12 dB could be assessed. CHL(DPOAE) correlated well with CHL(CAP) (R=0.741, p=0.0142). Mean difference between CHL(DPOAE) and CHL(CAP) was 4.2±2.6 dB. Estimation of minor CHL from DPOAE measurements might help to increase the diagnostic value of DPOAEs.     

Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles

We report on a lateral flow immunoassay (LFIA) for influenza A antigen using fluorescently-doped silica nanoparticles as reporters. The method is taking advantage of the high brightness and photostability of silica nanoparticles (doped with the dye Cy5) and the simplicity and rapidity of LFIA. The nucleoprotein of influenza A virion (one of its most abundant structural proteins) was used as a model to demonstrate a performance of the LFIA. Under optimized conditions and by using a portable strip reader, the fluorescence-based LFIA is capable of detecting a recombinant nucleoprotein as low as 250 ng?·?mL^-1 using a sample volume of 100 μL, within 30 min, and without interference by other proteins. The successful detection of the nucleoprotein in infected allantoic fluid demonstrated the functionality of the method. By comparison with a commercial influenza A test based on gold nanoparticles as reporters, the system provides an 8-fold better sensitivity.

Selective Oxidation of Ethylbenzene to Acetophenone Catalyzed by Mixed Oxide Spinels Derived from Layered Double Hydroxides

Mixed oxide spinels were prepared by the calcination of corresponding layered double hydroxides. Ethylbenzene was oxidized using tert-butyl hydroperoxide and H₂O₂. The catalytic activity of mixed oxide spinels increased in the order MgCuMnAl > MgCoMnAl > > MgMnAl > MgCrAl > CoMnAl oxides with high selectivity for acetophenone.     

Gastroprotective Activities of Ethanol Extract of Black Rice Bran (Oryza sativa L.) in Rats

Black rice is a type of rice in the Oryza sativa L. species. There are numerous reports regarding the pharmacological actions of black rice bran, but scientific evidence on its gastroprotection is limited. This study aimed to evaluate the gastroprotective activities of black rice bran ethanol extract (BRB) from the Thai black rice variety Hom Nil (O. sativa L. indica) as well as its mechanisms of action, acute oral toxicity in rats, and phytochemical screening. Rat models of gastric ulcers induced by acidified ethanol, indomethacin, and restraint water immersion stress were used. After pretreatment with 200, 400, and 800 mg/kg of BRB in test groups, BRB at 800 mg/kg significantly inhibited the formation of gastric ulcers in all gastric ulcer models, and this inhibition seemed to be dose dependent in an indomethacin-induced gastric ulcer model. BRB could not normalize the amount of gastric wall mucus, reduce gastric volume and total acidity, or increase gastric pH. Although BRB could not increase NO levels in gastric tissue, the tissue MDA levels could be normalized with DPPH radical scavenging activity. These results confirm the gastroprotective activities of BRB with a possible mechanism of action via antioxidant activity. The major phytochemical components of BRB comprise carotenoid derivatives with the presence of phenolic compounds. These components may be responsible for the gastroprotective activities of BRB. The 2000 mg/kg dose of oral BRB showed no acute toxicity in rats and confirmed, in part, the safe uses of BRB.     

Terpenoids with potent antimycobacterial activity against Mycobacterium tuberculosis from Trigonostemon reidioides roots

Phytochemical investigation of Trigonostemon reidioides roots led to the isolation of fourteen compounds. These included six new diterpenoids, trigonoreidons A?F (1?6), together with eight known diterpenoids 7?14. The structures of the new compounds were elucidated by spectroscopic techniques and the absolute configuration at the asymmetric carbon was determined by the modified Mosher's method. The structure of trigonoreidon B (2) was confirmed by X-ray crystallographic analysis. The known compounds were identified by comparison of the spectroscopic and physical data with those of reported values. The isolated compounds were evaluated for antimycobacterial activity against Mycobacterium tuberculosis. Among the compounds that exhibited antimycobacterial activity, the diterpenoids rediocide C (12) and rediocide G (14) were the most active compounds, with the MIC value of 3.84 μM.