Assessment of goat milk adulteration with a label-free monolithically integrated optoelectronic biosensor.

The label-free detection of bovine milk in goat milk through a miniaturized optical biosensor is presented. The biosensor consists of ten planar silicon nitride waveguide Broad-Band Mach-Zehnder interferometers (BB-MZIs) monolithically integrated and self-aligned with their respective silicon LEDs on the same Si chip. The BB-MZIs were transformed to biosensing transducers by functionalizing their sensing arm with bovine k-casein. Measurements were performed by continuously recording the transmission spectra of each interferometer through an external spectrometer. The amount of bovine milk in goat milk was determined through a competitive immunoassay by passing over the sensor mixtures of anti-k-casein antibodies with the calibrators or the samples. The output spectra of each BB-MZI recorded during the reaction were subjected to Discrete Fourier Transform in order to convert the observed spectral shifts to phase shifts in the wavenumber domain. The method had a detection limit of 0.04 % (v/v) bovine milk in goat milk, dynamic range 0.1-1.0 % (v/v), recoveries 93-110 %, and intra- and inter-assay coefficients of variation less than 12 and 15 %, respectively. The proposed biosensor compared well in terms of analytical performance with a competitive ELISA developed using the same monoclonal antibodies. Nevertheless, the duration of the biosensor assay was 10 min whereas the ELISA required 2 h. Thus, the fast and sensitive determinations along with the small size of the sensor make it ideal for incorporation into portable devices for assessment of goat or ewe's milk adulteration with bovine milk at the point-of-need.

Hydrolysis of 2-arachidonoylglycerol in Tetrahymena thermophila. Identification and partial characterization of a Monoacylglycerol Lipase-like enzyme.

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1µM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ∼45 and ∼40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.

Identification of endocannabinoids and related N-acylethanolamines in tetrahymena. A new class of compounds for Tetrahymena.

The endocannabinoid system is a lipid signaling system in mammalian cells. We reported that major components of the endocannabinoid system such as fatty acid amide hydrolase and monoacylglycerol lipase, are present in the protist Tetrahymena, with characteristics similar to those in mammals. Tetrahymena is a model organism for molecular and cellular biology studies as its genome sequence is available. Here we report the presence of N-acylethanolamines (AcEs) and their respective 2-acylglycerols (2-AcGs) in Tetrahymena thermophila for the first time; the former is a new lipid class for the protist. Using LC-MS/MS we identified, N y-linolenoyl, N-eicosenoyl, N-linoleoyl, N-palmitoyl, N-stearoyl and N-oleoylethanolamines as well as the corresponding monoacylglycerols. The levels of 2-acylglycerols were much higher than the corresponding N-acylethanolamines, as reported for mammals. To our knowledge, N-gamma-linolenoylethanolamine (GLEA) was found for the first time in nature. Anandamide and 2-AG were present in trace amounts. These results demonstrate the existence of a new lipid class in Tetrahymena, strengthen the conviction that the endocannabinoid system is present in this protist, verifying its importance throughout evolution. Tetrahymena could be used as a model for metabolic studies on the endocannabinoids, as well as for the study of drugs targeted towards biosynthetic and catabolic enzymes of AcEs and 2-AcGs.

Dual-cardiac marker capillary waveguide fluoroimmunosensor based on tyramide signal amplification.

The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin-horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction.

Metabolism of 2-acylglycerol in rabbit and human platelets. Involvement of monoacylglycerol lipase and fatty acid amide hydrolase.

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [(3)H]2-AG and [(3)H]2-oleoylglycerol (2-OG) were both metabolized to [(3)H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC(50) = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent K(M) of 0.11 microM and V(max) of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of approximately 33 kDa.

Anandamide increases the differentiation of rat adipocytes and causes PPARgamma and CB1 receptor upregulation.

Anandamide (N-arachidonoylethanolamine, AEA) or its metabolites participate in energy balance mainly through feeding modulation. In addition, AEA has been found to increase 3T3-L1 adipocyte differentiation process. In this study, the effect of AEA, R(+)-methanandamide (R(+)-mAEA), URB597, and indomethacin on primary rat adipocyte differentiation was evaluated by a flow cytometry method and by Oil Red-O staining. Reverse transcription-PCR and western blotting analysis were performed in order to study the effect of AEA on peroxisome proliferator-activated receptor (PPAR)gamma2, cannabinoid receptors (CBRs), fatty acid amidohydrolase (FAAH), and cyclooxygenase-2 (COX-2) expression, during the differentiation process. AEA increased adipocyte differentiation in primary cell cultures in a concentration- and time-dependent manner and induced PPARgamma2 gene expression, confirming findings with 3T3-L1 cell line. CB1R, FAAH, and COX-2 expression was also increased while CB2R expression was decreased. Inhibition of FAAH and COX-2 attenuated the AEA-induced differentiation. Our findings indicate that AEA regulates energy homeostasis not only by appetite modulation but may also regulate adipocyte differentiation and phenotype.

Capillary waveguide fluoroimmunosensor with improved repeatability and detection sensitivity.

An optical capillary waveguide fluoroimmunosensor based on glass capillaries internally coated with an ultrathin poly(dimethylsiloxane) (PDMS) film is presented. The evaluation of the capillaries developed was done in comparison with aminosilanized [3-(aminopropyl)triethoxysilane, APTES] glass and poly(methylpentene) (PMP) capillaries by immobilizing rabbit gamma-globulins on the internal capillary wall. Following reaction with (R)-phycoerythrin-labelled antibody, the capillary was scanned with a laser beam and the fluorescence waveguided through the capillary wall was detected by a photomultiplier placed at one of its ends. The capillaries developed provided considerably improved protein coating homogeneity (intracapillary coefficients of variation 2.9-6.6%) and repeatability (intercapillary coefficients of variation 2.1-5.0%) compared with APTES-treated ones (7.9-13.4 and 8.5-15.2%, respectively). With use of these capillaries in a sandwich-type immunosensor for the determination of rabbit gamma-globulins, the assay detection limit was improved eightfold (4.4 ng/mL) compared with that obtained using PMP capillaries (35.3 ng/mL), whereas the assay repeatability was improved threefold (intra-assay coefficients of variation 5.9-13.1%) compared with APTES-treated capillaries (15.6-36%).