Bioactive steroidal glycosides from the marine sponge Erylus lendenfeldi

Bioassay-guided fractionation of the methanol extract of Erylus lendenfeldi using engineered strains of budding yeast (Saccharomyces cerevisiae) has resulted in the isolation of the known compound eryloside A (1) and two new compounds, erylosides K (2) and L (3). The structures were established based mainly on 1D and 2D NMR data. The absolute stereochemistry of eryloside A, which had never been fully characterized, was determined using the modified Mosher's method. The absolute stereochemistry of eryloside K was determined by comparison with tetrahydroeryloside A. Compounds 1-3 exhibited selective cytotoxicity against a yeast strain (Δrad50) deficient in double strand break (DSB) repair.     

Polydiacetylene/Anti‐HBs Complexes for Visible and Fluorescent Detection of Hepatitis B Surface Antigen on a Nitrocellulose Membrane

The immunochromatographic assay (ICA) using a nitrocellulose (NC) membrane offers several advantages. This technique is a rapid and straightforward method in contrast to other immunoassays. Polydiacetylene (PDA) vesicles have unique optical properties, displaying red color and red fluorescence at the same time. In this system, red‐phase PDA vesicles are used as a fluorescent dye as well as a surface for immobilized hepatitis B surface antibody (HBsAb). PDA has a remarkable stability compared with other fluorescent dyes. In this study, the most suitable PDA/HBsAb complexes are introduced for detecting hepatitis B surface antigen (HBsAg). Then, the PDA/HBsAb complexes affixed antibody is attached to NC membrane, which has two lines to confirm detection of HBsAg. The main advantage of this system is that the detection of HBsAg can be observed in both visible and fluorescent images due to the optical properties of polydiacetylene. Detection of HBsAg is observed up to 0.1 ng mL⁻¹ by fluorescent analysis and confirmed by red line on the NC membrane up to 1 ng mL⁻¹ (HBsAg) using the naked eye. Consequently, these results show that PDA/HBsAb complexes were successfully applied to ICA for the diagnosis of hepatitis B.     

Direct and rapid electrochemical immunosensing system based on a conducting polymer

A system device using multifunctional conjugated copolymer poly(5-hydroxy-1,4-naphthoquinone-co-hydroxy-2-thioacetic acid-1,4-naphthoquinone) acting both as immobilizing and transducing element for reagentless immunosensor has been constructed. Its functionality was evaluated in an antigen-antibody interaction model using ovalbumin-anti-ovalbumin. It was shown that the system specifically detects via electrochemical signal the antigen-antibody immune interaction in a reagentless context. Comparison to the conventional ELISA technique relevant to performance and sensitivity was presented.     

Colorimetric enantiodiscrimination of mandelic acid by indicator displacement assay

The colorimetric enantiodiscrimination between mandelic acid and L-proline-Cu(II) is exploited to develop enantioselective indicator displacement assays. The sensitivity of the assay could be tuned by using a colorimetric indicator. The chromophoric ligand, pyrocatechol violet, effectively competes with the mandelic acid guest for open coordination sites on L-proline-Cu(II). The DA could be increased to 0.12 by changing the ratio of(+)- and(à)-mandelic acid concentrations that were found to be optimal from the displacement experiments. The resultant enantiomer excess versus DA relationship is linear. From the calibration curves, the absorbance values of the unknowns may be calculated for the enantiomeric excess value and the colorimetric enantiodiscrimination of mandelic acid can thus be obtained.     

Online Flowing Colloidosomes for Sequential Multi‐analyte High‐Throughput SERS Analysis

3D plasmonic colloidosomes are superior SERS sensors owing to their high sensitivity and excellent tolerance to laser misalignment. Herein, we incorporate plasmonic colloidosomes in a microfluidic channel for online SERS detection. Our method resolves the poor signal reproducibility and inter‐sample contamination in the existing online SERS platforms. Our flow system offers rapid and continuous online detection of 20 samples in less than 5 min with excellent signal reproducibility. The isolated colloidosomes prevent cross‐sample and channel contamination, allowing accurate quantification of samples over a concentration range of five orders of magnitude. Our system demonstrates high‐resolution multiplex detection with fully preserved signal and Raman features of individual analytes in a mixture. High‐throughput multi‐assay analysis is performed, which highlights that our system is capable of rapid identification and quantification of a sequence of samples containing various analytes and concentrations.     

Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection

Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample.In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L⁻¹ using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L⁻¹ and 0.123 μg L⁻¹ with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC₅₀ value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L⁻¹. This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish.     

Discovery of New Glucose Uptake Inhibitors as Potential Anticancer Agents by Non-Radioactive Cell-Based Assays

Tumor cells rely on aerobic glycolysis to support growth and survival, thus require more glucose supply. Glucose transporters GLUTs, primarily GLUT1, are overexpressed in various cancers. Targeting GLUTs has been regarded as a promising anticancer strategy. In this study, we first evaluated 75 potential GLUT1 inhibitors obtained from virtual screening of the NCI chemical library by a high-throughput cell-based method using a fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG) in COS-7 and SKOV3 cells that express high levels of GLUT1. Four compounds, #12, #16, #43 and #69, that significantly inhibited glucose uptake were further evaluated using flow cytometry directly measuring 2-NBDG uptake at the single-cell level and a Glucose Uptake-Glo^TM assay indirectly measuring 2-deoxy-d-glucose uptake in SKOV3, COS-7 or MCF-7 cells. The inhibitory effect on cancer cell growth was also determined in SKOV3 and MCF-7 cells, and #12 exhibited the best growth inhibitory effect equivalent to a known GLUT1 inhibitor WZB117. Although the anticancer effect of the identified potential GLUT1 inhibitors was moderate, they may enhance the activity of other anticancer drugs. Indeed, we found that #12 synergistically enhanced the anticancer activity of metformin in SKOV3 ovarian cancer cells.     

Spectroscopic information retained in screen photo-assisted techniques

The computer screen photo-assisted technique (CSPT), a recently reported method for characterization of colorimetric assays, has been modeled and the ability of the technique to retain key spectral features has been determined.The robustness of CSPT to operate with different kind of screens is demonstrated and a physical explanation of the result is provided.Simulated CSPT transmittances corroborate experimental results, and enable comparison with standard methods for qualitative and quantitative evaluation.