Design and validation of a novel immunological test for enterolactone.

Enterolactone (ENL) is produced by the gut microflora from lignans found in edible plants. ENL is estrogenic with no effect on the E-screen test and is a natural Selected Estrogen Receptor Modulator (SERM) with health interests that have to be checked in clinical studies with bioavailability assessment. Two haptens of ENL were synthesized, with a spacer arm at the C5 position having either 2 or 4 carbon atoms (ENLΔ2 and ENLΔ4, respectively). Hapten coupling to bovine serum albumin (BSA) was characterized by MALDI mass spectrometry. Polyclonal antibodies were obtained against the BSA conjugates. Additional conjugates were generated by coupling to swine thyroglobulin (Thyr). Homologous and heterologous competitive ELISAs were developed with Thyr or BSA conjugates as coating. The best assays were validated on biological samples from mice. Both antibodies exhibited the same IC50 at 1.5 ng mL(-1) with a detection limit below 0.5 ng mL(-1). Most cross-reactions with structurally related lignans were lower than 0.03%. This new assay type is faster, more specific and more reliable than existing ones.

Nanoscale structural features determined by AFM for single virus particles.

In this work, we propose "single-image analysis", as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis.

A mesofluidic multiplex immunosensor for detection of circulating cytokeratin-positive cells in the blood of breast cancer patients.

We have recently reported the analytical performance of an immunosensor comprising one mm-scale parallel plate laminar flow chamber and applied to capture MCF7 breast cancer cells (Ehrhart et al., Biosens. Bioelectr. 24, 467, 2008). Herein we present a new multiplex immunosensor embodying four parallel plate laminar flow chambers that fit onto a standard, functionalized, microscopy glass slide. The four surfaces are coated with long alkyl chain spacers of 21-aminohenicosyl trichlorosilane (AHTS) and then grafted with a monoclonal anti-human epithelial cell adhesion molecule (EpCAM) antibody specific of target cells to immobilize. We first demonstrate a significantly (P < 0.01) improved capacity of each of the four flow chambers of the multiplex immunosensor to capture MCF7 cells compared to the previous single chamber device. Second, in addition to an increase of cell immobilization, the multiplex device offers a versatile tool easily grafted with various purified antibodies onto the four surfaces. Third, we obtained high cell capture rate and efficiency of various numbers of MCF7 cells spiked in buffer containing an equal number of background leukocytes. And fourth, we demonstrate isolation efficiency of circulating tumor cells (CTCs) from peripheral blood drawn from a small cohort of patients with localized or metastatic breast cancer. This new multiplex immunosensor could be tested for its potential to capture different subpopulations of CTCs.

Aromatic potential of botrytized white wine grapes: identification and quantification of new cysteine-S-conjugate flavor precursors.

Sweet wines made from botrytized grapes contain much higher concentrations of volatile thiols, especially 3-sulfanylhexan-1-ol (3SH), than dry white wines. Three new specific volatile thiols (3-sulfanylpentan-1-ol (3SP), 3-sulfanylheptan-1-ol (3SHp), and 2-methyl-3-sulfanylbutan-1-ol (2M3SB) were recently identified in Sauternes wines. Like most volatile thiols, these compounds were almost totally absent from must, mainly being formed during alcoholic fermentation. In this work, we describe the identification and quantification of three new cysteine-S-conjugate precursors in must made from Botrytis-infected grapes. S-3-(pentan-1-ol)-L-cysteine (P-3SP), S-3-(heptan-1-ol)-L-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-L-cysteine (P-2M3SB) were identified by direct GC-MS analysis of their derivative forms obtained by silylation of an enriched fraction, isolated from must by affinity chromatography. Concentrations were considerably higher when Botrytis cinerea had developed on the grapes. In botrytized must, the mean levels of P-3SP, P-3SHp, and P-2M3SB were in the vicinity of 700, 50, and 500 nM, respectively, whereas concentrations in healthy must ranged from 0 to 50 nM. This indicated that these three new sulfanyl alcohols, responsible for the characteristic aroma of botrytized wines, were formed by the yeast metabolism during alcoholic fermentation from the corresponding non-volatile cysteine-S-conjugate precursors. Moreover, these results highlighted the predominant role of botrytization in developing grape aroma potential.

A new immunosensor for breast cancer cell detection using antibody-coated long alkylsilane self-assembled monolayers in a parallel plate flow chamber.

We designed a new efficient and reliable immunosensor and demonstrated its analytic performance to capture breast cancer MCF7 and T47D cells, under laminar flow, onto antibody-coated long alkylsilane self-assembled monolayers (SAMs) in a parallel plate flow chamber. The surface floor of the laminar flow chamber was grafted with an amino-terminated long alkyl chain spacer, 21-aminohenicosyl trichlorosilane (AHTS) followed by tethering a specific monoclonal antibody directed against the human epithelial cell adhesion molecule (EpCAM) antigen, which is overexpressed in primary breast cancer. Properties of the AHTS- and antibody-grafted surface floor were compared to that of surface floors coated with the short alkyl spacers 3-glycidoxy-propyl trimethoxysilane (GPTS) or 3-aminopropyl triethoxysilane (APTES) and antibodies. A theoretical model was constructed according to the geometry of the flow chamber in order to calculate the trajectories that would use cell flows. Cell capture experiments demonstrated that cell immobilization was optimized throughout the whole flow chamber. High cell capture was yielded on antibody-tethered long alkyl AHTS surface. This new procedure offers multiple advantages: a versatile tool readily applied to a panel of purified antibodies, an enrichment of cell immobilization using repetitive cell flow, and a stable capturing surface suitable for long term storage and handling.

Surprising structural lability of a cysteine-S-conjugate precursor of 4-methyl-4-sulfanylpentan-2-one, a varietal aroma in wine of Vitis vinifera L. cv. Sauvignon blanc.

4-Methyl-4-sulfanylpentan-2-one (1; 4MSP) provides a characteristic aroma compound of wines made from Vitis vinifera L. cv. Sauvignon blanc. 4MSP has a strong box-tree odor with a very low perception threshold and is derived from the cysteinylated precursor S-(1,1-dimethyl-3-oxobutyl)cysteine (4; P-4MSP). P-4MSP is transformed into 4MSP during alcoholic fermentation and is an excellent marker of varietal aroma potential. An improved synthesis of P-4MSP as well as of its deuterium-labeled analogue [D(6)]-P-4MSP is described. Several analytical methods (NMR, IR, LSI-MS, GC/MS, ESI-MS(n)) were combined to elucidate spontaneous reversible structural changes of P-4MSP at different pH values. At low pH, P-4MSP has a linear keto form. The keto-enol tautomerism was observed at neutral pH. At pH 8, the formation of N-substituted intramolecular hemiaminal was characterized by ESI-MS and ESI-MS(n) experiments. The hemiaminal loses H(2)O at high pH to produce a cycloimine, which is easily opened by acid hydrolysis. The keto-enol tautomerism explained the incorporation of only six D-atoms during the preparation of the P-4MSP deuterated standard even if [D(10)]mesityl oxide was used. Derivatization conditions for GC/MS analysis strongly affected the ratio of the monosilylated intramolecular cyclic form and the disilylated linear form of P-4MSP. The structural changes of P-4MSP may have a considerable impact on the development of methods of measuring varietal aroma potential.

Analysis of the diastereoisomers of the cysteinylated aroma precursor of 3-sulfanylhexanol in Vitis vinifera grape must by gas chromatography coupled with ion trap tandem mass spectrometry.

The diastereoisomeric distribution of S-3-(hexan-1-ol)cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexan-1-ol (3SH) in Vitis vinifera grape juice, was determined by a new method. This procedure is based on the purification of P-3SH in a small volume of must (500 microL) by affinity chromatography, followed by the separation of chiral molecules in derivative forms by gas chromatography coupled with ion trap tandem mass spectrometry (GC-MS/MS). The diastereoisomers were easily separated using heptafluorobutyric anhydride and heptafluorobutanol (HFBA and HFOH) as derivatization reagents. Method validation was conducted using samples of grape juice, synthetic must, fermenting must, and wine that were fortified with P-3SH at concentrations of 0.6 and 2.5 microM. The relative standard deviation (RSD) and limit of detection (LOD) of the GC-MS/MS method were 4.6% and 1.5 nM, respectively. P-3SH assays in Bordeaux white grape juice affected by Botrytis cinerea showed an unusually increased proportion of the RS form of the precursor (approximately RR:RS=30:70) as compared to a diastereoisomer ratio (in the vicinity of 50:50) in healthy grape juice.