Competitive, non-competitive, and mixed format cleavable tag immunoassays.

Immunoassays are one of the most useful diagnostic techniques in disease assessment, drug metabolite analysis, and environmental applications due largely in part to the selectivity and sensitivity provided by antibody-antigen interactions. Here, a multiplexed immunoassay termed cleavable tag immunoassay (CTI) was performed in competitive, non-competitive, and mixed formats for the analysis of proteins and small molecule biomarkers of inflammation and tissue damage. Microchip capillary electrophoresis (MCE) with fluorescence detection was employed for the analysis of fluorescently labeled tags corresponding to the analytes of interest cleaved from the detection antibodies. For this work we have selected 3-nitrotyrosine (3-NT) a molecule indicative of reactive nitrogen species (RNS), thyroxine (T4) a molecule used to monitor thyroid gland function, and C-reactive protein (CRP) a marker of chronic inflammation as model analytes to demonstrate the assay principles. The simultaneous detection of 3-nitrotyrosine (3-NT) and thyroxine (T4) was carried out as a proof-of-principle for the competitive CTI while non-competitive CTI performance was demonstrated via the analysis of C-reactive protein (CRP). Limit of detections (LOD) and dynamic ranges were investigated. LOD for 3-NT, T4, and CRP were 0.5µg/mL, 23nM, and 5µg/mL, respectively thus demonstrating the ability of the CTI to detect proteins and small molecules within clinical reference ranges. Moreover, this is the first report of the use of mixed format CTI chemistry for the simultaneous detection of proteins (CRP) and small molecules (3-NT) in a single assay. The success of this work demonstrates the ability of CTI to analyze intact proteins and small molecule biomarkers simultaneously.

Cell signaling pathways associated with a reduction in mammary cancer burden by dietary common bean (Phaseolus vulgaris L.).

Emerging evidence indicates that common bean (Phaseolus vulgaris L.) is associated with reduced cancer risk in human populations and rodent carcinogenesis models. This study sought to identify cancer-associated molecular targets that mediate the effects of bean on cancer burden in a chemically induced rat model for breast cancer. Initial experiments were conducted using a high dietary concentration of bean (60% wt/wt) where carcinoma burden in bean-fed rats was reduced 62.2% (P < 0.001) and histological and western blot analyses revealed that the dominant cellular process associated with reduced burden was induction of apoptosis. Further analysis of mammary carcinomas revealed changes in the phosphorylation states of mammalian target of rapamycin (mTOR) substrates (4E-binding protein 1 and p70S6 kinase) and mTOR regulators adenosine monophosphate-activated protein kinase and protein kinase B (Akt) (P < 0.001). Effects on mTOR signaling in carcinomas were also found at lower dietary concentrations of bean (7.5-30% wt/wt). Liquid chromatography-time of flight-mass spectrometry analysis of plasma provided evidence of altered lipid metabolism consistent with reduced mTOR network activity in the liver (P < 0.001). Plasma concentrations of insulin and insulin-like growth factor-1 were reduced by 36.3 and 38.9%, respectively, (P < 0.001), identifying a link to Akt regulation. Plasma C-reactive protein, a prognostic marker for long-term survival in breast cancer patients, was reduced by 23% (P < 0.001) in bean-fed rats. Identification of a role for the mTOR signaling network in the reduction of cancer burden by dietary bean is highly relevant given that this pathway is deregulated in the majority of human breast cancers.

Metabolomic analysis of the effects of edible dry beans (Phaseolus vulgaris L.) on tissue lipid metabolism and carcinogenesis in rats.

Metabolite profiling using liquid chromatography-time-of-flight MS was undertaken to identify candidate metabolic processes that account for dry bean effects on disease risk with a specific focus on the development of breast cancer. Normal mammary gland and mammary carcinomas from previously reported experiments were evaluated. Principal component analysis (PCA) of mass spectral data revealed that tissue of both types from control-fed v. bean-fed rats could be distinguished by their metabolomic profiles. Candidate ion identification using MassTRIX analysis software revealed that alterations in eicosanoid, fatty acid, TAG and steroid metabolism partially accounted for the differences observed in both PCA. In addition, evidence was obtained consistent with the hypothesis that the varying inhibitory effects on mammary carcinogenesis of genetically distinct dry bean types were mirrored by differential patterns of lipid metabolites in mammary carcinoma. The use of MassTRIX provided links for metabolite profile enrichment with metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes. Implicated pathways included a linkage between diacylglycerol and protein kinase C and eicosanoid metabolites and inducible cyclo-oxygenase-2 and/or eicosanoid degradation mediated via 15-PG dehydrogenase. These pathways have been reported to be misregulated during the development of cancer. The differences observed between control-fed and bean-fed rats in lipid metabolism require validation using targeted analytical methods and detailed analyses of how bean bioactive food components regulate genes that control lipid biosynthesis, interconversion and catabolism.

Poly(dimethylsiloxane) cross-linked carbon paste electrodes for microfluidic electrochemical sensing.

Recently, the development of electrochemical biosensors as part of microfluidic devices has garnered a great deal of attention because of the small instrument size and portability afforded by the integration of electrochemistry in microfluidic systems. Electrode fabrication, however, has proven to be a major obstacle in the field. Here, an alternative method to create integrated, low cost, robust, patternable carbon paste electrodes (CPEs) for microfluidic devices is presented. The new CPEs are composed of graphite powder and a binder consisting of a mixture of poly(dimethylsiloxane) (PDMS) and mineral oil. The electrodes are made by filling channels molded in previously cross-linked PDMS using a method analogous to screen printing. The optimal binder composition was investigated to obtain electrodes that were physically robust and performed well electrochemically. After studying the basic electrochemistry, the PDMS-oil CPEs were modified with multi-walled carbon nanotubes (MWCNT) and cobalt phthalocyanine (CoPC) for the detection of catecholamines and thiols, respectively, to demonstrate the ease of electrode chemical modification. Significant improvement of analyte signal detection was observed from both types of modified CPEs. A nearly 2-fold improvement in the electrochemical signal for 100 µM dithiothreitol (DTT) was observed when using a CoPC modified electrode (4.0 ± 0.2 nA (n = 3) versus 2.5 ± 0.2 nA (n = 3)). The improvement in signal was even more pronounced when looking at catecholamines, namely dopamine, using MWCNT modified CPEs. In this case, an order of magnitude improvement in limit of detection was observed for dopamine when using the MWCNT modified CPEs (50 nM versus 500 nM). CoPC modified CPEs were successfully used to detect thiols in red blood cell lysate while MWCNT modified CPEs were used to monitor temporal changes in catecholamine release from PC12 cells following stimulation with potassium.

Evaluation of diversity among common beans (Phaseolus vulgaris L.) from two centers of domestication using 'omics' technologies.

BACKGROUND: Genetic diversity among wild accessions and cultivars of common bean (Phaseolus vulgaris L.) has been characterized using plant morphology, seed protein allozymes, random amplified polymorphic DNA, restriction fragment length polymorphisms, DNA sequence analysis, chloroplast DNA, and microsatellite markers. Yet, little is known about whether these traits, which distinguish among genetically distinct types of common bean, can be evaluated using omics technologies. RESULTS: Three 'omics' approaches: transcriptomics, proteomics, and metabolomics were used to qualitatively evaluate the diversity of common bean from two Centers of Domestication (COD). All three approaches were able to classify common bean according to their COD using unsupervised analyses; these findings are consistent with the hypothesis that differences exist in gene transcription, protein expression, and synthesis and metabolism of small molecules among common bean cultivars representative of different COD. Metabolomic analyses of multiple cultivars within two common bean gene pools revealed cultivar differences in small molecules that were of sufficient magnitude to allow identification of unique cultivar fingerprints. CONCLUSIONS: Given the high-throughput and low cost of each of these 'omics' platforms, significant opportunities exist for their use in the rapid identification of traits of agronomic and nutritional importance as well as to characterize genetic diversity.

Spatially resolved electrochemical sensing of chemical gradients.

Chemical gradients drive a diverse set of biological processes ranging from nerve transduction to ovulation. At present, the most common method for quantifying chemical gradients is microscopy. Here, a new concept for probing spatial and temporal chemical gradients is reported that uses a multi-layer microfluidic device to measure analyte concentration as a function of lateral position in a microfluidic channel using electrochemistry in a format that is readily adaptable to multi-analyte sensing.

Metabolite profiling of a diverse collection of wheat lines using ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry.

Genetic differences among major types of wheat are well characterized; however, little is known about how these distinctions affect the small molecule profile of the wheat seed. Ethanol/water (65% v/v) extracts of seed from 45 wheat lines representing 3 genetically distinct classes, tetraploid durum (Triticum turgidum subspecies durum) (DW) and hexaploid hard and soft bread wheat (T. aestivum subspecies aestivum) (BW) were subjected to ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-TOF-MS). Discriminant analyses distinguished DW from BW with 100% accuracy due to differences in expression of nonpolar and polar ions, with differences attributed to sterol lipids/fatty acids and phospholipids/glycerolipids, respectively. Hard versus soft BW was distinguished with 100% accuracy by polar ions, with differences attributed to heterocyclic amines and polyketides versus phospholipid ions, respectively. This work provides a foundation for identification of metabolite profiles associated with desirable agronomic and human health traits and for assessing how environmental factors impact these characteristics.

Characterization of low molecular weight chemical fractions of dry bean (Phaseolus vulgaris) for bioactivity using Caenorhabditis elegans longevity and metabolite fingerprinting.

Dry bean consumption has been reported to be associated with reduced risk for a number of chronic diseases including cancer. The extent to which these benefits are associated with primary versus secondary plant metabolites is not known. The work reported herein focuses on low molecular weight secondary metabolites and uses longevity extension of wild-type Caenorhabditis elegans nematodes as a surrogate marker for human health benefits. A modified Bligh and Dyer technique was used to extract freeze-dried bean, and the resulting fractions were evaluated for longevity extension and metabolite fingerprinting using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Dry bean extracts extended adult C. elegans lifespan by as much as 16%. Hydrophilic fractions increased lifespan, whereas the hydrophobic fraction induced longevity reduction. Metabolite fingerprinting revealed distinguishing spectral differences among the four chemical fractions evaluated and demonstrated that within each fraction chemical composition differed significantly based on dry bean genetic heritage.