Physiological and Molecular Modulations to Drought Stress in the Brassica Species.

Climate change, particularly drought stress, significantly impacts plant growth and development, necessitating the development of resilient crops. This study investigated physiological and molecular modulations to drought stress between diploid parent species and their polyploid progeny in the Brassica species. While no significant phenotypic differences were observed among the six species, drought stress reduced growth parameters by 2.4% and increased oxidative stress markers by 1.4-fold. Drought also triggered the expression of genes related to stress responses and led to the accumulation of specific metabolites. We also conducted the first study of perfluorooctane sulfonic acid (PFOS) levels in leaves as a drought indicator. Lower levels of PFOS accumulation were linked to plants taking in less water under drought conditions. Both diploid and polyploid species responded to drought stress similarly, but there was a wide range of variation in their responses. In particular, responses were less variable in polyploid species than in diploid species. This suggests that their additional genomic components acquired through polyploidy may improve their flexibility to modulate stress responses. Despite the hybrid vigor common in polyploid species, Brassica polyploids demonstrated intermediate responses to drought stress. Overall, this study lays the framework for future omics-level research, including transcriptome and proteomic studies, to deepen our understanding of drought tolerance mechanisms in Brassica species.

Direct real-time measurements of superoxide release from skeletal muscles in rat limbs and human blood platelets using an implantable Cytochrome C microbiosensor.

Oxidative stress and excessive accumulation of the superoxide (O2.-) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O2.-) release is important to assess the extent of oxidative stress in these conditions. Herein, we present the design, fabrication and characterization of a robust (O2.-) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of L-Cysteine on a gold wire electrode to which Cytochrome C (Cyt c) was conjugated. The immobilized layers, studied using conductive Atomic Force Microscopy (c-AFM) revealed a stable and uniformly distributed redox protein on the gold surface, visualized as conductivity and surface topographical plots. The biosensor enabled detection of (O2.-) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/µM and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O2.-) release in activated human blood platelets and skeletal rat limb muscles following ischemia reperfusion injury (IRI), confirming the biosensor's stability and robustness for measurements in complex biological systems. The results demonstrate the ability of these biosensors to monitor real time release of (O2.-) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies.

Biosensors based on modularly designed synthetic peptides for recognition, detection and live/dead differentiation of pathogenic bacteria.

Rapid and sensitive detection of bacterial pathogens is critical for assessing public health, food and environmental safety. We report the use of modularly designed and site-specifically oriented synthetic antimicrobial peptides (sAMPs) as novel recognition agents enabling detection and quantification of bacterial pathogens. The oriented assembly of the synthetic peptides on electrode surfaces through an engineered cysteine residue coupled with impedimetric detection facilitated rapid and sensitive detection of bacterial pathogens with a detection limit of 10(2)CFU/mL for four bacterial strains including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The approach enabled differentiation between live and dead bacteria. The fabrication of the sAMPs functionalized surface and the importance of the sAMPs orientation for providing optimum recognition and detection ability against pathogens are discussed. The proposed methodology provides a universal platform for the detection of bacterial pathogens based on engineered peptides, as alternative to the most commonly used immunological and gene based assays. The method can also be used to fabricate antimicrobial coatings and surfaces for inactivation and screening of viable bacteria.

Effects of brewing conditions on the antioxidant capacity of twenty-four commercial green tea varieties.

A novel paper-based Nanoceria Reducing Antioxidant Capacity (NanoCerac) assay for antioxidant detection (Sharpe, Frasco, Andreescu, & Andreescu, 2012), has been adapted for the first time as a high-throughput method, in order to measure the effect of brewing conditions and re-infusion on the antioxidant capacity of twenty-four commercial green teas. The oxygen radical absorbance capacity (ORAC) assay, frequently applied to complex foods and beverages, was used as a comparator measure of antioxidant capacity. A novel measure of sustained antioxidant capacity, the total inherent antioxidant capacity (TI-NanoCerac and TI-ORAC) was measured by infusing each tea six times. Effects of brewing conditions (temperature, brew time, etc.) were assessed using one popular tea as a standard. Both NanoCerac and ORAC assays correlated moderately (R(2) 0.80 ± 0.19). The average first-brew NanoCerac, TI-NanoCerac, first-brew ORAC and TI-ORAC were: 0.73 ± 0.1 GAE/g tea; 2.4 ± 0.70 mmolGAE/g tea; 1.0 ± 0.3 mmolTE/g tea and 2.1 ± 0.71 mmolTE/g tea respectively. Brewing conditions including water temperature and infusion time significantly affected antioxidant capacity. The high-throughput adaptation of the original NanoCerac assay tested here offered advantages over ORAC, including portability and rapid analysis.

An acetylcholinesterase (AChE) biosensor with enhanced solvent resistance based on chitosan for the detection of pesticides.

Solvent tolerance of immobilized enzymes is important for many biosensing and biotechnological applications. In this paper we report an acetylcholinesterase (AChE) biosensor based on chitosan that exhibits high solvent resistance and enables sensitive detection of pesticides in presence of a high content of organic solvents. The solvent effect was established comparatively for the enzyme immobilized in chitosan and covalently cross-linked with glutaraldehyde. The activity of the immobilized AChE was dependent on the immobilization method and solvent type. The enzyme entrapped in chitosan fully conserved its activity in up to 25% methanol, 15% acetonitrile and 100% cyclohexane while the enzyme cross-linked with glutaraldehyde gradually lost its activity starting at 5% acetonitrile and methanol, and showed variable levels in cyclohexane. The detection limits of the biosensor for paraoxon were: 7.5 nM in 25% methanol, 100 nM in 15% acetonitrile and 2.5 µM in 100% cyclohexane. This study demonstrates that chitosan provides an excellent immobilization environment for AChE biosensors designed to operate in environments containing high amounts of organic solvents. It also highlights the effect of the immobilization material and solvent type on enzyme stability. These findings can enable future selection of the immobilization matrix and solvent type for the development of organic phase enzyme based systems.

Portable nanoparticle based sensors for antioxidant analysis.

Interest in portable sensing devices has increased throughout the past decade. Portable sensors are convenient for use in remote locations and in places with limited resources for advanced instrumentation. Often such devices utilize advanced technology that allows the final user to simply deposit the sample onto the sensing platform without preparation of multiple reagents. Herein, we describe preparation and characterization of a colorimetric paper-based metal oxide sensing array designed for the field detection of polyphenolic antioxidants. This sensor is a good candidate for use in analysis of the antioxidant character of food, drink, botanical medicines, physiological fluids, and more.

Metal oxide based multisensor array and portable database for field analysis of antioxidants.

We report a novel chemical sensing array based on metal oxide nanoparticles as a portable and inexpensive paper-based colorimetric method for polyphenol detection and field characterization of antioxidant containing samples. Multiple metal oxide nanoparticles with various polyphenol binding properties were used as active sensing materials to develop the sensor array and establish a database of polyphenol standards that include epigallocatechin gallate, gallic acid, resveratrol, and Trolox among others. Unique charge-transfer complexes are formed between each polyphenol and each metal oxide on the surface of individual sensors in the array, creating distinct optically detectable signals which have been quantified and logged into a reference database for polyphenol identification. The field-portable Pantone/X-Rite© CapSure® color reader was used to create this database and to facilitate rapid colorimetric analysis. The use of multiple metal-oxide sensors allows for cross-validation of results and increases accuracy of analysis. The database has enabled successful identification and quantification of antioxidant constituents within real botanical extractions including green tea. Formation of charge-transfer complexes is also correlated with antioxidant activity exhibiting electron transfer capabilities of each polyphenol. The antioxidant activity of each sample was calculated and validated against the oxygen radical absorbance capacity (ORAC) assay showing good comparability. The results indicate that this method can be successfully used for a more comprehensive analysis of antioxidant containing samples as compared to conventional methods. This technology can greatly simplify investigations into plant phenolics and make possible the on-site determination of antioxidant composition and activity in remote locations.

Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility.

The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a-/- embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a-/- embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34hpf and again between 64 and 74hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a-/- embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a-/- embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a-/- embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.

Portable ceria nanoparticle-based assay for rapid detection of food antioxidants (NanoCerac).

With increased awareness of nutrition and the advocacy for healthier food choices, there exists a great demand for a simple, easy-to-use test that can reliably measure the antioxidant capacity of dietary products. We report development and characterization of a portable nanoparticle based-assay, similar to a small sensor patch, for rapid and sensitive detection of food antioxidants. The assay is based on the use of immobilized ceria nanoparticles, which change color after interaction with antioxidants by means of redox and surface chemistry reactions. Monitoring corresponding optical changes enables sensitive detection of antioxidants in which the nanoceria provides an optical 'signature' of antioxidant power, while the antioxidants act as reducing agents. The sensor has been tested for the detection of common antioxidant compounds including ascorbic acid, gallic acid, vanillic acid, quercetin, caffeic acid, and epigallocatechin gallate and its function has been successfully applied for the assessment of antioxidant activity in real samples (teas and medicinal mushrooms). The colorimetric response was concentration dependent, with detection limits ranging from 20 to 400 µM depending on the antioxidant involved. Steady-state color intensity was achieved within seconds upon addition of antioxidants. The results are presented in terms of Gallic Acid Equivalents (GAE). The sensor performed favorably when compared with commonly used antioxidant detection methods. This assay is particularly appealing for remote sensing applications, where specialized equipment is not available, and also for high throughput analysis of a large number of samples. Potential applications for antioxidant detection in remote locations are envisioned.

Site-specific immobilization of a (His)6-tagged acetylcholinesterase on nickel nanoparticles for highly sensitive toxicity biosensors.

This paper reports site-specific affinity immobilization of (His)6-tagged acetylcholinesterase (AChE) onto Ni/NiO nanoparticles for the development of an electrochemical screen-printed biosensor for the detection of organophosphate pesticides. The method is based on the specific affinity binding of the His-tagged enzyme to oxidized nickel nanoparticle surfaces in the absence of metal chelators. This approach allows stable and oriented attachment of the enzyme onto the oxidized nickel through the external His residue in one-step procedure, allowing for fast and sensitive detection of paraoxon in the concentration range from 10(-8) to 10(-13) M. A detection limit of 10(-12) M for paraoxon was obtained after 20 min incubation. This method can be used as a generic approach for the immobilization of other His-tagged enzymes for the development of biosensors.

AChE biosensor based on zinc oxide sol-gel for the detection of pesticides.

Zinc oxide has been used as a matrix for immobilization of acetylcholinesterase (AChE) and detection of the pesticide paraoxon. The immobilized enzyme retained its enzymatic activity up to three months when stored in phosphate buffered saline (pH 7.4) at 4 degrees C. An amperometric biosensor for the detection of paraoxon was designed. The biosensor detected paraoxon in the range 0.035-1.38 ppm and can be used to detect other AChE inhibiting organophosphate pesticides.

Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish.

Metallic nanoparticles such as nickel are used in catalytic sensing, and electronic applications, but health and environmental affects have not been fully investigated. While some metal nanoparticles result in toxicity, it is also important to determine whether nanoparticles of the same metal but of different size and shape changes toxicity. Three different size nickel nanoparticle (Ni NPs) of 30, 60, and 100 nm and larger particle clusters of aggregated 60 nm entities with a dendritic structure were synthesized and exposed to zebrafish embryos assessing mortality and developmental defects. Ni NPs exposure was compared to soluble nickel salts. All three 30, 60, and 100 nm Ni NPs are equal to or less toxic than soluble nickel while dendritic clusters were more toxic. With each Ni NP exposure, thinning of the intestinal epithelium first occurs around the LD10 continuing into the LD50. LD50 exposure also results in skeletal muscle fiber separation. Exposure to soluble nickel does not cause intestinal defects while skeletal muscle separation occurs at concentrations well over LD50. These results suggest that configuration of nanoparticles may affect toxicity more than size and defects from Ni NPs exposure occur by different biological mechanisms than soluble nickel.

Studies of the binding and signaling of surface-immobilized periplasmic glucose receptors on gold nanoparticles: a glucose biosensor application.

Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.

Stable enzyme biosensors based on chemically synthesized Au-polypyrrole nanocomposites.

This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors. Three enzyme systems have been used as model examples: cytochrome c, glucose oxidase and polyphenol oxidase. The synthesis and deposition of the nanocomposite was first optimized onto a glassy carbon electrode (GCE) and then, the optimum procedure was used for enzyme immobilization and subsequent fabrication of glucose and phenol biosensors. The resulting nanostructured polymer strongly adheres to the surface of the GCE electrode, has uniform distribution and is very stable. The method has proved to be an effective way for stable enzyme attachment while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of pyrrole and enzyme and the Au-PPy matrix avoids enzyme leaking. The preparation conditions, Michaelis-Menten kinetics and analytical performance characteristics of the two biosensors are discussed. Optimization of the experimental parameters was performed with regard to pyrrole concentration, enzyme amount, pH and operating potential. These biosensors resulted in rapid, simple, and accurate measurement of glucose and phenol with high sensitivities (1.089 mA/M glucose and 497.1 mA/M phenol), low detection limits (2 x 10(-6)M glucose and 3 x 10(-8)M phenol) and fast response times (less than 10s). The biosensors showed an excellent operational stability (at least 100 assays) and reproducibility (R.S.D. of 1.36%).

Advanced electrochemical sensors for cell cancer monitoring.

The possibility of using minimally invasive analytical instruments to monitor cancerous cells and their interactions with analytes provide great advances in cancer research and toxicology. The real success in the development of a reliable sensor for cell monitoring depends on the ability to design powerful instrumentation that will facilitate efficient signal transduction from the biological process that occurs in the cellular environment. The resulting sensor should not affect cell viability and must function as well as adapt the system to the specific conditions imposed by the cell culture. Due to their performance, electrochemical biosensors could be used as an effective instrument in cell cancer research for studying biochemical processes, cancer development and progression as well as toxicity monitoring. Current research in this direction is conducted through high-throughput, compact, portable, and easy to use sensors that enable measurement of cells' activity in their optimum environment. This paper discusses the potential of a high-throughput electrochemical multisensor system, so-called the DOX system for monitoring cancerous cells and their interaction with chemical toxins. We describe the methodology, experiments, and the operation principle of this device, and we focus on the challenges encountered in optimizing and adapting the system to the specific cell-culture conditions. The DOX system is also compared with conventional cell-culture techniques.

Effect of natural and synthetic estrogens on a549 lung cancer cells: correlation of chemical structures with cytotoxic effects.

A series of synthetic (nonylphenol, diethylstilbestrol, and bisphenol A) and natural (quercetin, resveratrol, and genistein) phenolic estrogens were investigated for their ability to affect the viability and proliferation of A549 lung cancer cells. To assess and distinguish the cytotoxic effect of individual estrogens, we used both the MTT tetrazolium spectrophotometric method and the fluorescence assay, while the induction of the cell specific apoptotic process was examined by fluorescence microscopy after treatment of cells with SYTO 24 green fluorescent dye. A systematic study of interferences for both fluorescence and MTT methods is presented. The results showed that both natural and synthetic estrogens decreased the viability and proliferation of A549 lung cancer cells in a dose-dependent manner but at different sensitivities. Nonylphenol appeared very different as compared to the other estrogens, acting by inducing the higher inactivation rate of the cells within a very short time. The cytotoxic effect of the estrogens was directly related to their structural and conformational characteristics including chain length, number, and position of hydroxyl groups and degree of saturation.

Autonomous multielectrode system for monitoring the interactions of isoflavonoids with lung cancer cells.

The cytotoxic effect of isoflavonoids in the development of different forms of cancer has been reported by epidemiological and dietary studies. Consequently, there is a search for an accurate and reliable method for monitoring the interactions of these chemicals with cancerous cells. We have developed and optimized a fully autonomous electrochemical biosensor for studying the role of isoflavonoids on A549 lung adenocarcinoma cell line. This advanced biosensor uses a prototype 96-electrode (DOX-96) well-type device that allows the measurement of cell respiratory activity via the consumption of dissolved oxygen. The system provides a continuous, real-time monitoring of cell activity upon exposure to naturally occurring polyphenols, specifically resveratrol, genistein, and quercetin. The system is equipped with a multipotentiostat, a 96-electrode well for measurements and cell culturing with 3 disposable electrodes fitted into each well. A comparison with classical "cell culture" techniques indicates that the biosensor provides real-time measurement with no added reagents. A detection limit of 1 x 10(4) was recorded versus 200 and 6 x 10(3) cells/well for MTT and fluorescence assays, respectively. This method was optimized with respect to cell stability, reproducibility, applied potential, cell density per well, volume/composition of cell culture medium per well, and incubation. Others include total measuring time, temperature, and sterilization procedure. This study represents a basic research tool that may allow researchers to study the type, level, and specific influence of isoflavonoids on cells.

Correlation of analyte structures with biosensor responses using the detection of phenolic estrogens as a model.

The apparent increase in hormone-induced cancers and disorders of the reproductive tract in wildlife and humans has led to a search for an accurate and reliable method for monitoring endocrine-disrupting chemicals (EDCs). This study presents a generic approach that may allow researchers to establish screening procedures for potential EDCs by correlating the analyte structures with biosensor responses and explain possible reaction mechanisms. A simple amperometric tyrosinase-based biosensor (Tyr-CPE) has been developed for the detection of phenolic EDCs. The investigation of the enzymatic oxidation of selected phenolic estrogens was first carried out using UV-vis spectroscopy. The result was used to correlate sensor responses to enzymatic activity. Natural phytoestrogen polyphenols, including resveratrol (RES), genistein (GEN), and quercetin (QRC), were compared with synthetic estrogens, for example, bisphenol A (BPhA), nonylphenol (NPh), and diethylstilbestrol (DES). The Tyr-CPE biosensor resulted in rapid, simple, and accurate measurement of phenolic estrogens with varying degrees of sensitivity, selectivity, and response times. The sensor responses have been evaluated for the detection of binary and tertiary mixtures of EDCs and natural estrogens. The results showed that BPhA could be successfully discriminated in a composite mixture containing NPh and DES at various ratios. In the case of natural phenolic estrogens GEN, RES, and QRC, the sensor allows the determination of a total phenolic content. The sensor was also validated for the detection of BPhA in a real environmental water sample, and the results was compared with standard ASTM method 9065. Mechanistically, our results indicated that the number of OH groups, the nature and the position of aryl ring substituents, or both could affect the detection limit and the biosensor sensitivity.

Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents.

A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91x10(-8) M paraoxon and 1.24x10(-9) M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile.