Portable instruments based in multiband fluorescence transducers for biosensing applications

Dos prototipos de micro instrumentos ópticos basados en biosensores han sido diseñados y fabricados para mediciones de  fluorescencia de varios tipos de biomediadores en serie o individuales, con aplicaciones en monitoreo del medio ambiente y análisis  agroalimentario. Se trata de un sistema multiceldas, cada celda está compuesta por dos secciones modulares: en la parte inferior, un compartimiento con excitación  óptica de luz LED y un fotodiodo detector de emisiones de fluorescencia, y en la parte superior, un compartimiento  biocompatible donde se deposita la muestra biológica.  Los prototipos se han aplicado con algas genéticamente modificadas, que fueron empleadas en instrumentos para ensayos  experimentales en monitoreo de pesticidas en la contaminación del agua. Los pesticidas modifican la actividad en el foto sistema II (PSII), en términos de disminución de la intensidad de fluorescencia. Se presentan los resultados de las mediciones que emplean varios mutantes como el C251 y tres diferentes pesticidas en el aumento de las concentraciones y tiempos de incubación. Abstract Two prototypes of the miniaturized biosensor-based optical instrument has been designed and fabricated for  fluorescence  measurements of several biomediators in series or only one, with applications in environmental monitoring and agrofood  analysis. It is a multicell system, every cell is made up by two modular sections: the bottom compartment with optical LED light excitations and a photodiode  detector for fluorescence emission capture, and the top biocompatible compartment where the biosample is deposited.  The prototypes has been implemented with genetically modified algae that were employed in the instrument experimental testing by monitoring pesticide pollution in water. Pesticides modify the photosystem II (PSII) activity in terms of fluorescence quenching. Results from measurements employing several C251 mutants and three different pesticides at increasing concentrations and incubation times are presented and discussed.

Establishment of In Vitro Salvia Cell Biomass for the Controlled Production of Antioxidant Metabolites

Dietary consumption of foods rich in antioxidants has been gathering increasing support in recent years. The project NUTRASNACK (E.C. F.P. 6 contract No FOOD-CT-2005-023044), was developed to increase the antioxidant potential of food snacks. Several Salvia species and genotypes were tested for their total antioxidant content. Three genotypes with the highest antioxidant values were selected (S. officinalis 'SOF1', S. officinalis 'SOF2', and S. officinalis 'SOF3') and leaf explants of in vivo grown plants were cultured on modified MS media supplemented with 2,4-D, Kinetin and NAA in several combinations in order to induce friable callus. Friable green callus developed after five weeks in the presence of medium containing 0.5 mg L-1 2,4-D and 0.5 mg L-1 Kinetin. Callus was transferred several times to the same medium, and after three months, liquid primary cultures were established. After filtration, the synchronized cells grew rapidly and the growth parameters such as viability and growth curve in dry and fresh weight gave the essential information for the management of the cultures. The EC 50 of total antioxidant capacity was evaluated during all the growth phases of calli and suspension cultures

Establishment of in vitro salvia cell biomass for the controlled production of antioxidant metabolites

Dietary consumption of foods rich in antioxidants has been gathering increasing support in recent years. The project NUTRASNACK (E.C. F.P.6 contract No FOOD-CT-2005-023044), was developed to increase the antioxidant potential of food snacks. Several Salvia species and genotypes were tested for their total antioxidant content. Three genotypes with the highest antioxidant values were selected (S. officinalis ‘SOF1’, S. officinalis ‘SOF2’, and S. officinalis ‘SOF3’) and leaf explants of in vivo grown plants were cultured on modified MS media supplemented with 2,4-D, Kinetin and NAA in several combinations in order to induce friable callus. Friable green callus developed after five weeks in the presence of medium containing 0.5 mg L-1 2,4-D and 0.5 mg L-1 Kinetin. Callus was transferred several times to the same medium, and after three months, liquid primary cultures were established. After filtration, the synchronized cells grew rapidly and the growth parameters such as viability and growth curve in dry and fresh weight gave the essential information for the management of the cultures. The EC 50 of total antioxidant capacity was evaluated during all the growth phases of calli and suspension cultures

”LC-DADESI/MSn identification of active natural products from four plants species.”

In our work on the project NUTRASNACK (E.C. F.P.6 contract No FOOD-CT-2005-023044) composition of the extract from basil (Ocimium basilicum), dandelion (Taraxacum officinale), soybean (Glycine max), mint (Mentha piperica) was evaluated using hyphenated technique liquid chromatography combined with mass spectrometry LC-DAD-ESI/MSn. Applying this technique major consituents were preliminary identified or confirmed if appropriate standards were available. For basil, five phenolic constituents were identified: flavonoid (unknown), phenolic acid (unknown), chicoric acid and rosmarinic acid; minor peaks corresponding to quercetin glycosides were present. In dandelion extract cis- and tras-caftaric acids, two unknown phenolic acid, chlorogenic acid and chicoric acid were identified. Soybean extract contained 15 individual isoflavones with characteristic UV spectra. Based on their MSn their structures were confirmed. The mint phenolics contained rosmarinic acid, number of isoflavones and flavonols. Due to the fact that the structure of several compounds could not be confirmed because of their complexity and the lack of appropriate standards their preparative separation has been performed to produce reference substances

LC-DAD-ESI/MS identification of active natural products from four plants species

In our work on the project NUTRASNACK (E.C. F.P.6 contract No FOOD-CT-2005-023044) composition of the extract from basil (Ocimium basilicum), dandelion (Taraxacum officinale), soybean (Glycine max), mint (Mentha piperica) was evaluated using hyphenated technique liquid chromatography combined with mass spectrometry LC-DAD-ESI/MSn. Applying this technique major consituents were preliminary identified or confirmed if appropriate standards were available. For basil, five phenolic constituents were identified: flavonoid (unknown), phenolic acid (unknown), chicoric acid and rosmarinic acid; minor peaks corresponding to quercetin glycosides were present. In dandelion extract cis- and tras-caftaric acids, two unknown phenolic acid, chlorogenic acid and chicoric acid were identified. Soybean extract contained 15 individual isoflavones with characteristic UV spectra. Based on their MSn their structures were confirmed. The mint phenolics contained rosmarinic acid, number of isoflavones and flavonols. Due to the fact that the structure of several compounds could not be confirmed because of their complexity and the lack of appropriate standards their preparative separation has been performed to produce reference substances

Extraction and purification of active natural products from four plants species

The project NUTRASNACK (E.C. F.P.6 contract No FOOD-CT-2005-023044) has the main objective to produce by in vitro cultures secondary metabolites with health benefits, which can be used for fortification of some food products. On the of the key partner is ENERVIT®, a well-known firm in the development of nutrition programs to enhance the performances of athletes and sportsmen. For the purpose of the project a number of plant species have been selected for the study of the possibility of commercial application of their secondary metabolites. This includes basil (Ocimium basilicum), dandelion (Taraxacum officinale), soybean (Glycine max), mint (Mentha piperica) and Leguminosae species (genus Trifolium). For above species the conditions and protocols for the extraction of active principles need to be established. These protocols are aimed at the preparation of extracts for analytical purposes but also for commercial application for separation of large amounts of phytochemicals. For this purpose he extraction with different solvents and the purification of extracts by Reversed Phase C18 or Amberlite XAD4 Solid Phase Extraction. It was shown that in case of green samples two options should be considered. For samples used for analytical purposes the 70% EtOH should be a case of choice. For commercial purposes the water extract is more reasonable. Water extracts contain all active chemicals and allow to avoid chlorophyll presence in the extract. This substantially simplified the method of purification and enrichment. In case of soybean the EtOH extract is necessary as isoflavones are not so readily soluble in water. Further purification can be achieved by SPE

The ultra-performance liquid chromatography (UPLC) analysis of phenolics in four plant species

For the evaluation of the efficiency of in vitro systems and for standardization of commercial products the reliable fast analytical procedures are required. These are usually based on HPLC analysis. Regular HPLC separations are usually time and solvent consuming. The new achievements in analytical equipment allows to apply much faster technique UPLC for plant phytochemical analysis. The technology is quite new (developed in 2004) and in this respect there is no literature available. It was applied for separation of phenolics in the extracts of four plant species: basil (Ocimium basilicum), dandelion (Taraxacum officinale), soybean (Glycine max), mint (Mentha piperica) considered as a source of nutraceuticals researched under the project NUTRASNACK (E.C. F.P.6 contract No FOOD-CT-2005-023044). The Acquinity Ultra Performance Liquid Chromatograph (Waters) consisting of Binary Solvent Manager, Sample Manager, PDA detector and Empower Pro 2.0 software was used. The analyses were performed on an UPLC BEH C18 column (1.7mm, 50mm ´ 2.1mm) utilizing a gradient elution profile and a mobile phase consisting of 0,1% acetic acid in water and 40% AcN. The column was maintained at 50oC and at a flow rate was kept constant at 0.35 mL/min. The separation profiles obtained for four analysed species were of similar quality as the profiles obtained with HPLC. However, optimization of the separation conditions (water-acetonitrile gradient shape, column temperature) in UPLC allowed us to reduce separation time down to 5 min (basil, dandelion) and 6 min (mint and soybean); regular HPLC separation time was 50 min. The developed method simplified the analytical protocol and shortened the time of analysis just to few minutes. This an important achievement when big number of samples e.g. in vitro culture efficiency evaluation is necessary

Quantum dots functionalised artificial peptides bioinspired to the D1 protein from the Photosystem II of Chlamydomonas reinhardtii for endocrine disruptor optosensing

Herein we describe the design and synthesis of novel artificial peptides mimicking the plastoquinone binding niche of the D1 protein from the green photosynthetic alga Chlamydomonas reinhardtii, also able to bind herbicides. In particular, molecular dynamics (MD) simulations were performed to model in silico the behaviour of three peptides, D1Pep70-H, D1Pep70-S264K and D1Pep70-S268C, as genetic variants with different affinity towards the photosynthetic herbicide atrazine. Then the photosynthetic peptides were functionalised with quantum dots for the development of a hybrid optosensor for the detection of atrazine, one of the most employed herbicides for weed control in agriculture as well as considered as a putative endocrine disruptor case study. The excellent agreement between computational and experimental results self consistently shows resistance or super-sensitivity toward the atrazine target, with detection limits in the \u3bcg/L concentration range, meeting the requirements of E.U. legislation