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This paper reports on the development of a fluorescent label-free aptamer assay integrated in a lab-on-chip (LoC) system for the detection of Ochratoxin A (OTA). The detection system relies on the integration, on a single glass substrate, of an array of amorphous silicon photosensors and a long pass interferential filter. The aptamer assay, integrated into the microfluidic network, is an aptasensor having affinity versus OTA, selected as a case study. The fluorescent molecule is a "light switch" complex [Ru(phen)

A lab-on-chip system, integrating an all-glass microfluidics and on-chip optical detection, was developed and tested. The microfluidic network is etched in a glass substrate, which is then sealed with a glass cover by direct bonding. Thin film amorphous silicon photosensors have been fabricated on the sealed microfluidic substrate preventing the contamination of the micro-channels. The microfluidic network is then made accessible by opening inlets and outlets just prior to the use, ensuring the sterility of the device. The entire fabrication process relies on conventional photolithographic microfabrication techniques and is suitable for low-cost mass production of the device. The lab-on-chip system has been tested by implementing a chemiluminescent biochemical reaction. The inner channel walls of the microfluidic network are chemically functionalized with a layer of polymer brushes and horseradish peroxidase is immobilized into the coated channel. The results demonstrate the successful on-chip detection of hydrogen peroxide down to 18 μM by using luminol and 4-iodophenol as enhancer agent.

In this paper, we present the preliminary results of an ELISA-on-chip device, intended as a technological demonstrator of a novel analytical system suitable for the diagnosis and follow-up of celiac disease. The idea of the work is to combine an array of amorphous silicon photosensors with a pattern of a poly(2-hydroxyethyl methacrylate) polymer brush film, which acts as anchor for the immobilization of gliadin peptides containing the celiac disease epitopes. Recognition relies on a sandwich immunoassay between antibodies against the peptides and secondary antibodies marked with horseradish peroxidase to obtain a chemiluminescent signal. Detection is based on the measurement of photocurrent induced in the array of amorphous silicon photosensors by the chemiluminescent signal. An ad-hoc procedure has been developed in order to enable the fabrication of the photodiode array and the polymer brush pattern on the two sides of the same glass substrate ensuring the compatibility of the different technological steps. The sensitivity and the selectivity of the chip for multiplex immunoassays were demonstrated using two gliadin peptides (VEA and DEC). In particular, we found that the average amount of the bound HRP revealed by our analytical protocol is 3.5(±0.3) × 10−6 pg μm−2 and 0.85(±0.3) × 10−6 pg μm−2 for specific and non-specific interactions, respectively.

A multichannel glass-PDMS microfluidic chip functionalized with a novel aptasensor has been developed for the detection of Ochratoxin A (OTA). The aptasensor is built on a glass substrate covered with a poly(2-hydroxyethtyl methacrylate) brush layer, which is subsequently functionalized with an aptamer having high affinity towards OTA. The assay relies on an aptamer-linked immobilized sorbent assay (ALISA) using two different OTA aptamers, which, in presence of OTA, assemble to form a sandwich-like structure generating a chemiluminescent signal. The chip is combined with an array of amorphous silicon photosensors that transduce the chemiluminescent signal into an electrical signal that can be processed to detect and quantify the OTA in the sample. The successful detection of OTA has been demonstrated in standard solutions and in beer samples spiked with OTA. The results shows that the values measured for OTA, applying the ALISA assay, are comparable to those measured by the reference methods. The sensitivity of the proposed analytical method is 0.32 pA L/mg, while the LOD and the LOQ are 0.82 and 2.5 mg/L, respectively. Taking into account both the LOD of the ALISA and the method applied for OTA extraction from beer samples, we extrapolated that 1.1 g of beer sample is sufficient to detect OTA contamination under the regulatory limits.

The aim of this research was to verify the possibility of identifying and classifying maize seeds obtained from transgenic plants, in different classes according to the modification, on the basis of the concerted variation in metabolite levels detected by NMR spectra. It was possible to recognise the discriminant metabolites of transgenic samples as well as to classify non-a priori defined samples of maize. It is important to underline that the obtained results are useful to point out the metabolic consequences of a specific genic modification on a plant, without using a targeted analysis of the different metabolites, in fact it was possible to classify the seeds also without the complete assignment of the spectra. The analysis was performed by applying multivariate techniques (principal component analysis and partial least squares-discriminant analysis) to NMR data.

Metabolomics is the analysis of the concentration profiles of low molecular weight compounds present in biological fluids. Metabolites are nonpeptide molecules representing the end products of cellular activity. Therefore, changes in metabolite concentrations reveal the range of biochemical effects induced by a disease or its therapeutic intervention. Metabolomics has recently become feasible with the accessibility of new technologies, including mass spectrometry and high-resolution proton nuclear magnetic resonance, and has already been applied to several disorders. Indeed, it has the advantage of being a nontargeted approach for identifying potential biomarkers, which means that it does not require a preliminary knowledge of the substances to be studied. In this review, we summarize the main studies in which metabolomic approach was used in some allergic (asthma, atopic dermatitis) and rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus) to explore the feasibility of this technique as a novel diagnostic tool in these complex disorders.

A lab-on-chip for the diagnosis of celiac disease relying on the monitoring of patient-specific immune response to gliadin fractions has been developed. The detection is based on a chemiluminescent immunoenzymatic reaction that ensures high specificity and sensitivity. The chemiluminescent signal is monitored by hydrogenated amorphous silicon photosensors, fabricated on the same glass substrate hosting the biochemical recognition. The main challenge of the work has been the identification of the materials and the setup of the entire process that permitted the reliable fabrication of the device. Experiments performed with serum samples of rabbit immunized towards an epitope show a good specificity of the proposed technique, proving the feasibility of an integrated device for the patient-specific profiling.

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.

The copper (II) complex of amikacin in water solution at pH 5.5 was investigated by 13 C-NMR. The temperature dependence of spin–lattice relaxation rates was measured and fast exchange conditions were shown to apply. The motional correlation time of the complex was approximated by the pseudo-isotropic rotational correlation time of free amikacin in water solution ( τ c =0.17 ns at 300 K). Formation of a pseudo-tetrahedral 1:1 complex was demonstrated by relaxation rates analysis and also by UV–Vis spectrophotometry. Two amino nitrogens of amikacin, together with the amide nitrogen and the hydroxyl in the hydroxyl–aminopropyl carbonyl side chain, were assigned as the copper-binding sites and a model of the complex was built by using copper–carbon distances obtained by NMR analysis as input parameters.

We report here on the fabrication and the characterization of a new stationary phase for ultrathin layer chromatography (UTLC) based on a brush-gel polymer film obtained by cross-linking poly(glycidyl methacrylate) and di(ethylene glycole)dimethacrylate. The use of polymer brushes to design ultra thin layer chromatography plates opens interesting perspectives. Very thin polymer films can be covalently grafted on a glass surface with virtually any chemical functionality. By selecting and/or chemically modifying suitable monomers, stationary phases characterized by high affinity for specific substances can be accurately tailored. The structure of the brush gel formed on glass substrates was characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Atomic Force Microscopy (AFM) and Field-Emission Scanning Electron Microscopy (FESEM). The efficiency of poly(GMA-co-DEGDMA)-NH2 UTLC stationary phase compares very well with that of silica-gel TLC and other UTLC devices. However, the separation of a fluorescent dyes mixture with our UTLC device occurs faster and with less solvent compared to typical TLC methods. In addition the same plate can be re-used several times for multiple analyses without any reduction of the separation efficiency.

Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance.

We present an innovative thin layer chromatography plate, which integrates a linear array of amorphous silicon photodiodes for real-time qualitative and quantitative chromatographic analysis.

In this work, we present a novel thin layer chromatography system based on fluorescence detection by means of a linear array of amorphous silicon photodiodes. The photodiodes are optically coupled to a thin layer chromatography plate to monitor, in real-time, the separation of the components of a mixture during the chromatographic run. We designed a horizontal development chamber with UV transparent window and integrated eluent tank. The resulting system is extremely compact and ensures fast analysis using only small amounts of eluent. With our system, we analyzed different mixtures of fluorescent inks and other molecules such as fluorescein. Real-time data of sensors located in different position are used to detect the composition of the mixture. The various components can be determined from the time of the transit of the different species in front of the sensors. Furthermore, from the measured data it is possible to extract additional information as the transport properties of the stationary phase or the velocity profile along the run for each component of the mixture.

(13)C NMR spectroscopy associated with the use of (13)C-enriched substrates is a powerful tool to investigate intracellular metabolism because of the wealth of information contained in the distribution of isotopes in key metabolites. A new method of using (13)C label distribution measurements in carbon skeletons of metabolites to estimate metabolic fluxes through biochemical reaction networks is presented here. This method can be applied to metabolite synthesis occurring by condensation reactions of the type nA --> B, where n is the number of precursor A molecules needed to synthesize one molecule of product B. NMR isotopomer distribution analysis (NMR-IDA) involves the introduction of a (13)C-enriched precursor, and measurements of the (13)C positional enrichments at just one carbon atom position of the product B via (13)C NMR spectroscopy. Information on isotopomer distribution is obtained, and data are analyzed according to a mathematical model based on multinomial probability expressions to obtain the best fit between theoretical and experimental (13)C label distribution. The use of the NMR-IDA method allows for estimation of two key parameters representing the fractional flux of (13)C-enriched tracer A molecules to total precursor A pool and the fraction of product B synthesized in the presence of a (13)C-enriched source, respectively. A practical example of NMR-IDA application to fatty acid synthesis from [(1,2 (13)C(2))acetyl]-L-carnitine in cultured primary astrocytes is also presented.

Metabolic changes in the rabbit lens have been studied by means of nuclear magnetic resonance spectroscopy. These changes have been induced by prolonged topical treatment with dexamethasone. Our results demonstrate an increase in sorbitol, sorbitol-3-phosphate, fructose-3-phosphate, glycerol-3-phosphate and glucose-6-phosphate levels and a decrease in glutathione sulphate (GSH) and myo-inositol levels, in agreement with what was observed in lenses from streptozocin-diabetic rats before lens opacity. The hyperglycaemia can only partially explain all these observed biochemical variations. The lack of increase in the intermediates of pentose cycle, such as sedoheptulose-7-phosphate, seems to support the hypothesis of an inhibition of glucose-6-phosphate dehydrogenase by dexamethasone treatment. Finally dexamethasone treatment induces a decrease in GSH. The decreasing or the loss of GSH has been suggested as a possible pathogenic mechanism in the cataract formation.

Two naphthyridines interacting with Torpedo californica acetylcholinesterase (AChE) were investigated. 1 H NMR spectra were recorded and nonselective, selective, and double-selective spin–lattice relaxation rates were measured. The enhancement of selective relaxation rates could be titrated by different ligand concentrations at constant AChE (yielding 0.22 and 1.53 mM for the dissociation constants) and was providing evidence of a diverse mode of interaction. The double-selective relaxation rates were used to evaluate the motional correlation times of bound ligands at 34.9 and 36.5 ns at 300 K. Selective relaxation rates of bound inhibitors could be interpreted also in terms of dipole–dipole interactions with protons in the enzyme active site.

The interaction between Na-DNA and divalent metal cations (Mg2+, Mn2+, Co2+ and Ni2+) has been investigated by 23Na NMR relaxation. The experimental data have been interpreted in terms of Manning's counterion condensation theory and a two-site exchange for the sodium ions. The experiments showed that Mn2+ affects the polyion surface atmosphere in a different way from the other metal ions. This behaviour may be due to sugar puckering transition of the DNA induced by Mn2+.

The biochemical pathways involved in acetyl-l-carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4-h [(1,2-13C2)acetyl]-l-carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl-l-carnitine from tissues was observed. [(1,2-13C2)Acetyl]-l-carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3-hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2-13C2)acetyl]-l-carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C-labelling pattern in 3-hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl-l-carnitine-derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra- and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA-activated mitochondria-derived ketone bodies into 3-hydroxy-3-methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.

The effect of dexamethasone on lipid composition of Epstein-Barr virus transformed human B lymphocytes have been investigated by 31P- and 1H-NMR spectroscopy and compared to the effects due to exogenous sphingosine treatment. Furthermore, the effects of dexamethasone and sphingosine on membrane structure was evaluated by fluorimetry. No significant changes were evidenced in phospholipid composition and in the ratio of unsaturated to total fatty-acid chains. A significant increase in total cholesterol levels was evident at 30 min incubation with dexamethasone or sphingosine; a parallel increase in DPH polarization at 30 min was also demonstrated. TMA-DPH intensity measurements suggest a slowing of vesicular intracellular traffic due to the treatment. The results suggest a dexamethasone- and sphingosine-dependent inhibition of intracellular cholesterol transport.