1,720,970 research outputs found
Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase.
No full textWe have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized
Aptamer Microarray Development for Human Thrombin Detection: Interaction Analysis in Solution and in Solid Phase
No full textSewage monitoring for pathogenic microrganisms detection by using a low density microarray
No full textAims. Sewages are highly contaminated by numerous biological agents: bacteria, protozoa, fungi and viruses. Some of them can be very resistant to treatments and remain at high concentration in the effluent, thus representing an hazard especially in the case of water reuse for agriculture. Molecular methods are almost an important way to detect these pathogens, in particular the most applied techniques are based on protocols of nucleic acid amplification and quantification, of which the quantitative polymerase chain reaction (qPCR) is rapidly becoming established in the environmental sector for its higher sensitivity in comparison with cultural assays. Moreover the possibility in using molecular methods for simultaneous and rapid multiple detection of pathogens could be useful for risk assessment or safety purposes. This approach could be now possible using the technological advances of the DNA microarray, born for clinical analysis and based on the simultaneous qualitative analysis for multiple genera, species and strains. The large number of DNA sequences that can be spotted on a microarray together with the high specificity of binding to the immobilised sequence targets, allows the detection of a large range of microorganisms with high discriminatory ability.
Nevertheless the application of this technology requires a deep study of its sensitivity and specificity. In this work, an environmental monitoring of sewage, sampled from a Waste Water Treatment Plant (WWTP), was performed in order to detect some different viruses and bacteria frequently present: Human Adenovirus (HAdV), Norovirus GGII (NV), Hepatitis A Virus (HAV), Enterovirus (EV), Rotavirus (RV), Enterohemorrhagic E. coli (EHEC), Salmonella enterica (SE) using a low density microarray containing specific oligonucleotides sequences for each of them.
Methods. In the first phase of the study, a series of trials were performed to choose the sample treatment that was able to increase the sensitivity of molecular method. In particular 10 sewage samples (10 L) were treated with a first step based on tangential ultrafiltration followed by a second step in which the obtained eluates were further concentrated by ultracentrifugation until a final volume of 1 ml. After each step, the obtained eluates were treated with commercial kit (QIAgen) to extract viral and bacteria nucleic acids (NA) that were quantified by separate and specific qPCR reactions (Genomic Copies - GC) according to published protocols. In the second phase, an environmental monitoring was performed for 1 year from June 2013 to June 2014 by monthly sampling of 10 L of sewage from a WWTP in Northern Italy (13 samples). The concentrated samples, obtained by the previous phase chosen method, were treated to extract NA that were subsequently concentrated by speed VAC, labelled and overnight hybridised on the microarray slides. After that, a scanner read the positive signal. Parallel, aliquots of extracted NA of each samples were quantify (GC/10 L) by qPCR reactions.
Results. The data of the trials revealed that the combination of ultrafiltration-ultracentrifugation permitted to increase the mean concentration of target microorganisms of 1-2 Log in comparison with the use of only ultrafiltration method. The microarray analysis revealed the presence of HAdv, EV, EHEC and SE in tested samples, while no NV, HAV and RV were detected. In particular, 78% (10/13) of samples resulted positive for HAdV, 30% (4/13) for EV, 84% (11/13) for EHEH and 61% (8/13) for SE. The simultaneous presence in the same sample of all these 4 target was detected in 2 samples (15%), of three target in 6 samples (46%) and of two target in 2 of samples (15%). The mean concentration of positive target estimated by qPCR were 2 x 108 GC/10 L for HAdV, 4 x 108 CG/10 L for EV, 107 GC/10 L for EHEC and 3 x 105 GC/10 L for SE. These data are in according of sewage microbial concentration published by several authors (Figure 1). The absence of positive samples for NV, HAV and RV was probably due by epidemiological situation in the monitored region. The data, moreover, underlined a limit of sensitivity of the test: samples with a target concentration lower than 2,8 x 103 GC/10 L resulted negative to microarray.
Conclusion. The results of environmental monitoring were very promising for a multiple detection of pathogens in sewage confirming the possible use of microarray as a tool for screening. The major limitation of this technique was the scarce sensitivity that can be improved with specific sample treatments permitting also the purification of samples, as made in this study, by the combination of ultrafiltration and ultracentrifugation
Optical System Based on Nafion Membrane for the Detection of Ammonia in Blood Serum Samples
Full text linkThe blood ammonia (NH3) level is one of the most important hepatic biomarkers for the diagnosis and monitoring of liver pathologies and infections. In this work, we developed an optimized optical biosensing method to extract and quantify the ammonia contained in complex-matrix samples emulating the blood serum. First, the approach was tested with solutions of phosphate-buffered saline (PBS) and ammonia chloride. Then, further trials were carried out with solutions of fetal bovine serum (FBS). The ammonia was extracted from the tested samples through a customized cell, and it was optically quantified by exploiting the indophenol reaction. The extraction cell included a cation-exchange membrane in Nafion, which was chemically pre-treated through cleaning procedures of sulfuric acid and hydrogen peroxide to keep a basic pH in the ammonia solution and to avoid contaminants in the membrane. From the NH3 solution, the indophenol reaction produced light-reactive indophenol dye molecules, which were used as colorimetric indicators. Through absorbance measurements of the indophenol dye solution at 670 nm wavelength, we were able to detect and quantify the ammonia level in the samples both with a spectrophotometer and a customized miniaturized read-out system, obtaining a detection limit of 0.029 mu mol/mL
Electrochemical Biosensor for Timely Detection of Lactococcus Lactis Bacteriophage in Milk Samples
No full textMilk contaminations by Lactococcus lactis bacteriophages prevent the proper lactic fermentation, causing large economic losses in the dairy industry. This work presents a novel cost-effective biosensor for monitoring the presence of L. lactis phages in milk samples in less than 4 hours. The detection relies on the parametric variations in the electrochemical impedance spectroscopy response of the proposed biosensor. Differences of more than one order of magnitude is measured in the charge transfer resistance when the solution under test is contaminated by phages, due to the phage lytic activity on the L. lactis bacteria. The sensing approach is validated in milk samples by comparing the proposed electrochemical sensing with the optical absorbance of the solutions at 600 nm. Finally, the biosensor performance is improved by treating the milk samples with calcium chloride which enhances the phage activity, thus leading to larger shifts in the charge transfer resistance. Tests at different phage concentrations reveal minimum detecting capabilities of 105 PFU/mL of phages in milk
Ricerca di microrganismi patogeni enterici in matrici idriche mediante l'utilizzo della tecnologia del microarray
No full textObiettivi. Il monitoraggio ambientale basato sulla ricerca di indicatori rappresenta un importante strumento per la valutazione del rischio biologico nelle matrici idriche. Tuttavia per la scarsa correlabilità tra questi ed alcuni patogeni, soprattutto di natura virale, è stata suggerita anche a livello internazionale (OMS, EPA), la ricerca di 'patogeni indice' soprattutto per alcune matrici come ad esempio le acque di sorgente, quelle reflue trattate riemesse nell'ambiente e quelle destinate ad un riutilizzo agricolo o industriale. Uno dei problemi più rilevanti per tale scopo è la possibilità di avere un elevato numero di microrganismi patogeni e la difficoltà di eseguire dei test che permettano di identificarli simultaneamente. Questo approccio è ora possibile grazie ai progressi nel campo della biologia molecolare, che permettono un'analisi microbiologica più sofisticata riuscendo a risolvere molti problemi associati anche alla non colturabilità. In particolare, la "nanotecnologia" del DNA microarray, potrebbe essere applicata per valutare il rischio biologico nei campioni di acqua. L'elevato numero di sequenze sonde di DNA che possono essere inserite unitamente alla specificità di legame tra i target e queste, permettono il rilevamento simultaneo di una vasta gamma di generi, specie e ceppi microbici con elevata capacità discriminatoria. Tuttavia l'applicazione di questa tecnologia in campo ambientale richiede uno studio approfondito della sua sensibilità e specificità, nonché il confronto con le tecniche tradizionali. In questo lavoro, è stato sviluppato un oligonucleotide microarray per lo screening di microrganismi enterici (virus e batteri) legati all'inquinamento idrico: adenovirus, norovirus GGII, virus dell'epatite A, enterovirus, rotavirus, E.coli O157H7 e Salmonella enterica . Metodi. Dopo una prima fase, dove sono state identificate per ciascun microrganismo le regioni genomiche più conservate per la sintesi del microarray, sono stati allestiti una serie di test per verificare la specificità con campioni ad alto titolo per ciascun target. Quindi la metodica è stata testata con campioni di acqua reflua contaminata artificialmente per verificare la sensibilità con matrici simili a quelli "naturali". Dopo concentrazione, gli acidi nucleici sono stati contemporaneamente quantificati, mediante real time PCR, per stimare la contaminazione (copie genomiche/μl) e analizzati con la piattaforma microarray. Conoscendo la concentrazione iniziale di ciascun microrganismo ed il volume dei campioni depositato è stato possibile avere una stima della sensibilità. Risultati. I risultati dei test su campioni ad alto titolo hanno confermato la specificità della piattaforma microarray: ciascun microrganismo bersaglio è stato identificato. Il test sui campioni artificialmente contaminati hanno rivelato un limite di sensibilità media per ciascun target microbico di 1000-10000 copie genomiche/μl, corrispondente a 10^9-10^10 copie genomiche/L. Conclusioni. I dati ottenuti sono stati molto promettenti per l'applicazione della tecnica su matrici idriche dove la problematica correlazione indicatori-patogeni è ben nota e dove la concentrazione dei patogeni che si raggiunge è costantemente conforme al livello di sensibilità ottenuta, come nei reflui. Tuttavia per poterne auspicarne una sua applicazione in altre matrici come acque superficiali o potabili dove la contaminazione è molto più bassa, risulta necessario effettuare ulteriori studi per implementarne la sensibilità soprattutto nella fase di concentrazione e purificazione del campione dalla presenza di inibitori
Electrochemical Biosensor for the Monitoring of Phages of in Milk-Based Samples
No full textLactococcus lactis (LL) bacteriophage infections in milk prevent proper lactic fermentation, leading to the production of unsaleable low-quality products and great economic losses in the dairy industry. In this work, we present an innovative biosensing approach for the cost-effective detection of LL phages (PGs) in milk-based samples through electrochemical impedance spectroscopy (EIS). The detection is based on the evident parametric shifts in the charge transfer resistance and the impedance phase at 100 Hz caused by different bacteria proliferation due to PG activity. The EIS results are compared with optical absorbance measurements at 600 nm, in order to validate the proposed method. Preliminary experimental tests with filtered milk-based samples confirm the sensor capability to detect PGs at different concentrations in milk-based solutions in less than 4 h. In order to reach a higher sensitivity, we propose a new milk pretreatment adding calcium chloride (CaCl2) to the samples. The EIS results with CaCl2-treated milk evidence an enhanced PG activity, which leads to a much larger parametric shift. Lastly, the sensor is tested with CaCl2-treated milk-based samples at different PG concentrations, obtaining an enhanced performance reaching the limit of detection (LOD) of 103 PFU/mL
SPECTRA: A Novel Compact System for Surface Plasmon Resonance Measurements
Full text linkSurface plasmon resonance (SPR) is a common and useful measurement technique to perform fast and sensitive optical detection. SPR instrumentations usually comprise optical systems of mirrors and lenses which are quite expensive and impractical for point-of-care applications. In this work, we presented a novel and compact SPR device called SPECTRA, designed as a spectrophotometer add-on with a grating coupling configuration. The device is conceived as a marketable solution to perform quick SPR measurements in grating configuration without the requirement of complex instrumentation. The device can be customized either in a vertical structure to reach lower incident light angles, or in a horizontal configuration, which is suitable for SPR analysis using liquid solutions. The SPECTRA performance was evaluated through SPR measurements in typical applications. The vertical SPECTRA system was employed to detect different functionalization molecules on gold 720 nm-period grating devices. Meanwhile, the horizontal SPECTRA configuration was exploited to carry out fluid-dynamic measurements using a microfluidic cell with glycerol solutions at increasing concentrations to account for different refractive indexes. The experimental tests confirmed that the SPECTRA design is suitable for SPR measurements, demonstrating its capability to detect the presence of analytes and changes in surface properties both in static and dynamic set-ups
Screen-printed Electrochemical Biosensor for the Detection of Bacteriophage of Lactococcus Lactis for Dairy Production
Full text linkIn the dairy industry, the spreading of phages of Lactococcus lactis (LL) prevents the proper lactic fermentation, causing waste of contaminated products and economic losses. This work presents a cheap biosensing method for rapidly detecting the LL phages. The detection is based on live LL bacteria covering the sensor electrodes, whose electrical response is measured by electrochemical impedance spectroscopy (EIS). Solutions contaminated by phages induce bacteria lysis, clearly reducing the bacteria coverage over the electrodes and leading to evident parametrical shifts in the charge transfer resistance and in the impedance phase at 400 Hz. Experimental tests with laboratory contaminated samples confirm the better detection capability of screen-printed gold sensors compared to the interdigitated gold electrodes. Two measurement protocols, called spill-out and drop-in methods, are evaluated to optimize the sensor detection capability and time. The EIS results are compared with optical absorbance measurements at 600 nm, in order to validate the proposed detection method with 107-PFU/mL phages and with a detection time of about 5 h. Finally, the proposed method is tested successfully with milk-based solutions. Evident shifts between phage-contaminated and non-contaminated sensors are measured in the charge transfer resistance of more than one order of magnitude with impedance phase differences of 43°
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