Evaluation of new biorecognition elements for environmental monitoring

To date, environmental monitoring is mainly focused on traditional chemical techniques, or on the assessment of specific biomarkers. However, these analyses are affected by several limitations: mainly, they are expensive, spot-sampling and time-consuming. In order to overcome these drawbacks, new biological monitoring methods, such as biosensors and biological early warning system (BEWS) are under development. These kinds of devices, built around whole cells, enzymes and antibodies, are well-suited to cooperatively and continuously monitor the environmental conditions. The key-factor of this very promising approach is the biological sensing element. Whole cell systems and enzymes are well suited for environmental monitoring: they are able to determine the bioavailable and toxic concentration of xenobiotics, especially if the source and nature of the compound cannot be predicted. Microorganisms usually detect a broad spectrum of chemicals, and represent a good opportunity for low cost, long shelf-life, and wide range of conditions in which they can be applied. Besides, enzymes are effective when a particular kind of pollutant would be detected because is possible to fine tune their metabolic behaviour by means of protein engineering. In this work, three biological sensing elements, related to three different index of toxicity were evaluated, in order to develop new biosensors for environmental monitoring: a broad toxicity index associated to the decrease of light emission (EC50 or half effective concentration) of a bioluminescent bacterium, Vibrio fischeri, a metal toxicity connected to the metal-regulated production of a siderophore (pyoverdine) by the soil and water microorganism Pseudomonas fluorescens, and finally an index of toxicity given by PAHs, was related to the metabolization of these compounds by laccase of Trametes versicolor. One of the first step during the assessment of a new biological sensing element is the study of the effect of physical-chemical parameters. The tested physical-chemical parameters (temperature, pH, inoculum percentage (v/v) and carbon source) influenced both microbial sensible elements (V. fischeri and P. fluorescens), therefore, these sensible elements can be used in a whole-cell biosensor for in-situ application, even if the response is affected by the environmental variables. Furthermore, the light emission of V. fischeri was highly variable, although a more stable bioluminescence was obtained by means of a glucose fed-batch: this is one step towards the direct application of this system, usually tailored for laboratory assays, to estimate the broad acute toxicity directly in situ in a portable device. Regarding the interaction between P. fluorescens and Fe3+, Cu2+, and Zn2+, the minimum inhibitory concentration (MIC) and the pyoverdine critical concentration (PCC) obtained values were compared to those indicated in the WHO Guidelines for drinking water quality and in European directive 98/83/EC: MICs of Fe3+, Cu2+ and Zn2+ are always above the threshold specified, whilst PCCs are very near to the recommended thresholds for iron and copper. The PCC was not determined for zinc in the tested range of concentration and conditions. These results highlighted that this sensible element should be further investigated for the development of a biosensor able to monitor metals in the environment. The last and most promising sensing element assessed in this work was the lccβ laccase of T. versicolor. A combination of computational docking (SwissDock) and molecular biology techniques was used to generate rationally engineered laccases with increased ability to process large and persistent PAHs. These mutated isoforms were produced by heterologous expression in P. pastoris, successfully purified, and characterized by means of biochemical assays. The activity of the enzymes was initially tested and characterized with phenolic and non phenolic substrates at different pH (3.0-8.0): the best mutated enzyme F162A/L164A (M1) showed an increased specific activity (UI/mg) in comparison with the wild type, in every tested condition. This result was in agreement with those obtained by computational docking simulations (estimated free binding energy), validating the rational design approach. Moreover, decolourization assays of large aromatic dyes, used as model compounds, have shown that the mutated enzymes are reactive towards molecules with chemical structure resembling that of aromatic organic pollutants. By means of example, enzyme mutants with a larger binding pocket (e.g. M1) showed higher activity against triphenylmethane dyes (e.g. Methyl Green), especially without a mediator of the reaction (HBT), and high stability under a variety of temperature conditions (4, 22 °C, room temperature). Therefore, the best enzyme should be integrated on an appropriate transducer (e.g. electrode), and coupled to a wireless platform generating a BEWS for environmental monitoring

SERS-active metal-dielectric nanostructures integrated in microfluidic devices for ultra-sensitive label-free miRNA detection

In this work, silver decorated porous silicon membranes integrated in a polydimethylsiloxane multi-chamber microfluidic chip were functionalized with DNA-probes and used for the detection of miRNA by Surface-enhanced Raman Scattering analysis. An innovative biological protocol has been designed: the probe was divided in two short pieces that interact before and after the miRNA incubation. The optofluidic biosensor was applied for the label-free detection of miRNA sequences at in vivo concentrations

Graphene-metal nanostructures as surface enhanced Raman scattering substrates for biosensing

Multilayered structures composed by Single Layer Graphene (SLG), silver nanoparticles and polydimethylsiloxane membranes were used as SERS substrates for the analysis of porphyrins and hemoproteins (e.g. Myoglobin). The transfer process of SLG from its Cu growth substrate to the Ag-decorated polydimethylsiloxane membrane was optimized. A Limit of Detection (LOD) of 10^-8 M was found for ethanolic solutions of Rhodamine 6G and the efficient detection of porphyrins and Myoglobin, adsorbed on SLG surface, was achieved. This study evidenced the potentialities of plasmonic graphene-based chips for biosensing

Rational modification of estrogen receptor by combination of computational and experimental analysis

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known ER-ligand complexes with computational analysis, we were able to predict ER mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an IC50 value of 2 nM for the 17α−Ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated ERs instead of the wild type ER as bio-recognition element would be beneficial in an assay or biosensor.JRC.I-Institute for Health and Consumer Protection (Ispra

Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100-10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays

Detection methodologies for microRNA biomarker profiling

Owing to their pivotal role as expression regulators, microRNAs (miRNAs) have different physiological roles and can be involved in the onset and progression of several diseases. For this reason, their altered presence can be predictive of a pathological state. Furthermore, the ubiquitous presence of these short RNA sequences in basically all body tissues and fluids makes them elite candidates as biomarkers. With this concept in mind, it is fundamental to have effective, sensitive, and selective techniques to perform their accurate detection and profiling. The goal of this chapter is to summarize the pros and cons of some of the available detection approaches used for miRNA profiling, starting from the most common unamplified/amplified probe-based methods to the advanced techniques based on biosensors and bioassays