Browsing by Author "Mascini, Marco"
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Item Open Access Approaches to allergy detection using aptasensors(Taylor and Francis, 2007) Brys, Emile; Tombelli, Sara; Minunni, Maria E.; Mascini, Marco; Turner, Anthony P. F.1. Immunoglobulins and IgE Antibodies are glycoprotein molecules which are produced by plasma cells in response to an immunogen (1). They belong to a class of spherical proteins called globulins and are hence known as immunoglobulins (Igs). Their primary function is to mediate the host immune response by binding to antigens.Item Open Access Aptamers for biosensors(Cranfield University and University of Firenze., 2008) Bini, Alessandra; Mascini, Marco; Turner, Anthony P. F.Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface Plasmon Resonance as transducers. The work was focused on two case studies, i.e. the Thrombin-binding aptamer and the aptamer against C-Reactive Protein. From an analytical point of view, the work was devoted to the optimisation of the analytical performance of a piezoelectric and an optical aptasensor for Thrombin and C-Reactive Protein detection, respectively. Efforts towards the application of these aptasensors in complex matrices, such as human plasma and serum, were also undertaken, in order to demonstrate the wide applicability of aptamers, as an alternative to antibodies. In this work, the possibility of introducing a computationally-assisted method to study aptamer-protein interaction and aptamer selection was also evaluated. For this purpose, the Thrombin-binding aptamer was chosen as a model and a retrospective docking study was performed by comparing the affinity of mutated sequences for thrombin with that of the Thrombin-binding aptamer, on the basis of a computationally-derived binding score. Finally, the reliability of computational results was tested by experimental measurements. For this purpose, the Thrombin-binding aptamer and other mutated sequences, selected on the basis of their binding score, were employed for the development of optical biosensors and the resulting analytical performances were compared. Even if further studies should be carried out in order to validate the proposed computational approach to aptamer selection, this work can have a significant impact on future aptamers selection for sensors and diagnostics.Item Open Access Detection of TP53 mutation using a portable Surface Plasmon Resonance DNA-based biosensor(Elsevier, 2005-02-02T17:36:22Z) Jiang, Tieshan; Minunni, Maria E.; Wilson, P. K.; Zhang, Jian; Turner, Anthony P. F.; Mascini, MarcoA DNA-based Surface Plasmon Resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETATM SPR-EVM-BT, from Texas Instruments. A direct immobilization procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridization reactions between the immobilized probe and a short sequence (26 mer) complementary, non-complementary and one point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR) amplified real samples, DNA extracted from both normal, wild type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base and hence it appears to be a strong candidate technique for the detection of gene mutation.Item Open Access Evaluation of an FIA Operated Amperometric Bacterial Biosensor, Based on Pseudomonas Putida F1 for the Detection of Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX)(Taylor and Francis, 2005) Rasinger, Josef D.; Marrazza, Giovanna; Briganti, Fabrizio; Scozzafava, Andrea; Mascini, Marco; Turner, Anthony P. F.Recently, the development and optimization of a flow injection analysis (FIA) operated bacterial biosensor based on the aerobic catabolism of Pseudomonas putida ML2 was reported in the literature (Lanyon et al. 2004, 2005). By adapting information from these reports, we investigated whether operating parameters and procedures of the benzene biosensor could be directly applied to a new system based on a different bacterial strain for the detection of the whole benzene, toluene, ethylbenzene, and xylenes range. Cells of the investigated bacterial strain, Pseudomonas putida F1, were immobilized between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen electrode. The P. putida F1 aerobically degrades benzene, toluene, and ethylbenzene (BTE) (Cho et al. 2000). The BTE biosensor in kinetic mode FIA displayed a linear range of 0.02-0.14 mM benzene (response time: 5 min, base-line recovery time: 15 min), 0.05-0.2 mM toluene (response time: 8 min, baseline recovery time: 20 min), and 0.1-0.2 mM ethylbenzene (response time: 12 min, baseline recovery time: 30 min), respectively. Due to the differences in sensitivity, response, and baseline recovery times for BTE, it was possible to differentiate each compound in mixtures of these volatile organic compounds (VOCs). No response for xylenes could be obtained since they cannot be completely metabolized by this bacterial strain. However, it was reported that the range of compounds degradable by P. putida F1 can possibly be expanded by cultivating the cells on different carbon sources (Choi et al. 2003). The sensor showed good intra- and interassay reproducibility, and all obtained results were comparable with those reported in the literature. The demonstrated reproducibility and the simplicity and ease of use as well as the portability for in situ measurements indicates that the biosensor could be suitable as a reliable initial warning device for elevated BTE levels in indoor and outdoor environments.Item Open Access Improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals(Elsevier, 2002-12) Tombelli, S.; Mascini, Marco; Turner, Anthony P. F.The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide).The four coating methods were all based on the use of self-assembled monolayers. Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe.Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step.Item Open Access A novel optical chip for affinity biosensors based on fluorescence anisotropy(Cranfield University, 2008-07) Carloni, Adolfo; Mascini, Marco; Turner, Anthony P. F.The subject of this thesis relates to the realisation of a novel biochip for sensors based on optical principles. In particular, affinity biosensors for antigens (IgG/anti-IgG), for a transcription factor, and for mRNA were studied and developed. The interrogation of the biosensors implied the development of a novel and innovative optical platform based on fluorescence which was designed, implemented and thoroughly characterised. The biosensors developed were integrated within an optical biochip connected to an innovative platform. The optical biochip was fabricated from polymethylmethacrylate (PMMA) formed by two pieces of PMMA complementary shaped in order to obtain four micro-channels. The lower part included the micro-channels and the inlet and outlet for the fluidic, while the sensing biolayer was immobilised on the upper part. The optical signal comprised the fluorescence emitted by the layer, which was anisotropically coupled to the cover and suitably guided by the chip. Several chemical treatments of the surface were investigated to obtain the most effective distribution of carboxylic groups for the covalent immobilisation of antibodies. Deposition of the polymers, in particular by Langmuir-Blodgett method, can enhance the performances of the chip going through lower detection limits. The potential of the optical chip as a biosensor was investigated in depth by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. Following this, bioassays for the determination of the NF-kB transcription factor and for the mRNA that codifies for MGMT protein were implemented on the optical platform. The development and characterisation of the biosensors, the identification of the protocol for the bioassays and the design and characterisation of the optical platform were performed at the Institute of Physics Applied Carrara of National Research Council (IFAC-CNR), Firenze, Italy. The research devoted to the development of biosensing surfaces for the realisation of affinity biosensors able to detect and quantify antigens, transcription factors and RNA sequences were carried out at the laboratories of the Institute of Clinical Physiology of National Research Council (IFC-CNR), in Pisa, Italy. Langmuir-Blodgett deposition of polymers, Atomic Force Microscopy imaging and further fluorescence measurements of proteins covalently bound on the surfaces were performed at Cranfield University in Silsoe, England. The overall project starts from a first characterisation of the optical system, and comprehend the enhanced optimisation of the biochip performances until the realisation of the biosensors previously mentioned.Item Open Access A novel optical biosensor format for the detection of clinically relevant TP53 mutations(Elsevier, 2005-05-15) Wilson, P. K.; Jiang, T.; Minunni, Maria E.; Turner, Anthony P. F.; Mascini, MarcoThe TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However, the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta™ chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes, which had not hybridised to the target, resulting in a three-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.Item Open Access Surface plasmon resonance imaging for affinity-based biosensors(Elsevier, 2010-01-15) Scarano, Simona; Mascini, Marco; Turner, Anthony P. F.; Minunni, Maria E.SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity.