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Title: Biosensors for the forensic detection of body fluids
Author: Gooch, James Philip
ISNI:       0000 0004 8500 0543
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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Body fluids may be considered the most important form of trace evidence within forensic analysis. The detection and identification of fluids, such as blood, semen and saliva, can often help advance criminal investigations by providing information on the nature of an offence, as well as a source of genetic material that may be used to construct a scientific link between an individual and a crime through DNA profiling. However, current techniques used to locate and attribute the identity of fluids deposited at crime scenes are often disadvantaged by issues of low analytical specificity, detrimental effects on DNA recovery and an inability to be performed simultaneously as part of multiplex assays. This project therefore explores the use of fluorescent biosensors as novel strategies for the detection and identification of body fluids. Such sensors may have the potential to reduce overall fluid screening times by producing 'turn-on' fluorescence emissions in response to specific biological interactions with intra-fluidic targets. As previous investigations into the ability of biosensors to detect analytes in situ remain limited, efforts were first made to establish the fluorescent sensing mechanisms exhibiting greatest potential for use as novel fluid analysis strategies. This was achieved by assessing the performance of four commercially available biosensing assays against targets deposited on a range of surfaces commonly encountered within forensic casework. With biosensors based on enzymatic substrate digestion demonstrating ideal in situ detection ability during commercial assay testing, explorations were made into the application of previously reported fluorogenic peptide substrates specific to prostate specific antigen (PSA), kallikrein 8 and thrombin towards the detection of semen, saliva and blood respectively. Attempts to improve upon these assays were then conducted through the construction of custom substrates containing rhodamine-110 and cresyl violet fluorophores, using a novel solid-phase peptide synthesis route. A displacement immunosensor specific to PSA was also developed via the conjugation of anti-PSA antibodies to highly-fluorescent quantum dot nanoparticles. Emissions from this antibody-nanoparticle complex were initially quenched through the moderate binding of a quencher-labelled peptide analogue to PSA. In the presence of the native PSA protein, this analogue is displaced, relieving quenching effects and restoring quantum dot fluorescence. This immunosensor complex was subsequently used to successfully detect nanomolar amounts of PSA within solution. Attempts were also made in the construction of a multiplex fluid detection biosensor based on the displacement of fluorescently-labelled aptamers from graphene oxide. In this assay, emissions from three single-stranded DNA sequences, each labelled with a different fluorophore, were initially quenched upon adsorption to the surface of graphene nanosheets. These sequences, specific to the proteins thrombin, PSA and lysozyme, are then individually displaced as a result of target binding, producing emissions at particular wavelengths to indicate the type of target present. Efforts to increase the specificity of this aptasensor were then undertaken through the selection of novel aptamer sequences towards human sperm cells. Analysis of obtained aptamer pools using next generation sequencing and a novel bioinformatics pipeline successfully identified a number of promising sperm-cell binding ligands. Forensic taggants, materials containing unique coding elements used for the monitoring of contact transfer during a criminal offence, were lastly developed using the fluorophore and peptide components employed for fluorogenic substrate construction. These materials exploit the particular amino acid sequence of incorporated peptides as identifiable 'codes' that may be registered to a specific taggant formulation. It is hoped that such components will eventually allow for the production of 'reactive' taggants containing biosensor molecules, which become fluorescent as a result of handling contact.
Supervisor: Frascione, Nunzianda ; Daniel, Barbara Elizabeth Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available