Nunzianda Frascione

Enabling fluorescent biosensors for the forensic identification of body fluids

The search for body fluids often forms a crucial element of many forensic investigations. Confirming fluid presence at a scene can not only support or refute the circumstantial claims of a victim, suspect or witness, but may additionally provide a valuable source of DNA for further identification purposes. However, current biological fluid testing techniques are impaired by a number of well-characterised limitations; they often give false positives, cannot be used simultaneously, are sample destructive and lack the ability to visually locate fluid depositions. These disadvantages can negatively affect the outcome of a case through missed or misinterpreted evidence. Biosensors are devices able to transduce a biological recognition event into a measurable signal, resulting in real-time analyte detection. The use of innovative optical sensing technology may enable the highly specific and non-destructive detection of biological fluid depositions through interaction with several fluid-endogenous biomarkers. Despite considerable impact in a variety of analytical disciplines, biosensor application within forensic analyses may be considered extremely limited. This article aims to explore a number of prospective biosensing mechanisms and to outline the challenges associated with their adaptation towards detection of fluid-specific analytes.

The use of silsesquioxane cages and phage display technology to probe silicone–protein interactions

A combination of two model approaches was used to explore the nature of silicone–protein interactions. Phage display technology was used to present a combinatorial library of peptides, in this case the phage library PhD.-12, to a series of model silicone surfaces. Solid, molecular cage silsesquioxanes were used as the models for silicone surfaces. The silsesquioxanes were octamethyloctasilsesquioxane (methyl8-T8), octaphenyloctasilsesquioxane (phenyl8-T8), octahydridooctasilsesquioxane (H8-T8) and dodecatrifluoropropyldodecasilsesquioxane (trifluoropropyl12-T12). The first two silsesquioxanes bear simple aliphatic (Me) and aromatic (Ph) pendant groups, and are simple silicone analogues. The second two silsesquioxanes have functionalised pendant groups, H and CF3CH2CH2. The panning results, using a wild-type phage as a control, show that the phage library binds to the simple aliphatic and aromatic silsesquioxanes strongly but non-specifically, and largely through the protein coat on the phage. The functionalised silsesquioxanes (H and CF3CH2CH2) are bound specifically by the phages. A statistical analysis of the DNA sequences of the strongly binding phages was carried out. The peptides that bind to H8-T8 are strongly enriched in proline content at positions 7 and 8, with enhanced histidine at positions 5 and 9, and enhanced threonine at position 11. The proline residues presumably induce a favourable conformation in the peptide for binding to the silsesquioxane surface. The enhanced histidine content is significant. It has been known for many years that imidazole has a particular affinity for electrophilic silanes. The amino acid distribution for trifluoropropyl12-T12 binding peptides is markedly different from that of H8-T8 silsesquioxane. Proline is again enhanced, but in positions 4, 11 and 12, and there are also very high concentrations of serine in positions 1, 9 and 11, and threonine in positions 1, 2 and 12. The highly polar nature of the CF3 group is likely to engage in hydrogen bonding with the OH groups of the serine and threonine side chains, accounting for the tight binding to trifluoropropyl12-T12 silsesquioxane.

Development of a biosensor for human blood: new routes to body fluid identification.

The identification of human blood at a crime scene can provide crucial information to an investigation whilst also providing a source of nuclear material which can be targeted for DNA profiling. Here, we report on the development of an immunofluorescent biosensor for the identification of human blood which has the potential to overcome the drawbacks of the current body fluid identification techniques. An antibody (Ab) raised against human erythrocytes was conjugated to fluorescent semiconductor quantum dots (QDs) by sulfhydryl chemistry. The conjugation was verified by agarose gel electrophoresis and immunohistochemistry. Incubation of liquid blood samples with the conjugated nanocrystals was shown to quench the fluorescence emission spectra in a concentration-dependent manner. A different effect was observed with unconjugated QDs incubated in blood. Full profiles were obtained from blood samples previously treated with the Ab–QDs, demonstrating that the method does not interfere with DNA profiling. To our knowledge, this is the first example of a hybrid Ab–QD sensor that has the potential to be employed for the identification of human blood. The results of this study are expected to open up a new research direction in the field of body fluid detection.

Touch DNA—The prospect of DNA profiles from cables

Metal theft in the railroad industry poses significant challenges to transport investigators. Cable sheaths left behind at crime scenes, if appropriately analysed, could provide valuable evidence in a forensic investigation, but attempts at recovering DNA are not routinely made. Experiments were set up to ascertain the success in DNA recovery from the surface of cable sheaths after deposition of (a) sweat, (b) extracted DNA and (c) fingermarks. Since investigators try to collect fingermarks and often treat the cables with cyanoacrylate fuming (CNA fuming) or wet powder suspensions (WPS) to enhance the marks this study investigated the recovery of DNA from fingermarks pre- and post-enhancement. The double-swab technique and mini-taping were compared as options to recover DNA from the cable sheaths. Results demonstrate that generally, there is no significant difference between using swabs or mini-tapes to recover the DNA from the non-porous cables (p>0.05). It was also illustrated that CNA fuming performed better than WPS in terms of subsequent recovery and profiling of DNA. CNA fuming resulted in an average increase in DNA recovered via swabbing and taping (more than 4× and 8×, respectively), as compared to no treatment, with 50% of the DNA recovered after CNA fuming generating full DNA profiles.

Fluorogenic displacement biosensors for PSA detection using antibody-functionalised quantum dot nanoparticles

Novel quantum dot conjugated immunosensors are presented for the accurate identification of seminal fluid in forensic casework. The production of a fluorescence signal upon the displacement of moderately bound quencher-labelled peptide analogues by Prostate Specific Antigen (PSA) overcomes many of the practical disadvantages associated with traditional lateral flow cartridge testing.

Development of an immunoassay for the differentiation of menstrual blood from peripheral blood.

Forensic identification of body fluids is a crucial aspect of the discipline. To date, there are no robust tests to make a distinction between menstrual blood and peripheral blood. Past techniques have fallen short of producing clear and consistent results while present mRNA techniques are still in their infancy. The aim of this study was to develop of an accurate and rapid immunoassay based identification method. Three different targets were evaluated: matrix metalloproteinase 14 (MMP14), oestrogen receptor α (ERα), and fibrinogen. Cellular or membrane bound antigens were analyzed using immunocytochemical staining while soluble antigens were analyzed by indirect ELISA. Results showed that ERα and MMP14 are present in the endometrial cells of menstrual blood but absent in peripheral blood. At a total protein concentration of 10 μg/mL or lower, fibrinogen was detected in menstrual blood but absent in peripheral. If evaluated on a larger scale, immunoassays may prove to be beneficial in discriminating menstruum and peripheral blood.

The applicability of fluorescence lifetime to determine the time since the deposition of biological stains

Age estimation of bloodstains is of importance to a forensic investigation in that it gives an indication as to when the crime was committed and can discern whether the bloodstains resulted from a single event or were deposited on different occasions. To date, there is no established method for determining the age of bloodstains and therefore this work investigates the potential of using fluorescence lifetime measurements to determine the time since the deposition of bloodstains. Experiments were carried out using human blood obtained from 6 donors. The results showed a decrease in the fluorescence lifetime within the first 91 hours of deposition, after which the fluorescence lifetime plateaued, illustrating a clear distinction between fresh and old bloodstains. The results also show the applicability of this method to stains originated from a blood/saliva mix (5 : 1 ratio). Saliva stains aged with the same parameters and conditions as blood showed no change in the fluorescence lifetime, suggesting that this phenomenon only takes place in blood and not saliva. Further analysis on test stains revealed that measuring the fluorescence lifetime over a period of time provides information on their freshness and can therefore form the basis of a valuable bloodstain dating technique.

Application of fluorescent substrates to the in situ detection of prostate specific antigen.

The forensic identification of body fluids frequently presents an important source of genetic material and investigative interpretation. However, presumptive testing techniques presently employed in the discrimination of biological fluids are subject to criticism for poor specificity, lack of fluid localisation ability and detrimental effects on DNA recovery rates. The recognition of fluid-specific biomarkers by fluorogenic substrates may provide a novel resolution to these issues but research has yet to establish any pertinent in situ fluid detection applicability. This study therefore utilises a fluorogenic substrate (Mu-HSSKLQ-AFC) specific to the seminal protein prostate specific antigen in an effort to detect human semen deposited on a number of surfaces typical to criminal investigation. The ability of fluorescent fluorogenic substrates to simultaneously identify and visualise biological fluids in situ is demonstrated for the first time, whilst the production of complete STR profiles from fluid sources is also confirmed to be completely unaffected by substrate application.

Analytical characterization of three cathinone derivatives, 4-MPD, 4 F-PHP and bk-EPDP, purchased as bulk powder from online vendors

Novel emerging drugs of abuse, also referred as new psychoactive substances, constitute an ever-changing mixture of chemical compounds designed to circumvent legislative controls by means of chemical modifications of previously banned recreational drugs. One such class, synthetic cathinones, namely β-keto derivatives of amphetamines, has been largely abused over the past decade. A number of new synthetic cathinones are detected each year, either in bulk powders/crystals or in biological matrices. It is therefore important to continuously monitor the supply of new synthetic derivatives and promptly report them. By using complementary analytical techniques (i.e. one- and two-dimensional NMR, FT-IR, GC-MS, HRMS and HPLC-UV), this study investigates the detection, identification and full characterization of 1-(4-methylphenyl)-2-(methylamino)pentanone (4-methylpentedrone, 4-MPD), 1-(4-fluorophenyl)-2-(pyrrolidin-1-yl)hexanone (4F-PHP) and 1-(1,3-benzodioxol-5-yl)-2-(ethylamino)-1-pentanone (bk-EPDP), three emerging cathinone derivatives.

Enhancing in vitro selection techniques to assist the discovery, understanding and use of inorganic binding peptides

Reflecting the increasing interest in combinatorial approaches, peptide phage display has seen an unprecedented expansion in a wide range of research areas. Its application to the discovery and analysis of metal binding peptides has opened up new research directions and largely contributed to the nanotechnology field. The rationale behind the need to identify such peptides varies depending on the final aim of the research and its application. Therefore, the possibility to modify the selection technique according to the different requirements would allow for a more systematic approach to be adopted and would ultimately provide substantial benefits. Although the standard panning method can be virtually applied to any target, its use for the identification of metal binding peptides does not provide the characteristics and the flexibility required for an efficient and tailored selection. Here we report on the development of a new panning method that can contribute to a faster, versatile and more informative analysis. Through the use of rolling-circle amplification, polymerase reaction and wild type phage, we have converted the standard selection technique into a more dynamic process in which adjustments can be evaluated and made consistently with the need of the experiment. The successfulness of the improved method is demonstrated in a number of panning experiments with different inorganic targets. The modifications applied to each selection are described and comparisons between the results obtained are made in order to extensively assess and evaluate the impact of the new process. The importance of tailoring the screening method to the specific objectives of a study is also considered. New binder sequences for the materials included in the investigation are identified; their sequences and distinctive characteristics are reported and their ability to act as templates for the nucleation of inorganic material is demonstrated and discussed.