The Development and Characterisation of ssDNA Aptamers via a Modified Cell-SELEX Methodology for the Detection of Human Red Blood Cells.

Blood is one of the most commonly found biological fluids at crime scenes, with the detection and identification of blood holding a high degree of evidential value. It can provide not only information about the nature of the crime but can also lead to identification via DNA profiling. Presumptive tests for blood are usually sensitive but not specific, so small amounts of the substrate can be detected, but false-positive results are often encountered, which can be misleading. Novel methods for the detection of red blood cells based on aptamer-target interactions may be able to overcome these issues. Aptamers are single-stranded DNA or RNA sequences capable of undergoing selective antigen association due to three-dimensional structure formation. The use of aptamers as a target-specific moiety poses several advantages and has the potential to replace antibodies within immunoassays. Aptamers are cheaper to produce, display no batch-to-batch variation and can allow for a wide range of chemical modifications. They can help limit cross-reactivity, which is a hindrance to current forensic testing methods. Within this study, a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process was used to generate aptamers against whole red blood cells. Obtained aptamer pools were analysed via massively parallel sequencing to identify viable sequences that demonstrate a high affinity for the target. Using bioinformatics platforms, aptamer candidates were identified via their enrichment profiles. Binding characterisation was also conducted on two selected aptamer candidates via fluorescent microscopy and qPCR to visualise and quantify aptamer binding. The potential for these aptamers is broad as they can be utilised within a range of bioassays for not only forensic applications but also other analytical science and medical applications. Potential future work includes the incorporation of developed aptamers into a biosensing platform that can be used at crime scenes for the real-time detection of human blood.

Accumulation of endogenous and exogenous nucleic acids in "Touch DNA" components on hands.

Successful forensic DNA profiling from handled items is increasingly routine in casework. This "touch DNA" is thought to contain both cellular and acellular nucleic acid sources. However, there is little clarity on the origins or characteristics of this material. The cellular component consists of anucleate, terminally differentiated corneocytes (assumed to lack DNA), and the occasional nucleated cell. The acellular DNA source is fragmentary, presumably cell breakdown products. This study examines the relative contributions each component makes to the hand-secretions (endogenous) and hand-accumulations (exogenous) by recovering rinses from the inside and outside of worn gloves. Additionally, cellular and acellular DNA was measured at timepoints up to 2 h after hand washing, both with and without interim contact. Microscopic examination confirmed cell morphology and presence of nucleic acids. Following the novel application of a hair keratinocyte lysis method and plasma-DNA fragment purification to hand rinse samples, DNA profiles were generated from both fractions. Exogenous cell-free DNA is shown to be a significant source of touch DNA, which reaccumulates quickly, although its amplifiable nuclear alleles are limited. Endogenous DNA is mostly cellular in origin and provides more allelic information consistently over time.

Generating aptamers towards human sperm cells using massively parallel sequencing.

Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection.

The use of the M-Vac® wet-vacuum system as a method for DNA recovery.

Collecting sufficient template DNA from a crime scene sample is often challenging, especially with low quantity samples such as touch DNA (tDNA). Traditional DNA collection methods such as double swabbing have limitations, in particular when used on certain substrates which can be found at crime scenes, thus a better collection method is advantageous. Here, the effectiveness of the M-Vac® Wet-Vacuum System is evaluated as a method for DNA recovery on tiles and bricks. It was found that the M-Vac® recovered 75% more DNA than double swabbing on bricks. However, double swabbing collected significantly more DNA than the M-Vac® on tiles. Additionally, it was found that cell-free DNA is lost in the filtration step of M-Vac® collection. In terms of peak height and number of true alleles detected, no significant difference was found between the DNA profiles obtained through M-Vac® collection versus double swabbing of tDNA depositions from 12 volunteers on bricks. The results demonstrate that the M-Vac® has potential for DNA collection from porous surfaces such as bricks, but that alterations to the filter apparatus would be beneficial to increase the amount of genetic material collected for subsequent DNA profiling. These results are anticipated to be a starting point to validate the M-Vac® as a DNA collection device, providing an alternative method when DNA is present on a difficult substrate, or if traditional DNA collection methods have failed.

Monitoring Criminal Activity through Invisible Fluorescent "Peptide Coding" Taggants.

Complementing the demand for effective crime reduction measures are the increasing availability of commercial forensic "taggants", which may be used to physically mark an object in order to make it uniquely identifiable. This study explores the use of a novel "peptide coding" reagents to establish evidence of contact transfer during criminal activity. The reagent, containing a fluorophore dispersed within an oil-based medium, also includes a unique synthetic peptide sequence that acts as a traceable "code" to identify the origin of the taggant. The reagent is detectable through its fluorescent properties, which then allows the peptide to be recovered by swabbing and extracted for electrospray ionization-mass spectrometry (ESI-MS) analysis via a simple liquid-liquid extraction procedure. The performance of the reagent in variable conditions that mimic the limits of a real world use are investigated.

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.

Analysis of drugs seized from amnesty bins at two major United Kingdom summer music festivals using two portable gas chromatography-mass spectrometry (GC-MS) instruments.

Globally, the number of drug users and the proportion of the drug using population has increased from 210 million in 2009 to 269 million in 2019. Several studies suggest that music festival attendees are more likely to abuse illicit substances and have a high-risk profile. Consequently, it is crucial to develop robust field drug analysis methods that facilitate harm reduction and drug monitoring. The work presented in this report aimed at developing and validating qualitative analytical methods for 3,4-methylenedioxymethamphetamine, 4-bromo-2,5-dimethoxyphenethylamine (2C-B), ketamine and N-ethylpentylone on two portable gas chromatography-mass spectrometry (GC-MS) systems: Griffin G510 (Teledyne FLIR, West Lafayette, IN) and Torion T-9 (PerkinElmer, Shelton, CT). The diagnostic ability of the mobile GC-MS units was assessed on 200 samples in total, seized at two large summer music festivals in the United Kingdom. The method validation process included selectivity/specificity, limit of identification, carry-over, ruggedness/robustness, and inter- and intra-day precision (repeatability and reproducibility). The Griffin G510 demonstrated a limit of identification from 1 mg/mL for 2C-B to 0.063 mg/mL for ketamine and good ruggedness and precision results. The precision for 2C-B using the Torion T-9 was poorer than for the Griffin G510, but equivalent for the other compounds tested. Correct identifications (versus benchtop GC-MS) for the two festivals were 85%-86% and 74%-83% for the Griffin G510 and the Torion T-9, respectively. The two portable instruments were able to adequately cover current on-site drug-testing analytical gaps and proved to be a powerful addition to the on-site drug analysis techniques.

Assessment of a Single Quadrupole Mass Spectrometer Combined with an Atmospheric Solids Analysis Probe for the On-Site Identification of Amnesty Bin Drugs.

The surging number of people who abuse drugs has a great impact on healthcare and law enforcement systems. Amnesty bin drug analysis helps monitor the "street drug market" and tailor the harm reduction advice. Therefore, rapid and accurate drug analysis methods are crucial for on-site work. An analytical method for the rapid identification of five commonly detected drugs ((3,4-methylenedioxymethamphetamine (MDMA), cocaine, ketamine, 4-bromo-2,5-dimethoxyphenethylamine, and chloromethcathinone)) at various summer festivals in the U.K. was developed and validated employing a single quadrupole mass spectrometer combined with an atmospheric pressure solids analysis probe (ASAP-MS). The results were confirmed on a benchtop gas chromatography-mass spectrometry instrument and included all samples that challenged the conventional spectroscopic techniques routinely employed on-site. Although the selectivity/specificity step of the validation assessment of the MS system proved a challenge, it still produced 93% (N = 279) and 92.5% (N = 87) correct results when tested on- and off-site, respectively. A few "partly correct" results showed some discrepancies between the results, with the MS-only unit missing some low intensity active ingredients (N-ethylpentylone, MDMA) and cutting agents (caffeine, paracetamol, and benzocaine) or detecting some when not present. The incorrect results were mainly based on library coverage. The study proved that the ASAP-MS instrument can successfully complement the spectroscopic techniques used for qualitative drug analysis on- and off-site. Although the validation testing highlighted some areas for improvement concerning selectivity/specificity for structurally similar compounds, this method has the potential to be used in trend monitoring and harm reduction.

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.

Nanomaterials for optical biosensors in forensic analysis.

Biosensors are compact analytical devices capable of transducing a biological interaction event into a measurable signal outcome in real-time. They can provide sensitive and affordable analysis of samples without the need for additional laboratory equipment or complex preparation steps. Biosensors may be beneficial for forensic analysis as they can facilitate large-scale high-throughput, sensitive screening of forensic samples to detect target molecules that are of high evidential value. Nanomaterials are gaining attention as desirable components of biosensors that can enhance detection and signal efficiency. Biosensors that incorporate nanomaterials within their design have been widely reported and developed for medical purposes but are yet to find routine employment within forensic science despite their proven potential. In this article, key examples of the use of nanomaterials within optical biosensors designed for forensic analysis are outlined. Their design and mechanism of detection are both considered throughout, discussing how nanomaterials can enhance the detection of the target analyte. The critical evaluation of the optical biosensors detailed within this review article should help to guide future optical biosensor design via the incorporation of nanomaterials, for not only forensic analysis but alternative analytical fields where such biosensors may prove a valuable addition to current workflows.

Fingermarks in wildlife forensics: A review.

Wildlife forensics is defined as providing forensic evidence to support legal investigations involving wildlife crime, such as the trafficking and poaching of animals and/ or their goods. While wildlife forensics is an underexplored field of science, the ramifications of poaching can be catastrophic. The consequences of wildlife crime include disease spread, species and habitat loss, human injury, and cultural loss. Efforts to use forensic science to combat poaching are currently limited to DNA-based techniques. However, fingermark analysis for the identification of perpetrators of wildlife crimes has not been explored to the same extent, despite being a cost-effective, simple-to-use forensic method that is easy to deploy in-field. This review covers literature that has explored fingermark examination techniques used on wildlife-related samples, such as pangolin scales, ivory-based substances, bone, and eggs, as well as feathers and skins, among more obscure trafficked items. Useful preliminary work has been conducted in this subject area, demonstrating that commonly used fingermark analysis techniques can be applied to wildlife-based items. However, many of these studies suffer from limitations in terms of experimental design. More work should be done on creating studies with larger sample sizes and novel approaches should be validated under environmental conditions that mimic real crime scenes. Further research into determining the forensic fingermark analysis techniques that perform the most efficiently in the environmental conditions of the countries where they are needed would therefore benefit legal investigations and help to reduce instances of poaching.

Detection and identification of body fluid stains using antibody-nanoparticle conjugates.

Body fluids are considered one of the most important evidence types in forensic casework. The presence and location of blood, semen and saliva can provide crucial information to investigators. Current practice relies on an accurate visual examination followed by the use of presumptive tests to determine the identity of the body fluid type. Further laboratory based tests are required to unequivocally confirm the identity of a stain. Body fluid stains can be difficult to detect with the naked eye, particularly on dark backgrounds and hence vital evidence may be overlooked. Current methods are fluid-type specific, with a separate, and different, test required for each body fluid. The laborious nature of such analysis and the impossibility of being carried out at the crime scene, leads to a delay in the investigation process that could prove detrimental to the solving of the case. Hence, there is a need for sensitive, specific and direct methods which can simultaneously detect, differentiate, and locate human fluids on items of forensic evidence. Here, we describe the preparation of functionalized iron oxide nanoparticles conjugated to antibodies specific to blood and saliva components and their use in detecting small traces against non-contrasting substrates including glass, ceramic tile, paper and black fabric. The advantage of our technique is that it can simultaneously detect blood and saliva and can spatially locate and differentiate these body fluid types. Most importantly, our technology, which exploits the superparamagnetic properties of iron oxide nanoparticles, works in situ with no need to remove the body fluid stains for testing and with no washing steps and does not interfere with downstream DNA profiling. Thus, our technology represents a novel and effective alternative to existing methods.

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.

Unlocking the potential of forensic traces: Analytical approaches to generate investigative leads.

Forensic investigation involves gathering the information necessary to understand the criminal events as well as linking objects or individuals to an item, location or other individual(s) for investigative purposes. For years techniques such as presumptive chemical tests, DNA profiling or fingermark analysis have been of great value to this process. However, these techniques have their limitations, whether it is a lack of confidence in the results obtained due to cross-reactivity, subjectivity and low sensitivity; or because they are dependent on holding reference samples in a pre-existing database. There is currently a need to devise new ways to gather as much information as possible from a single trace, particularly from biological traces commonly encountered in forensic casework. This review outlines the most recent advancements in the forensic analysis of biological fluids, fingermarks and hair. Special emphasis is placed on analytical methods that can expand the information obtained from the trace beyond what is achieved in the usual practices. Special attention is paid to those methods that accurately determine the nature of the sample, as well as how long it has been at the crime scene, along with individualising information regarding the donor source of the trace.

The classification of handwriting features of the Kazakh language written in Latin script.

In 2017, the Republic of Kazakhstan began the phased transition of its alphabet from Cyrillic to Latin script. This transition has presented significant challenges to Kazakhstani document examiners, who have yet to develop appropriate methodologies for the analysis of handwriting samples written in the Kazakh language using Latin letters. This study aims to identify distinguishing macro and micro features of letters within Kazakh writing samples produced using the Latin alphabet and determine their frequencies of occurrence and discriminating power indices. Micro features were examined using the four most frequently appearing letters: "a", "y", "e" and "n". A comparative analysis of tested Latin letters with those of a similar configuration in Cyrillic demonstrated differences in the number of distinguishing features, as well as in the frequency of occurrence and discriminating power indices of similar features. These results show that separate statistical bases should be used for Latin and Cyrillic letters when analysing handwriting samples based on the frequencies of occurrence of micro and macro writing features.

Technical Note: Lysis and purification methods for increased recovery of degraded DNA from touch deposit swabs.

Touch deposits are a routine yet challenging sample type in forensic casework and research. Recent work investigating their contents has indicated corneocytes to be the major cellular constituent while cell-free DNA is present at significant levels. Prolonged incubation including a reducing agent such as DTT has been shown to lyse corneocytes; a plasma cfDNA recovery kit which targets shorter DNA fragments has been demonstrated to improve cfDNA recovery from hand rinses. Herein these methods are combined and tested on mock casework touch deposit swabs from communal surface areas. Both fluorescence- and qPCR-based quantification methods are used and their results compared to query DNA degradation levels. Both proposed lysis and purification methods demonstrate increased recovery of DNA detectable with fluorescence quantification and some additional alleles at short loci, indicating high levels of fragmented DNA in these samples.

3,4-Methylenedioxymethamphetamine quantification via benchtop 1H qNMR spectroscopy: Method validation and its application to ecstasy tablets collected at music festivals.

We describe a method validation for the quantification of 3,4-methylenedioxymethamphetamine (MDMA) in tablets based on the United Nations Office on Drugs and Crime (UNODC) guideline for quantitative Nuclear Magnetic Resonance analysis (qNMR). qNMR experiments were carried out on a 60 MHz benchtop NMR spectrometer employing ethylene carbonate as an internal calibrant. A series of 'ecstasy' tablets seized at music events were quantified and the results discussed regarding their within-batch variation and yearly median dose. The method showed good specificity and selectivity, with linearity, precision, accuracy, and recovery well within the UNODC recommended criteria. The limit of detection and quantification are 0.33 mg/mL and 0.10 mg/mL respectively, proving the method works well on small amounts of MDMA. Overall, the lowest amount of MDMA free base detected in this study was 9.35 mg in a piperazine mix, while the highest dosed tablet contained 237.55 mg MDMA free base, with a 9.1% decrease in median amount compared to the pre-pandemic data (2019), but still higher than the data collected in a previous study (105 mg median amount of MDMA free base in 2018). The within-batch variation was insignificant for one of the seizures but showed greater variation for the other, which confirmed that the MDMA content of a single tablet may not reflect that of the whole batch. This dynamic upward change in tablet dosage highlights the importance of ongoing trend monitoring and specific prevention intervention to counteract the negative consequences associated with MDMA use. Benchtop NMR has been successfully employed in quality control, material science and more recently, drug analysis. The present study demonstrates its beneficial application in forensic science overcoming the limitations of currently available instruments and techniques employed in harm reduction and field testing.

Profiling and imaging of forensic evidence - A pan-European forensic round robin study part 1: Document forgery.

The forensic scenario, on which the round robin study was based, simulated a suspected intentional manipulation of a real estate rental agreement consisting of a total of three pages. The aims of this study were to (i) establish the amount and reliability of information extractable from a single type of evidence and to (ii) provide suggestions on the most suitable combination of compatible techniques for a multi-modal imaging approach to forgery detection. To address these aims, seventeen laboratories from sixteen countries were invited to answer the following tasks questions: (i) which printing technique was used? (ii) were the three pages printed with the same printer? (iii) were the three pages made from the same paper? (iv) were the three pages originally stapled? (v) were the headings and signatures written with the same ink? and (vi) were headings and signatures of the same age on all pages? The methods used were classified into the following categories: Optical spectroscopy, including multispectral imaging, smartphone mapping, UV-luminescence and LIBS; Infrared spectroscopy, including Raman and FTIR (micro-)spectroscopy; X-ray spectroscopy, including SEM-EDX, PIXE and XPS; Mass spectrometry, including ICPMS, SIMS, MALDI and LDIMS; Electrostatic imaging, as well as non-imaging methods, such as non-multimodal visual inspection, (micro-)spectroscopy, physical testing and thin layer chromatography. The performance of the techniques was evaluated as the proportion of discriminated sample pairs to all possible sample pairs. For the undiscriminated sample pairs, a distinction was made between undecidability and false positive claims. It was found that none of the methods used were able to solve all tasks completely and/or correctly and that certain methods were a priori judged unsuitable by the laboratories for some tasks. Correct results were generally achieved for the discrimination of printer toners, whereas incorrect results in the discrimination of inks. For the discrimination of paper, solid state analytical methods proved to be superior to mass spectrometric methods. None of the participating laboratories deemed addressing ink age feasible. It was concluded that correct forensic statements can only be achieved by the complementary application of different methods and that the classical approach of round robin studies to send standardised subsamples to the participants is not feasible for a true multimodal approach if the techniques are not available at one location.

Exploration of cell-free DNA (cfDNA) recovery for touch deposits.

Although touch deposit DNA is widely used in forensic casework, its cellular and acellular contents and their biological origins are poorly understood. There is evidence that the cell-free component of DNA deposited by handling may contribute substantial genetic information; however, most research into touch DNA recovery does not separate cellular and cell-free fractions or seek to characterize their contents. This work is an important early step in developing methods to isolate the cfDNA from biological material deposited by handling. Size-filtration as a separation technique was determined to be prone to DNA loss, even on optimized control samples of pure ladder DNA. Centrifugal separation was optimized to determine minimum speed and time required to reliably remove all cellular debris from the material collected by rinsing donor hands. To determine if the centrifugal force risked rupturing shed corneocyte cells and releasing cellular DNA into the supernatant, DNA levels were measured, and cells were visualized microscopically before and after centrifugation of hand rinses. Heated buccal cells were used as a positive control to demonstrate cell rupture would be detected with these methods. Following the determination of a suitable separation technique, an investigation into purification methods for cfDNA was conducted. DNA recovery using three kits for plasma cfDNA, one for PCR clean-up and one for genomic DNA were assessed on both ladder DNA to simulate cfDNA fragments and on collected hand deposit supernatants from both unwashed and washed hands. Purification methods designed for recovery of short DNA fragments from plasma yielded the highest recovery percentage across sample types, with BioChain cfPure performing the best. Donors' hands were shown to shed high levels of cfDNA, which were better recovered with a method for short fragments than with a traditional genomic technique often used on touch DNA samples.

The forensic investigation of vehicle-pedestrian collisions: A review.

Rapid urbanisation, a steady increase in the number of vehicles, speeding, negligence in road safety, and other factors have led to the inevitable worldwide growth of road traffic accidents involving pedestrians. According to the 'Global Status Report on Road Safety' released by the World Health Organization, road traffic collisions are one of the leading causes of death for people of all ages, with approximately 1.35 million road fatality deaths occurring globally each year. Figures from the report also highlight that a large part of road deaths involves pedestrians as the most vulnerable road users. Therefore, forensic examination of vehicle-pedestrian collisions has become increasingly important in the detection, investigation and reduction of road casualties and permanent development of this discipline is urgently needed. Thus, this article aims to review the capability and effectiveness of forensic examination in tackling road fatalities and explores the most important aspects of this discipline, such as nature of a vehicle-pedestrian collision, common issues resolved by this type of examination and typical physical evidence used in the reconstruction of vehicle-pedestrian collisions. Moreover, the paper outlines the latest advances and approaches in the field.