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.

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.

Aptamers are short, single-stranded DNA or RNA oligonucleotides that can specifically bind to their target with high affinity and specificity. Aptamers have gained widespread attention in recent years as possible replacements for antibodies within many analytical fields, due to their high chemical and thermal stability and relative low cost of production. Red blood cells are of interest within not only the medical field, but also are of interest within forensic science. Few aptamers have been reported that can specifically detect human red blood cells, or surface proteins of, but they have great potential for use as biorecognition elements within immunoassays or biosensors. Three aptamers have been identified from recent literature that have been designed to bind to human red blood cells as a whole cell target or glycophorin A as a protein-based target. However, they are yet to be fully characterised for their binding affinity to red blood cells, and no sequence optimisation has been conducted. Within this work, a comprehensive characterisation of three reported aptamers has been conducted. In silico modelling has been explored as a means to better understand the 3D structures and the target ligand of each aptamer. The 3D structures of these aptamers have been reported and utilised within the HDOCK server to predict the docking of the aptamers to red blood cell-specific surface proteins. Both enzyme-linked oligonucleotide assays and microscale thermophoresis have been used to characterise aptamer-target biding, with dissociation constants being predicted in the nanomolar to low micromolar range for each aptamer. Additionally, sequence optimisation has been conducted to enhance the binding of the sequences to human red blood cells through sequence truncation mechanisms. To the best of our knowledge, this work represents the first characterisation of these aptamers and will guide future use of these aptamers as analytical probes.

Blood is a common biological fluid in forensic investigations, offering significant evidential value. Currently employed presumptive blood tests often lack specificity and are sample destructive, which can compromise downstream analysis. Within this study, the development of an optical biosensor for detecting human red blood cells (RBCs) has been explored to address such limitations. Aptamer-based biosensors, termed aptasensors, offer a promising alternative due to their high specificity and affinity for target analytes. Aptamers are short, single-stranded DNA or RNA sequences that form stable three-dimensional structures, allowing them to bind to specific targets selectively. A nanoflare design has been employed within this work, consisting of a quenching gold nanoparticle (AuNP), DNA aptamer sequences, and complementary fluorophore-labelled flares operating through a fluorescence resonance energy transfer (FRET) mechanism. In the presence of RBCs, the aptamer–flare complex is disrupted, restoring fluorescence and indicating the presence of blood. Two aptamers, N1 and BB1, with a demonstrated binding affinity to RBCs, were selected for inclusion within the nanoflare. This study aimed to optimise three features of the design: aptamer conjugation to AuNPs, aptamer hybridisation to complementary flares, and flare displacement in the presence of RBCs. Fluorescence restoration was achieved with both the N1 and BB1 nanoflares, demonstrating the potential for a functional biosensor to be utilised within the forensic workflow. It is hoped that introducing such an aptasensor could enhance the forensic workflow. This aptasensor could replace current tests with a specific and sensitive reagent that can be used for real-time detection, improving the standard of forensic blood analysis.