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.

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.

Nucleic acid aptamers as aptasensors for plant biology.

Our knowledge of cell- and tissue-specific quantification of phytohormones is heavily reliant on laborious mass spectrometry techniques. Genetically encoded biosensors have allowed spatial and some temporal quantification of phytohormones intracellularly, but there is still limited information on their intercellular distributions. Here, we review nucleic acid aptamers as an emerging biosensing platform for the detection and quantification of analytes with high affinity and specificity. Options for DNA aptamer technology are explained through selection, sequencing analysis and techniques for evaluating affinity and specificity, and we focus on previously developed DNA aptamers against various plant analytes. We suggest how these tools might be applied in planta for quantification of molecules of interest both intracellularly and intercellularly.

Polymer nanoparticles pass the plant interface.

As agriculture strives to feed an ever-increasing number of people, it must also adapt to increasing exposure to minute plastic particles. To learn about the accumulation of nanoplastics by plants, we prepared well-defined block copolymer nanoparticles by aqueous dispersion polymerisation. A fluorophore was incorporated via hydrazone formation and uptake into roots and protoplasts of Arabidopsis thaliana was investigated using confocal microscopy. Here we show that uptake is inversely proportional to nanoparticle size. Positively charged particles accumulate around root surfaces and are not taken up by roots or protoplasts, whereas negatively charged nanoparticles accumulate slowly and become prominent over time in the xylem of intact roots. Neutral nanoparticles penetrate rapidly into intact cells at the surfaces of plant roots and into protoplasts, but xylem loading is lower than for negative nanoparticles. These behaviours differ from those of animal cells and our results show that despite the protection of rigid cell walls, plants are accessible to nanoplastics in soil and water.