{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T20:19:34Z","timestamp":1791404374905,"version":"4.3.3"},"reference-count":62,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T00:00:00Z","timestamp":1706832000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2013target 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.<\/jats:p>","DOI":"10.3390\/ijms25031814","type":"journal-article","created":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T09:24:08Z","timestamp":1706865848000},"page":"1814","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["The Development and Characterisation of ssDNA Aptamers via a Modified Cell-SELEX Methodology for the Detection of Human Red Blood Cells"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9675-5018","authenticated-orcid":false,"given":"Hayley","family":"Costanzo","sequence":"first","affiliation":[{"name":"Department of Analytical, Environmental & Forensic Sciences, Faculty of Life Sciences & Medicine, King\u2019s College London, London SE1 9NH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James","family":"Gooch","sequence":"additional","affiliation":[{"name":"Department of Analytical, Environmental & Forensic Sciences, Faculty of Life Sciences & Medicine, King\u2019s College London, London SE1 9NH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sireethorn","family":"Tungsirisurp","sequence":"additional","affiliation":[{"name":"Department of Analytical, Environmental & Forensic Sciences, Faculty of Life Sciences & Medicine, King\u2019s College London, London SE1 9NH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nunzianda","family":"Frascione","sequence":"additional","affiliation":[{"name":"Department of Analytical, Environmental & Forensic Sciences, Faculty of Life Sciences & Medicine, King\u2019s College London, London SE1 9NH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"100096","DOI":"10.1016\/j.fsir.2020.100096","article-title":"Body fluid identification: A case for more research and innovation","volume":"2","author":"Williams","year":"2020","journal-title":"Forensic Sci. Int. Rep."},{"key":"ref_2","unstructured":"Gooch, J.P. (2019). Biosensors for the Forensic Detection of Body Fluids. [Ph.D. Thesis, King\u2019s College London]."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/j.scijus.2022.12.006","article-title":"From crime scene to courtroom: A review of the current bioanalytical evidence workflows used in rape and sexual assault investigations in the United Kingdom","volume":"63","author":"Dawnay","year":"2023","journal-title":"Sci. Justice"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"7279","DOI":"10.1039\/c3an01372c","article-title":"Enabling fluorescent biosensors for the forensic identification of body fluids","volume":"138","author":"Frascione","year":"2013","journal-title":"Analyst"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.scijus.2012.09.004","article-title":"The acid phosphatase test two minute cut-off: An insufficient time to detect some semen stains","volume":"53","author":"Redhead","year":"2013","journal-title":"Sci. Justice"},{"key":"ref_6","first-page":"1","article-title":"Wrongful convictions and DNA exonerations: Understanding the role of forensic science","volume":"279","author":"Laporte","year":"2018","journal-title":"Natl. Inst. Justice J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.fsigen.2016.02.015","article-title":"Analysis and implications of the miscarriages of justice of Amanda Knox and Raffaele Sollecito","volume":"23","author":"Gill","year":"2016","journal-title":"Forensic Sci. Int. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1007\/s12024-010-9198-1","article-title":"Blood stain pattern analysis","volume":"7","author":"Peschel","year":"2011","journal-title":"Forensic Sci. Med. Pathol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1111\/1556-4029.12720","article-title":"Evaluation of a visualization assay for blood on forensic evidence","volume":"60","author":"Vandewoestyne","year":"2015","journal-title":"J. Forensic Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1097\/JFN.0000000000000116","article-title":"Alternate light source findings of common topical products","volume":"11","author":"Pollitt","year":"2016","journal-title":"J. Forensic Nurs."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Kobilinsky, L.F., Liotti, T.F., and Oeser-Sweat, J. (2005). DNA: Forensic and Legal Applications, Wiley Online Library.","DOI":"10.1002\/0471681911"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1016\/j.talanta.2014.02.021","article-title":"Application of fluorescent substrates to the in situ detection of prostate specific antigen","volume":"125","author":"Gooch","year":"2014","journal-title":"Talanta"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1111\/j.1556-4029.2006.00324.x","article-title":"Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA","volume":"52","author":"Tobe","year":"2007","journal-title":"J. Forensic Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.forsciint.2009.02.013","article-title":"Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene","volume":"188","author":"Virkler","year":"2009","journal-title":"Forensic Sci. Int."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Harris, H.A., and Lee, H.C. (2019). Introduction to Forensic Science and Criminalistics, CRC Press.","DOI":"10.4324\/9781315119175"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2424","DOI":"10.1016\/j.bios.2004.11.006","article-title":"Analytical applications of aptamers","volume":"20","author":"Tombelli","year":"2005","journal-title":"Biosens. Bioelectron."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.trac.2017.07.019","article-title":"Developing aptasensors for forensic analysis","volume":"94","author":"Gooch","year":"2017","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1002\/elsc.202100148","article-title":"Aptasensors versus immunosensors\u2014Which will prevail?","volume":"22","author":"Heuer","year":"2022","journal-title":"Eng. Life Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Roxo, C., Kotkowiak, W., and Pasternak, A. (2019). G-quadruplex-forming aptamers\u2014Characteristics, applications, and perspectives. Molecules, 24.","DOI":"10.3390\/molecules24203781"},{"key":"ref_20","unstructured":"Gooch, J., Morillas, A.V., and Frascione, N. (2019). Emerging Technologies for the Analysis of Forensic Traces, Springer."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1178","DOI":"10.1016\/j.bios.2010.07.033","article-title":"Recent advances in recognition elements of food and environmental biosensors: A review","volume":"26","author":"Mehta","year":"2010","journal-title":"Biosens. Bioelectron."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1169","DOI":"10.1038\/nprot.2010.66","article-title":"Development of DNA aptamers using Cell-SELEX","volume":"5","author":"Sefah","year":"2010","journal-title":"Nat. Protoc."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"The Galaxy Community (2022). The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 Update. Nucleic Acids Res., 50, W345\u2013W351.","DOI":"10.1093\/nar\/gkac610"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1016\/j.omtn.2018.04.006","article-title":"AptaSUITE: A full-featured bioinformatics framework for the comprehensive analysis of aptamers from HT-SELEX experiments","volume":"11","author":"Hoinka","year":"2018","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5821","DOI":"10.1007\/s00216-021-03562-7","article-title":"Generating aptamers towards human sperm cells using massively parallel sequencing","volume":"413","author":"Gooch","year":"2021","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"H786","DOI":"10.1152\/ajpheart.00349.2008","article-title":"Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP","volume":"295","author":"Hanson","year":"2008","journal-title":"Am. J. Physiol.-Heart Circ. Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.cll.2014.10.002","article-title":"Red blood cell population dynamics","volume":"35","author":"Higgins","year":"2015","journal-title":"Clin. Lab. Med."},{"key":"ref_28","unstructured":"Dean, L., and Dean, L. (2005). Blood Groups and Red Cell Antigens, NCBI."},{"key":"ref_29","first-page":"280","article-title":"Fundamental Issues in Forensic Semen Detection","volume":"59","author":"Stefanidou","year":"2010","journal-title":"West Indian Med. J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1186\/2041-2223-3-25","article-title":"Isolating DNA from sexual assault cases: A comparison of standard methods with a nuclease-based approach","volume":"3","author":"Garvin","year":"2012","journal-title":"Investig. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.ajhg.2009.06.022","article-title":"Massively parallel sequencing: The next big thing in genetic medicine","volume":"85","author":"Tucker","year":"2009","journal-title":"Am. J. Hum. Genet."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1007\/s00414-020-02294-0","article-title":"Massive parallel sequencing in forensics: Advantages, issues, technicalities, and prospects","volume":"134","author":"Ballard","year":"2020","journal-title":"Int. J. Leg. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e20220077","DOI":"10.1590\/1678-4685-gmb-2022-0077","article-title":"Applications of massively parallel sequencing in forensic genetics","volume":"45","author":"Carratto","year":"2022","journal-title":"Genet. Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Lee, S.J., Cho, J., Lee, B.H., Hwang, D., and Park, J.W. (2023). Design and Prediction of Aptamers Assisted by In Silico Methods. Biomedicines, 11.","DOI":"10.3390\/biomedicines11020356"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3406","DOI":"10.1093\/nar\/gkg595","article-title":"Mfold web server for nucleic acid folding and hybridization prediction","volume":"31","author":"Zuker","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1002\/jcc.21596","article-title":"NUPACK: Analysis and design of nucleic acid systems","volume":"32","author":"Zadeh","year":"2011","journal-title":"J. Comput. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Abreu, R.D.S., Antunes, D., Moreira, A.D.S., Passetti, F., Mendon\u00e7a, J.B., de Ara\u00fajo, N.S., Sassaro, T.F., Alberto, A.V.P., Carrossini, N., and Fernandes, P.V. (2023). Next Generation of Ovarian Cancer Detection Using Aptamers. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24076315"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Ilgu, M., Yan, S., Khounlo, R.M., Lamm, M.H., and Nilsen-Hamilton, M. (2019). Common secondary and tertiary structural features of aptamer\u2013ligand interaction shared by RNA aptamers with different primary sequences. Molecules, 24.","DOI":"10.3390\/molecules24244535"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"W676","DOI":"10.1093\/nar\/gkl253","article-title":"QGRS Mapper: A web-based server for predicting G-quadruplexes in nucleotide sequences","volume":"34","author":"Kikin","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5402","DOI":"10.1093\/nar\/gkl655","article-title":"Quadruplex DNA: Sequence, topology and structure","volume":"34","author":"Burge","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_41","first-page":"17","article-title":"Quadruplex DNA structure characterization by circular dichroism","volume":"68","author":"Gray","year":"2017","journal-title":"Curr. Protoc. Nucleic Acid Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1016\/j.omtn.2019.12.034","article-title":"Binding and structural properties of DNA aptamers with VEGF-A-mimic activity","volume":"19","author":"Yoshitomi","year":"2020","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1713","DOI":"10.1093\/nar\/gkp026","article-title":"Circular dichroism and conformational polymorphism of DNA","volume":"37","author":"Kypr","year":"2009","journal-title":"Nucleic Acids Res."},{"key":"ref_44","first-page":"381","article-title":"How to study G-quadruplex structures","volume":"93","author":"Gudanis","year":"2012","journal-title":"Biotechnologia"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1089\/nat.2017.0680","article-title":"Effect of chemical modifications on aptamer stability in serum","volume":"27","author":"Kratschmer","year":"2017","journal-title":"Nucleic Acid Ther."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"20979","DOI":"10.3390\/molecules201219739","article-title":"DNA aptamers in the diagnosis and treatment of human diseases","volume":"20","author":"Zhu","year":"2015","journal-title":"Molecules"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"34","DOI":"10.32607\/20758251-2013-5-4-34-43","article-title":"Aptamers: Problems, solutions and prospects","volume":"5","author":"Lakhin","year":"2013","journal-title":"Acta Naturae"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.talanta.2014.09.036","article-title":"Diagnostic applications of gastric carcinoma cell aptamers in vitro and in vivo","volume":"134","author":"Ding","year":"2015","journal-title":"Talanta"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"12743","DOI":"10.1021\/acs.analchem.7b02862","article-title":"Aptamer-based ELISA assay for highly specific and sensitive detection of Zika NS1 protein","volume":"89","author":"Lee","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1002\/asia.201000242","article-title":"In vivo fluorescence imaging of tumors using molecular aptamers generated by cell-SELEX","volume":"5","author":"Shi","year":"2010","journal-title":"Chem. Asian J."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Aljohani, M.M., Cialla-May, D., Popp, J., Chinnappan, R., Al-Kattan, K., and Zourob, M. (2022). Aptamers: Potential diagnostic and therapeutic agents for blood diseases. Molecules, 27.","DOI":"10.3390\/molecules27020383"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"4126","DOI":"10.2174\/092986711797189565","article-title":"Improving the stability of aptamers by chemical modification","volume":"18","author":"Wang","year":"2011","journal-title":"Curr. Med. Chem."},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Siddiqui, S., and Yuan, J. (2021). Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7. Molecules, 26.","DOI":"10.3390\/molecules26010204"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1007\/s11033-022-07991-0","article-title":"DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX","volume":"50","author":"Ferreira","year":"2023","journal-title":"Mol. Biol. Rep."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"19311","DOI":"10.1038\/s41598-019-55892-3","article-title":"Real-time PCR for direct aptamer quantification on functionalized graphene surfaces","volume":"9","author":"Almeida","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"El-Husseini, D.M., Sayour, A.E., Melzer, F., Mohamed, M.F., Neubauer, H., and Tammam, R.H. (2022). Generation and selection of specific aptamers targeting Brucella species through an enhanced cell-SELEX methodology. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23116131"},{"key":"ref_57","unstructured":"World Heath Organisation (WHO) (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen."},{"key":"ref_58","first-page":"1841","article-title":"Single-strained DNA aptamers mask RhD antigenic epitopes on human RhD+ red blood cells to escape alloanti-RhD immunological recognition","volume":"21","author":"Zhang","year":"2020","journal-title":"Mol. Med. Rep."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"e1657","DOI":"10.31661\/gmj.v9i0.1657","article-title":"Identification of Aptamers that Specifically Bind to A1 Antigen by Performing Cell-on Human Erythrocytes","volume":"9","author":"Hosseini","year":"2020","journal-title":"Galen Med. J."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"5419","DOI":"10.1021\/acs.analchem.3c00115","article-title":"Aptamer-Mediated Enrichment of Rare Circulating Fetal Nucleated Red Blood Cells for Noninvasive Prenatal Diagnosis","volume":"95","author":"Li","year":"2023","journal-title":"Anal. Chem."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"11347","DOI":"10.1038\/srep11347","article-title":"Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)","volume":"5","author":"Birch","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_62","unstructured":"Bushnik, E. (2018). Partially Robotic Selection of Aptamers to Red Blood Cell Protein Glycophorin A. [Master\u2019s Thesis, Universit\u00e9 d\u2019Ottawa\/University of Ottawa]."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/3\/1814\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:53:36Z","timestamp":1760104416000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/3\/1814"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,2]]},"references-count":62,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2024,2]]}},"alternative-id":["ijms25031814"],"URL":"https:\/\/doi.org\/10.3390\/ijms25031814","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,2]]}}}