References & Contact Details An Investigation into the use of FTIR Spectroscopy in the Age Determination of Biological Materials. Charlotte-Maria Orphanou BSc (Hons), Dr. Harry Mountain, & Dr. Laura Walton. Forensic & Crime Science, Staffordshire University. Introduction Objectives The most commonly encountered biological materials at the scene of a crime are blood, saliva, semen and vaginal secretions. Biological materials often play an important role within criminal investigations of violent crime and sexual assault. The ability to determine the age of biological materials is the next significant step to be researched by forensic scientists [1-4]. The significance of the age determination of a biological sample within criminal investigations is that it can place individuals at the scene of a crime at identifiable time intervals. It can also allow a chronological order of evidence, allowing evidence analysis to be prioritised. The current accepted method of age determination involves the use of messenger RNA (mRNA), which is an expensive technique and requires specialist equipment and interpretational skills. To date, the research investigating the use of forensic techniques to estimate the age of biological material since deposition has not yet produced a definitive method to be used routinely within criminal investigations by forensic practitioners [5-7]. Attenuated total reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a non-destructive forensic technique that examines the reflectance of component vibrations within the infrared region of the electromagnetic spectrum. ATR-FTIR is a commonly utilised in the analysis of chemicals, such as solvents, drugs, and inks and has previously been successfully utilised in the presumptive testing of biological materials [8]. The objectives of this investigation were to determine: The degree to which it is possible to differentiate between blood, saliva and vaginal secretions using ATR-FTIR. The extent to which is it possible to identify changes within the biological components of blood, saliva and vaginal secretions that are aged from 24 hours to one month. Method Blood, saliva and vaginal secretions were deposited independently in volumes of 20µl on to individual sterile 100% cotton cloth 4x4cm swatches, which were stored in individual sterile petri dishes. Samples were then placed in to storage at room temperature, in accordance with the Human Tissue Act (2004) for pre-determined time intervals. The time intervals investigated were 24 hours up to one month. Samples were analysed using the Nicolet 380 Fourier Transform Infrared (FTIR) Spectrometer with a Smart Orbit, diamond crystal attenuated total reflectance (ATR) accessory and Omnic software. Samples were scanned 32 times per analysis at 4cm-1 resolution within the 400-4000cm-1 infrared region. The measurement format utilised was %Reflectance with data spacing at 1.929 cm-1. Each biological material sample was analysed in 4 different positions within the stained area on the cotton cloth. Results & Discussion a) Blood b) Saliva c) Vaginal Secretions Figure 1: Neat air dried sample spectra after 5 hours a) blood, b) saliva, c) vaginal secretions. Blood Wavenumber (cm-1) Component Identification 1300 Albumin 1395 Amino acid side chains 1507-1557 Amide II 1652 Amide I (α helix) 2956 CH3 stretches of lipids in plasma 3292 Amide A Saliva Wavenumber (cm-1) Component Identification 1075 Sugar moieties 1241 Amide III 1396 Amino acid side chains 1543 Amide II (β sheets) 1649 Amide I (α helix) 2057 Thiocyanate anions 2924 CH2 stretches of lipid acyls in oral mucosa 3283 Amide A Vaginal Secretions Wavenumber (cm-1) Component Identification 1078 Glycogen from human cervical epithelia 1231 Nucleic acid phosphate/Amide III 1540 Amide II 1646 Amide I (α helix) 2922 CH2 stretches of lipid acyls 3273 Amide A Tables 1-3: Peak component identification 1) blood, 2) saliva, 3) vaginal secretions Figure 1 (a-c) demonstrates the components detectable within neat blood, saliva and vaginal secretions, respectively ,when analysed using ATR-FTIR. Tables 1-3 list the key components identified within neat samples of the biological materials. All three biological materials obtain visibly different chemical profiles, although they do all each contain peaks representative of Amide I, II and A. It is the shapes and sizes of these peaks that differ between the samples which is a result of the secondary structures of the proteins specific to each biological material [9]. The Amide peaks have demonstrated the greatest variation in their intensities over time in blood samples aged from 24 hours to one month. Significant differences have been demonstrated between the peak intensities for the Amide I, II and A peaks when analysed using a two-factor independent measures analysis of variance (ANOVA ) statistical test. However, this result is not reflected within the saliva and vaginal secretion samples. To date, both biological materials have been undetectable when deposited on to cotton cloth when carrying out analysis using ATR-FTIR. Reasons for this may include the sample volume deposited being below the limit of detection when applied to cotton cloth, or that the areas analysed are not concentrated enough with biological material once the sample has been absorbed into the cotton and dried. Further Work References & Contact Details The results presented here are part of a wider study that is exploring the age determination of biological materials with alternative forensic techniques. The technique sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is also being explored with samples of blood, saliva and vaginal secretions. Method development is currently on going in the use of ATR-FTIR for age determination for saliva and vaginal secretion samples. In addition to the above mentioned, longer term aging of biological materials is underway , with samples being aged up to 18 months. An exploration of biological materials deposited and aged on to different surfaces is also being looked into. 1. Bauer, M. Polzin, S. & Patzelt, D. (2003) For. Sci. Int,138, 94-103. 2. Anderson, S. et al. (2005) For. Sci. Int. 148, 37-45. 3. Strasser, S et al. (2007) For. Sci. Int. 170, 8-14. 4. Arany, A. Ohtani, S. (2011) For. Sci. Int. 212, 36-39. 5. Li, B et al. (2013) Science and Justice, 53 270-277. 6. Botonjic-Sehic, E. et al. (2009) Spectroscopy, 24 42-48 7.Bremmer, R.H. et al. (2011) For. Sci. Int. 216 (1-3) 1-11 8. Elkins, K.M. (2011) J. For. Sci. 56 (6) 1580-1587. 9. Garidel , P. & Schott, H. (2006) Bio Process Technical, 48-55 Charlotte-Maria Orphanou Tel: (+44) 01782 295742 Email: c.orphanou@staffs.ac.uk