FTIR Analysis for Identification of Semen in Mixed Biological Fluids

Introduction

Forensic science is the application of all-natural sciences, in solving the mystery of a crime. Crime is any activity which leads to the breach of law. A crime scene can never be perfect as a criminal always left something at crime scene and take something from crime scene. This “something” become a crucial evidence which helps in solving the case. Some crucial evidence that are commonly found at crime scene are fingerprints, blood stain, biological fluids like semen: in case of assaults, hair, fibers, footprints etc. and has a great impact in forensic investigation. Biological evidences are mostly transfer evidences as they are transferred either from suspect to any possible place on crime scene or from victim such as blood stain can be found at bed, walls, floor etc., semen transferred from assailant to victim etc. The

major significance of these evidences is that these types of evidences are confirmatory in nature due to the presence of DNA [1].

In cases of assaults, rapes biological fluids are the most crucial evidence for identification of suspect and to prove that assault has taken place. During investigation and examination of body fluids like blood, semen plays a very important role in proving guilty or innocence in rape cases where it is very problematic to justify whether the assault has taken place or it’s just a false accusation for personal rivalry. There are various preliminary tests and confirmatory techniques are available for conforming the presence these body fluids at scene of crime. The biological fluids, such as seminal fluid, fluoresce under Ultra-Violet light, so it has become a practice to study bodily fluids under UV light and many researchers have buoyed this method [2]. Polilight with xenon arc lamp aids in detection of seminal stain on fabrics [3]. A study of Rehdorf, et al. [4] demonstrated that a forensic light source Lumatec superlite detects semen and saliva between an excitation range of 415-490nm.

Acid phosphatase is a lysosomal enzyme which works at an acidic pH by hydrolyzing organic phosphates [5]. It is present in body fluids and tissues such as serum, red blood cells, blood, vaginal secretion, urine, plasma, kidney, liver etc. but the concentration is highest in seminal fluid. The increased concentration of acid phosphatase in serum is used to detect various disease or abnormal functioning of tissues [6]. It is also present in urine where its concentration revealed glomular damage in adult females and children [7]. Periodontitis disease in human can be identified by increase in salivary acid phosphatase concentration. It is secreted by prostate gland in seminal fluid where its concentration is 400 times greater than other body fluids. The positive result of acid phosphatase test is used as screening test for identification of semen, also it fluoresces under UV light. Semen is secreted from male reproductive tract and contain sperm cells along with seminal plasma which keeps the sperms viable. There are about 200-300 million sperm count in each ejaculation and in each ejaculation 2-5 ml of semen is released. In appearance it looks like greyish white fluid with chlorine like smell. It has slightly alkaline pH ranges from 7-7.5. Raman spectroscopy prove to be a confirmatory technique in characterizing and discriminating many body fluids of forensic interest including semen [8]. In forensic investigations, detecting the types of biological fluids found on different evidences aids in determining if an offence was committed or not and can serve with the details for reconstructions of the sequence of events [9].

Semen is the most important evidence in case of sexual assaults as it helps in proving that whether the rape has actually occurred or not and also as DNA profiling can be performed by sperm which helps in identification of suspect. Seminal plasma consists of secretions originated from epididymis, seminal vesicles, the prostate, vasa-differentia, bulbourethral and urethral glands [10]. Sometimes it is found in mixed state with other biological fluids like saliva, urine, blood, vaginal fluid etc. The analysis of semen is also known as “Seminogram” which is the detection of ejaculated seminal stain received from a crime scene [11]. The role of forensic expert in solving the sexual assault cases is to prove any ciphers of sexual intercourse, by examining evidence of penetration and ejaculation which can be established by explicit constituent from the seminal fluid. The cellular constituent of semen that characteristically investigated is spermatozoa, whereas the components that contribute to plasma part are crystal choline and spermine, acid phosphatase, and zinc [12]. A valuable oral fluid, saliva is often taken for granted, but it proves to be very crucial in personal identification because of its noninvasive systemic sampling measure for medical diagnosis and research [13].

Fourier transfer infrared spectroscopy is the widely used form of infrared spectroscopy. FT-IR spectroscopy is an idyllic technique for the identification of possible bodily fluids as evidence prior to any expensive DNA analysis [14]. The infrared radiations passes through the sample some of it get absorbed and some is transmitted through the sample. The transmitted IR radiations reaches to the detector where the spectra are formed which is the molecular fingerprint of the sample. Thus, it is used as a confirmatory technique for identifying the biological fluids by their standard fingerprint pattern. Spectroscopic techniques provide the signature spectra of bodily fluids and present striking advantages such as fast results, solvents-free, cost-effective and easy-to-use [15]. ATR-FTIR is a label-free, non-destructive analytical technique used widely to study a vast type of different molecules in a range of different conditions [16].

Biological evidence with forensic aspect may be found in quite a lot of assault cases, being principally pertinent for sexually related ones [17]. Body fluids are one of the most common and crucial evidence found at scene of crime which helps in proving or disproving whether crime has actually taken place or not. These are mostly found in assault cases such as semen, urine, blood etc. There are various chemical tests for body fluids which serve the purpose of screening test such as acid phosphatase test for semen, luminol for blood, phabedas test for saliva etc. Positive results of presumptive test do not account for the confirmation of specific fluids as acid phosphatase test give positive result for semen as well as vaginal secretion, urine etc. To evade false positive test results, confirmatory tests like GC-MS, spectroscopic techniques are applied for detection. These body fluids are a good source of DNA and thus help in individualization and aid in solving criminal cases.

FTIR Analysis

ATR FTIR spectra are obtained for all the samples (M1 to M50) and. Spectra for cotton fiber is considered as a positive control and is represented in figure 3 with main peaks at 3288.41 cm-1, 2890.2 cm-1, 1314cm-1,1027.83cm-1, 556.21cm-1.

The FTIR spectra of semen mixed with urine and mixed with saliva on cotton cloth are compared with spectra of individual body fluids, for identification of seminal peak.

The spectra of seminal stain on cotton cloth for M1 show absorbance at wavelength 3276.48cm-1, 2885cm-1, 1635.8 cm-1, 1537.8 cm-1, 1314.49 cm-1, 1028 cm-1, 557cm-1, which are clearly illustrated in figure 4. Similarly, the spectrum of urine and saliva on cotton fabric for M1 shows multiple peaks at 3275.81cm-1, 2890.2cm-1,1631cm-1, 1314cm-1 ,115.8cm-1 ,1108.57cm-1, 1053.62cm-1,1028.73cm-1 ,434cm-1,557cm-1 and 3276.42cm-1,2899.5cm-1 ,1314cm-1 ,1160.13cm-1 ,1108.47cm-1, 1053.77cm-1 ,1029.55cm-1,434cm-1 ,557cm-1 respectively observed from figures 5 and 6.

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All the three body fluids have the unique characteristic spectra which can be used for their detection. This is in accordance with the recent work of Takakura, et al. [23] where they identify the five different biological fluids with their unique FTIR spectra. In the spectra of semen, excluding the wavelength of cotton fabric, the two wavelength 1635.8 cm-1 and 1537.8 cm-1 corresponds to Amide I band and Amide II band respectively, which is also proved by the study of Berthomieu C, [24] that peaks ranging from 1680cm-

1-1620cm-1 contribute to the (C=O) of peptide backbone forming the Amide I band and peak ranging from 1560cm-1- 1529cm-1 contributing to the (CN)+(NH) forming the Amide II band.

Figure 7 indicate the spectra of mixed sample of semen- urine on cotton fiber for M1 with absorbance at wavelength 3272.5cm-1, 2890.2cm-1, 1626.4cm-1, 1537554.31cm-1, 1314cm-1, 1029cm-1, and the mixed sample of semen- saliva on cotton fiber shown by figure 8 have the peaks at wavelength 3272.5cm-1, 2890.2cm-1, 1631cm-1, 1318.7cm-1, 1029.52cm-1, 558.58cm-1.

When the spectrum of mixed sample semen-urine on cotton piece are compared with the spectrum of semen on cotton fabric, urine on cotton fabric and cotton fabric then it is observed that peaks of cotton fabric are found in all spectrum but in mixed sample a peak observed at 1537.8cm-

1 is exactly as that of semen spectrum excluding peaks of cotton fabric, which confirms the presence of semen in the sample. The study of Zapata F, et al. [25] also proved that vaginal fluid, semen, and urine on fabrics can be identified and characterized by external reflection FT-IR, further comparison of the FT-IR spectra obtained, revealed that they were unique so this technology has interesting efficiency to detect stains of bodily evidences. Similarly, the spectrum of semen-saliva are compared with the spectra of cotton, semen and saliva and it is analyzed that in mixed sample the peak 1631cm-1 is close to the peak of semen spectrum which is 1635.8cm-1 and in this range no peak corresponds to the spectra of saliva which confirms the presence of semen on cotton with mixed stain of semen-saliva. So, it can be concluded that ATR FTIR can be used as a confirmatory technique for detection of semen mixed with other biological fluids.

Conclusion

The use of forensic science evidence such as semen in criminal justice systems can be considered to be, if not actually at a crossroads, then fast approaching one. Identification of biological fluids is an important aspect in forensic field, as it is a principal approach in a criminal investigation [26]. The past few decades have seen the use of this important and persuasive evidence attracting increasing criticism across many jurisdictions, not only over the way such evidence is admitted into courts but also regarding more fundamental doubts over the validity of the techniques, the probative value of the outcomes and the organizational structure of forensic market places. Now-a-days the crime against women is increasing at an alarming rate in all over the world. Crimes like sexual assaults, rapes, molestations occur in every one hour. The problem with investigation of these cases is collection and examination of evidences which are generally body fluids, present as a satin on fabric or sometimes gets mixed with each other such that their isolation and identification become a serious problem in solving the cases. The present study was commenced to address the question of distinguishing the seminal fluid from urine and saliva in mixed samples, further the main focus is on detection of semen mixed with urine and saliva. Fifty samples each of semen, urine, saliva and their mixed samples are first tested for activity of acid phosphatase and then FT-IR analysis are performed. The acid phosphatase shows its presence in all three concerned body fluids and mixed samples with highest degree of activity in semen, followed by urine and saliva respectively. The acid-phosphatase test produces the intense purple color if semen is present, have advantage of getting the result within 1-3 minutes, and can be directly performed on the spot, further helps in localization of seminal stain. The main disadvantage of the AP test is that it also gives false positive test for saliva and urine, but the intensity of color is very low in these two body fluids in contrary to the color intensity on semen samples. The intensity of purple color developed can be used to distinguish seminal stain with other stains, further it provides a lead for detection of seminal stain and can only be used as preliminary test. The main advantage of FTIR analysis is that it provides the fingerprint spectra of the substance which is to be studied and data can be obtained at multiple wavelengths simultaneously. The FT- IR spectra obtained for all samples and compared leading to the conclusion that ATR FT-IR can be used as a confirmatory technique for identifying seminal stain mixed with other body fluids. ATR FTIR spectroscopy has noteworthy advantages in that there are no expendable charges or chemical elements, rapid, and it is explicit in discerning biological materials [27]. It was proven that FT-IR spectroscopy is appropriate and efficient in distinguishing between body fluids and different substances which could be erroneous as body fluid [28]. The onuses and farm duties of a forensic expert in an investigation are very important as it encompasses the vigilant inspection of evidences whereas making firm that it is not meddled with anything. A countless pool of forensic souls and tools go in the enquiry of an offence [29].

We recommend that more extensive studies should be accomplished on validation parameters and pertinency of current methodology to evaluate the effects of environmental factors by feigning actual crime scene surroundings on body fluid investigation. In the future, researches on the pertinency of ATR FT-IR spectroscopy for the examination of other biological fluid traces with imitated criminal casework situations will be undertaken. All-embracing, this method has an enormous scope of application in body fluid analysis because of its prompt, confirmatory, non-destructive nature and requirement of nominal sample preparation [30, 31].