Author’s Accepted Manuscript

Forensic application of fluorescence spectroscopy:

an efficient technique to predict the presence of
human saliva

Saira Elizabeth Denny, Shaiju S. Nazeer, T.T.

Sivakumar, Bindu. J. Nair, Ramapurath S. Jayasree
www.elsevier.com/locate/jlumin

PII: S0022-2313(17)31782-9
DOI: https://doi.org/10.1016/j.jlumin.2018.07.022
Reference: LUMIN15769
To appear in: Journal of Luminescence
Received date: 23 October 2017
Revised date: 2 July 2018
Accepted date: 18 July 2018
Cite this article as: Saira Elizabeth Denny, Shaiju S. Nazeer, T.T. Sivakumar,
Bindu. J. Nair and Ramapurath S. Jayasree, Forensic application of fluorescence
spectroscopy: an efficient technique to predict the presence of human saliva,
Journal of Luminescence, https://doi.org/10.1016/j.jlumin.2018.07.022
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 Forensic application of fluorescence spectroscopy: an efficient technique to predict
the presence of human saliva

Saira Elizabeth Denny2, Shaiju S Nazeer1#, T T Sivakumar2, Bindu.J.Nair3,Ramapurath S

Jayasree1*

1
Division of Biophotonics and Imaging, Biomedical Technology Wing, Sree Chitra Tirunal
Institute for Medical Sciences & Technology, Poojappura, Thiruvananthapuram - 695 012,
Kerala, India.
2
Department of Oral pathology, PMS Dental College, Thiruvananthapuram
3
Dr.Vivek’s Dental Clinic, Vazhuthacadu, Thiruvananthapuram

Abstract

In this study, we aim to use the fluorescence spectroscopic data as a preliminary forensic
evidence for the detection of saliva stains from the surface of drinking glass, to see the potential
of utilizing a comparatively simple method to trace the presence of saliva from inanimate
objects such as cigars, papers, cloths and envelopes. Since dried stains of saliva are invisible to
human eyes, detection of saliva from such sources is a challenge in forensic analysis. The
fluorescence emission spectra of dried saliva samples collected from drinking glass were
compared to that of undiluted liquid saliva of the same volunteers. The deposition of saliva was
predicted based on the emission spectra of the enzyme amylase around 350 nm, which is present
in high concentrations in saliva. The dried saliva stains and undiluted liquid saliva showed an
emission peak at 350 ± 5 nm and 345 ± 5 nm, respectively. The fluorescence emission spectra
obtained from the dried saliva samples confirmed well to those of undiluted liquid saliva and
amylase.The fluorescence intensity and area underneath the fluorescence peak of undiluted liquid
saliva as well as dried saliva were also studied. The study concludes that correlating fluorescence
emission peak, fluorescence intensity and area under the curve, saliva can be detected from dried
stains. The potential of fluorescence spectroscopy is also emphasised to detect the presence of
saliva from inanimate objects like that of drinking glass, by identifying the emission peak of
amylase, one of the prominent saliva components. Thus by using this simple technique, the best
possible samples for a detailed DNA analysis may be screened and selected, which could
significantly contribute to forensic identification.

Introduction

Body fluid traces recovered at crime scenes are among the most important types of
evidence to forensic investigators[1,2]. When we think of biological traces found at a crime
scene, blood comes to mind first. Besides blood, there are numerous types of bodily fluids that
may be found at a crime scene or on a victim, e.g., semen, saliva, vaginal fluid, and urine, all of
which have the potential to be analysed and used in the identification and incrimination of the
perpetrator. The examination of such substances not only can provide clues to the identity of the
offender, but also help investigators to develop a detailed picture of the sequence of events which
occurred. The analysis of bodily fluids may also determine the presence of quantities of certain
substances in the body, such as alcohol or toxins[3,4].

Saliva is one of the vital fluids secreted in human beings. It is a clear liquid produced in
the mouth for various purposes, primarily to act as lubrication for food and provide the enzyme
amylase to begin the breakdown of this food. Composed of water, enzymes, various electrolytes,
mucus, and epithelial cells, saliva is ideal for DNA profiling [1–3,5]. Saliva can be found in
many areas inside a crime scene such as in bite marks left in objects or victims of violent crimes,
cigars, papers, cloths and other inanimate objects.The presence of saliva may act as supportive
evidence providing a link to a potential suspect through the recovery and analysis of
DNA[6].Unfortunately, dried saliva stains are invisible to the human eye, which adds to the
difficulty of recognizing and collecting it. Saliva is often sampled on objects where its presence
is suspected rather than visually confirmed, e.g. drinking glass, bottles and cans.

Successful identification of saliva traces from inanimate objects can be a significant lead
to DNA analysis. Testing trace swabs for the presence of saliva would provide a method of
reducing the proportion of samples giving negative DNA results[2]. Some crime laboratories will
analyse the DNA only if the presumptive assays are positive, some will proceed with analysis
regardless of the results, and some proceed straight to DNA analysis without performing a
presumptive assay[7]. Therefore, an ideal detection method is required first to identify the
presence of invisible saliva stain and then proceed with other methods of extracting DNA for
detailed investigation.

The potential of fluorescence spectroscopy in biomedical applications ranging from cancer

detection to treatment follow up has been established.[8-16] Fluorescence spectroscopy can be
used to detect inorganic or organic fluorophores from any body fluids. It is based on the principle
that when a fluorescent material is excited at a particular wavelength, it emits radiation of longer
wavelength[2]. The aromatic amino acids, tyrosine and tryptophan, which are the important
amino acids in α salivary amylase, an enzyme present in saliva, gives combined characteristic
emission spectrum at 345–355 nm when excited at a particular wavelength of 282 nm or lesser,
whereas excitations above which are reported excite tryptophan selectively[17].The fluorescence
profile of dried saliva samples are expected to be similar to those obtained from aqueous samples
of amylase and tryptophan[1,2].Recently, Mc Shine et al have demonstrated that the
fluorescence lifetime measurement of blood stains can help forensic investigation by estimating
the age of the bloodstain [18]. In the present study, we propose to exploit the advantage of using
a very basic technique rather than a complicated, expensive and time consuming one for the
detection of saliva. We propose this technique as an initial screening method for the presence of
saliva on a crime site like bite mark which is expected in case of several situations of murder or
other crimes like rape. This is a simple study that takes only less than 5 minutes. Likewise, the
present study hypothesizes that, the detection of fluorescence emission spectra of materials
collected by swabbing suspected inanimate objects like that of drinking glass could give an
emission fluorescence at 345-355 nm characteristics of saliva components, when excited at 282
nm, suggesting a strong presumptive indication of saliva deposition. The objective of the work
is to compare the fluorescence emission spectra of dried human saliva with undiluted liquid
saliva and to find out the prediction potential of fluorescence spectroscopy as a screening
technique to identify dried human saliva.

Materials & Methods

Saliva samples were collected from 56 volunteers of PMS College of Dental Science and
Research, Trivandrum. Volunteers who were willing to participate were included in the study.
Volunteers who had severe alcoholism, poorly controlled diabetes, undergoing or undergone
chemotherapy and radiation therapy and those under medications such as antidepressants,
amphetamines and antihistamines were excluded from the study. The study was reviewed and
approved by the institutional ethical committee of PMS College of Dental Sciences &
Research,Thiruvananthapuram. Informed consent was obtained from all volunteers.0.1M
phosphatebuffer with pH 7.4 was used for swabbing the saliva samples. Fluorolog-3 Model
FL3-22 (JobinYvon, Edison, NJ, USA) spectro-fluorimeter was used for recording the
fluorescence spectra (Fig 1 A).

Collection of undiluted liquid saliva sample:

The volunteers were asked to refrain from eating, drinking or performing oral hygiene
procedures for at least 1 hour prior to saliva collection. They were given drinking water and
asked to rinse their mouth out well for 1 min. Five minutes after this oral rinse, 2 ml of
unstimulated whole saliva were collected into a vial and stored at 4°C.

Collection of dried saliva sample:

Volunteers were made to drink water from glass to obtain saliva stains and the surface of
the glasses were allowed to under normal conditions for 30-45 minutes. No precautionary storage
measures were taken in this case so as to mimic the exact condition of collecting dried saliva
from forensic scenes.A fibre-free cotton dipped in pH 7.4 phosphate buffer 0.1M KCl with
excess solution removed was rubbed over the suspected area of drinking glass. Each swab was
mixed in a separate cuvette containing 2 ml KCl solution for 10 seconds. This solution, collected
in quartz cuvette was used for fluorescence spectroscopic analysis.

Fluorescence spectroscopic Analysis

The undiluted liquid saliva sample and dried saliva sample swabbed using PBS were
transferred into separate quartz cuvettes. Each cuvette was then placed on to the sample holder in
the spectrofluorimeter one after the other. The excitation wavelength of 282nm was selected
using a double-grating monochromator.The excitation light from the 450W xenon lamp was
directed perpendicularly to the cuvette containing sample. The emission light of the selected
wavelength range was then passed into the photomultiplier tube and the emission spectra were
recorded. All spectra were recorded using Data Max™ software (Data-Max, Round Rock, TX,
USA) and further evaluated by means of Microcal Origin 6.0 software.

Data analysis

Comparative analysis of the fluorescence emission spectra of undiluted liquid saliva and
dried saliva was done. The fluorescence emission peak, fluorescence intensity and area under the
fluorescence curve of undiluted liquid saliva, dried saliva and waterwere evaluated.The
fluorescence emission peaks of undiluted liquid saliva and dried saliva were correlated. Curve
fitting analysis was done to find any hidden peaks in the spectra of undiluted liquid saliva and
dried saliva.

Statistical analysis:

Statistical analysis was done with computer software using Statistical Package for Social
Sciences (SPSS) version 16, and for data entry software Micro Excel was used.

Results and Discussion

In the present study, 56 samples of dried saliva swabbed from the drinking glass and
another 56 samples of undiluted liquid saliva were excited at 282 nm and fluorescence spectra
were acquired in the 300-500 nm region. The excitation wavelength was chosen as 282 nm after
ascertaining the excitation spectra of both liquid and dried saliva separately. Both the samples
showed excitation maximum around 282 nm(Fig 1 D & E). Additionally, the absorption
spectrum of saliva showed a sharp absorption at 282 nm (Fig 1C). The fluorescence emission
spectra of undiluted liquid saliva had an average emission maximumat 345 nm (Fig 2A). In the
case of dried saliva, majority of the 56 samples showed two peaks with a blue shifted component
at 340 nm and another red shifted peak at 350 nm (Fig 2B). These prominent peaks in both
samples corresponds to the collective emission of aromatic amino acids, tryptophan and tyrosine,
with more contribution from tryptophan which is one of the important amino acids of the
salivary enzyme amylase. Tyrosine has three strong fluorescence areas, with λ ex/λem=202/304nm,
λex/λem=220/304nm and λex/λem=274/304nm, with the emission peaking at 304 nm, in all the cases. Additionally,
the amylase peak is very broad, covering a wide area. Considering these facts, we assigned the observed
emission as that from the collective emission of both Tyrosine and tryptophan. The observation was
partially in accordance with the study by Soukas et al[1] and Nanda et al[2] where dried saliva
stains swabbed from human skin has seen to produce a fluorescence emission in the range of 345
-355 nm when excited at 282 nm. The other fluorophores expected in this region are mucin and
lysozymes whose emission falls at 310 and 353 nm respectively. As the amylase peak of this
study is very broad and covers a wide range including the emission profiles of mucin and
lysozymes, they are not considered separately.The correlation coefficient (r) and the p value of
the fluorescence emission peak position of undiluted liquid saliva and dried saliva obtained were
0.044 and 0.750 respectively. As there is no statistically significant difference in the fluorescence
emission peak between the two samples, this could be used as a predictor of saliva. Blue-shifted
emission peaks from the dried saliva is expected to be due to the presence of folded state of the
proteins due to the absence of water compared to that of the solution spectra [19]. On drying the
saliva, the removal of water reduces the polarity around typtophan. Whereas, on rehydration,
protein unfolding occurs with complete exposure of tryptophan to water, making it highly
polarized. Unfolding of proteins in aqueous solutions usually results in a change in the
microenvironment of trpyptophan from a relatively hydrophobic core to a polar aqueous
environment. Therefore, a red shift in the fluorescence emission is observed when it is in the
solution phase. The hydrophobic component gives a blue shifted peak whereas the rehydrated
component give rise to the red shifted component. Hence, the appearance of doublet peak in the
dried saliva sample predicts the existence of a mixture of both undiluted saliva and dried saliva
in this sample.

The average fluorescence intensity of dried saliva samples were five fold less than that of
undiluted liquid saliva. The t-test shows significant difference in the fluorescence intensity of
undiluted liquid saliva and dried saliva (p< 0.01). Similarly, the average area underneath the
fluorescence peak of undiluted liquid saliva was found to be 4.5 times greater than that obtained
from dried saliva. The difference between the average area under the fluorescence curve of
undiluted liquid saliva and dried saliva was also significant as per the t- test (p<0.01). The
reason for these observations are attributed to the dilution factor since the dried saliva samples
from the drinking glass were diluted using PBS. The fluorescence intensity also showed minimal
variation within the samples of dried saliva and undiluted liquid saliva, which probably would be
due to the difference in the protein content of saliva. Certain enzymes like amylase, lysozyme,
lactoferrine, lactoperoxidasis, cystatins, histatins, mucins, lipase, proteinase, DNAse, and
RNAse, electrolytes and other components like bacteria, epithelial cells, erythrocytes and
leukocytes as well as food debris which may be present in saliva could also be responsible for
the fluorescence and may vary from case to case, slightly[20].

Curve fitting analysis of normalized fluorescence emission spectra of undiluted liquid

saliva and dried saliva showed an additional peak at 380 nm (Figures 3 A & B). The
fluorescence intensity obtained for the amylase peak and the additional peak at 380nm showed a
strong positive correlation (r = 0.966, p <0.01 0) in both undiluted liquid saliva and dried saliva
samples (Figures 3 C&D). The observed positive correlation between the amylase peak and
additional peak at 380 nm in both undiluted liquid saliva and dried saliva indicates that the
tryptophan protein with conformational changes or emission due to the presence of ATP, ADP,
adenine and adenosine in the epithelia cells and bacteria in saliva[20], would have contributed to
the second peak[22]. However, the amylase peak at 345 nm alone is considered for the
identification of the presence of saliva, in this study.

Spectrofluorometric analysis of water was also done in our study. Water showed a very
weak signal centred at 345 nm which is negligible in intensity and hence will not interfere with
the fluorescence results. Origin of this peak could be from the fluorescence of minute impurities
present in the water. Peak due to Raman scattering of water is ruled as it is expected as a well
defined peak at 311 nm. The water signal is not interfering with the results of saliva because
fluorescence intensity of water is much lower than that of both undiluted and diluted saliva
samples (Fig 1B). Dried saliva samples showed a fluorescence intensity which is five times
higher than that of water. The area under the fluorescence peak of water was found to be 4.3
times lesser than that of dried saliva.

In the study by Auvdel et al., saliva stains were detected in 30%, 21% and 48% of test
items using argon laser, UV light and high intensity quartz arc tube respectively[23], whereas
the present study yielded 100% detection using fluorescence spectroscopy. The present study for
the detection of amylase in samples from both liquid saliva and dried saliva is a quick procedure
and takes approximately 3-5 minutes when compared to the other chemical-based saliva
detection tests like Phadebas which takes much longer time[2]. In the study of Vandenberg et
al[24] using Polilight (a versatile light source), to examine saliva stains on fabrics, the
detectability of the stains were affected by the colour of the materials on which the saliva was
deposited. Also, this technique required the use of different coloured goggles to observe the
fluorescence. In their study, one false negative result was observed among 40 case works. The
UV-VIS light source detection system developed by Fiedleret al also required the use of different
coloured goggles and filters to detect saliva stains from fabrics. Fiedler et al also found that
darker the colour, the lower the chance to detect biological traces using the UV-VIS light source.
This method detected saliva stains in 60% of cases, whereas our study using fluorescence
spectroscopy exhibited 100% positivity [20].

In a study using ELISA method and monoclonal anti-human salivary amylase antibody
for the detection of salivary amylase on dried stains, Quarino et al have found that along with
100% positivity with saliva samples, 13% of non saliva human body fluids also showed
positivity[25]. Moreover, it required the use of many reagents and had a lengthy procedure.
Although salivary genes such as statherin (STATH) and HTN3 developed by Juusola J et al[26]
and the salivary markers (SPRR3, SPRR1A, KRT4, KRT6A, and KRT13) developed by
Zubakovet al[27] are highly specific, differentiating saliva from other amylase containing body
fluids like that of vaginal fluid was not possible. Even though mRNA markers show high tissue-
specificity and adequate sensitivity for forensic analysis, mRNA profiling is not yet widely used
in forensic laboratories since it does not allow for the simultaneous analysis of DNA profiling
and body fluid identification. In addition, heat and humidity remained a threat to the stability of
mRNA markers.

Amylase is not only found in saliva, it is also present in other body fluids in the human
beings. However it is understood that there is huge difference in the quantity of amylase present
in saliva when compared to other body fluids[28]. Since concentration of amylase is positively
correlated with the intensity of fluorescence emission, this variation in concentration can be an
indicator for identification of specific body fluids. Also, when amylase detected from stains on
bite marks and other inanimate objects like cigars, envelopes and drinking glass, suspicion of
saliva is more likely than other body fluids. Spectrofluorometric studies on the activity of
amylase in various body fluids other than saliva need to be experimented for further
confirmation.
 Another possible complication is the deposition saliva from animals. Study using RSID-
saliva kit by Old et al[6] showed positive reactions in samples containing alpha-amylases from
mammals such as gorillas and rats. Animal bite marks can be distinguished from human bite
marks by its characteristic pattern. However, it may be difficult to distinguish between the two, if
saliva is deposited without leaving any bite marks. Thus, positive amylase result is indicative,
not conclusive, for human saliva. Saliva mixed with liquid beverages such as aerated drinks, fruit
juices, alcohol, tea, coffee etc. can present occasionally as dried stains. The cross reactivity of
these non-biological fluids to the salivary amylase with regard to fluorescence activity is yet to
be studied.

By correlating different parameters such as fluorescence emission peak, fluorescence

intensity and area under the curve, fluorescence spectroscopy proves to be a rapid and non-
destructive technique for identification of saliva from dried stains. Our study is a pioneer work
on the detection of saliva stain from inanimate objects using fluorescence spectroscopy. It was
successful in detecting dried saliva stains from drinking glass by using the excitation – emission
principle of fluorescence spectroscopy. Since amylase activity in saliva stains is stable for
several months, this technique can be used to detect dried saliva stains from immediate as well as
long term evidences obtained for forensic analysis.

Conclusion

Identification of human saliva stain plays a significant role in forensic investigation as it

is an important source of DNA. Detection of salivary amylase can be used as a presumptive test
to locate saliva stains on the surfaces of both animate as well as inanimate objects. Our study
using fluorescence spectroscopy was performed as an attempt to detect saliva stains deposited on
the surface of drinking glass by evaluating the fluorescence emission spectra of amylase.
Emission peak of amylase around 350 nm is explored for this. The fluorescence emission spectra
obtained from dried saliva confirmed well to those of undiluted liquid saliva and amylase.
Thereby our study shows that fluorescence spectroscopy can be used for the prediction of saliva
deposited on inanimate objects. The fluorescence intensity and area underneath the fluorescence
peak of undiluted liquid saliva was found to be nearly 4.5 times greater than that obtained from
dried saliva stains. By correlating different parameters such as fluorescence emission peak,
fluorescence intensity and area under the curve, salivary amylase in saliva may be differentiated
from amylase in other body fluids. The study also highlights the efficiency of fluorescence
spectroscopy as an ideal tool for rapid identification of saliva from dried stains. It also has the
advantage of using the same samples for further DNA analysis as it is a non-destructive
procedure. However, further studies using larger sample size and study on cross reactivity with
contaminants will enable us in better defining the usefulness of fluorescence spectroscopy as a
diagnostic tool. This study concludes that by using this technique, the best possible samples for
DNA analysis may be selected, which could significantly contribute to forensic identification.

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C D E

Fig 1: Experimental set used for recording the fluorescence spectra (A), Average fluorescence
emission spectra of undiluted liquid saliva, dried saliva and water(B), Absorption spectrum of
saliva (C), Excitation spectrum of dried and liquid saliva respectively (D&E)
 A B

Fig 2 Compiled fluorescence emission spectra of undiluted (liquid)saliva and diluted (dried)
saliva
 B
A

C
D

Fig 3: Curve fitting of normalized fluorescence emission spectra of undiluted (A) and dried(B)
saliva. C & D shows the correlation plot between normalized fluorescence intensity at the
amylase peak and the 380 nm peak in undiluted liquid saliva and dried saliva respectively. In
both cased r = 0.966, p p <0.01