Optik 127 (2016) 2086–2088

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Optik
journal homepage: www.elsevier.de/ijleo

Raman spectroscopic analysis of dengue virus infection in human

blood sera
Saranjam Khan a,∗ , Rahat Ullah a , Muhammad Saleem a , Muhammad Bilal a ,
Rashad Rashid a , Inamullah Khan b , Arshad Mahmood a , Muhammad Nawaz a
a
National Institute for Lasers and Optronics, Nilore, Islamabad 45650, Pakistan
b
Nuclear Institute of Foods and Agriculture, PO Box 446, Peshawar, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: We are presenting Raman spectroscopic analysis of dengue virus infection in the human blood sera. Blood
Received 28 April 2015 samples from 40 individuals infected with dengue virus and 25 healthy volunteers have been used in this
Accepted 10 November 2015 study. Raman spectra of all these samples have been acquired in the spectral range from 600 cm−1 to
1750 cm−1 using 532 nm laser as an excitation source. These Raman spectra have been used to analyze
Keywords: the biochemical changes appeared in the blood caused by dengue virus infection. Two Raman lines at
Dengue virus
750 cm−1 and 850 cm−1 found in all spectra of dengue infected sera, indicate the presence of adenosine
Thrombocytes
diphosphate (ADP), which is expected to be excreted due to rupturing of thrombocytes.
Adenosine diphosphate
Raman spectroscopy © 2015 Elsevier GmbH. All rights reserved.
Immunoglobulins (G&M) IgG, IgM

1. Introduction
Dengue virus infection is a global health issue affecting millions
of people worldwide each year. It is a mosquito-borne infectious
disease transmitted by the bite of a mosquito from one of the
four dengue virus serotypes [1]. In recent years, transmission has
increased predominantly in urban and semi-urban areas and has
become a major international public health concern. In some cases,
the disease developed into life-threatening dengue hemorrhagic
fever (DHF) or dengue shock syndrome (DSS) resulting in bleeding,
low levels of blood platelets, blood plasma leakage, and danger-
ously low blood pressure. In Asia, the risk of severe dengue infection
is greater in children (≤15 years) than in adults [2–5].
An early and efficient diagnosis of dengue virus itself or its
associated biochemical changes is of critical importance for the
treatment of patients and controlling epidemics. A variety of labo-
ratory diagnostic methods have already been in practice includes
virus isolation in cell culture, RNA detection and anti-bodies detec-
tion in serological tests, etc. [6,7]. The diagnostic modalities with
high sensitivity and specificity such as RCR and ELISA are most
favorable, but such methods are normally more complex, labori-
ous and expensive. An early diagnosis of the dengue virus infection,
necessary for the treatment and to control epidemic, still remains
a challenging task. A situation where the rapid detection is needed,
compromises have to be made on sensitivity and specificity. In
the recent years, Raman spectroscopy has proved itself an emerg-
ing tool for the characterization of biomedical media and achieved
significant progress in clinical diagnostics [8,9].
Raman spectroscopy is based on the molecular vibration that
originates from an inelastic scattering of light by the sample.
As a result of interaction of light with intra-molecular bonds,
Raman scattered photons comes outside with different energy.
This difference in energy (wavelength) between incident and emit-
ted photons, termed as Raman shift, represented as cm−1 . There
are different chemical bonds inside the body fluids and tissue,
each produces its own characteristics spectra. Therefore Raman
spectrum provides a fingerprint from which information about
molecular composition can be obtained that forms the basis for
the diagnosis of the infectious diseases.
In this article, we are presenting the use of Raman spectroscopy
for the diagnosis of dengue virus infection in the human blood sera.
This technique may be more efficient and useful, as it does not
require any complicated and lengthy procedure.
2. Materials and methods

∗ Corresponding author at: Agri-Biophtonics Division, National Institute for Lasers

2.1. Sample collection and preparation
and Optronics (NILOP), Nilore, Islamabad 45650, Pakistan. Tel.: +92 519248671;
fax: +92 512208051. The entire blood samples used in this study have been obtained
E-mail address: k.saranjam@yahoo.com (S. Khan). from Swat region, the northern part of Pakistan. These samples have

http://dx.doi.org/10.1016/j.ijleo.2015.11.060
0030-4026/© 2015 Elsevier GmbH. All rights reserved.
 S. Khan et al. / Optik 127 (2016) 2086–2088
(a)
Raman signal intensity (normalized)
(b)
Raman shift (cm-1)
Fig. 1. Raman spectra of human blood sera: (a) shows the Raman spectra of blood
sera of 25 normal healthy volunteers; (b) shows the Raman spectra of 40 confirmed
dengue virus infected sera.
been confirmed for dengue virus infection on the basis of serologi-
cal tests. In addition, 25 samples have been collected from healthy
volunteers of the same region to make a comparison with the dis-
eased one. The blood samples of about 3 ml have been collected
from each individual with the help of a sterile syringe. All the sam-
ples (healthy and infected) have been centrifuged at 3500 rpm for
10 min using Hittich Centrifuge D-7200. The obtained sera have
been aliquoted in different tubes and stored at −80 ◦ C till use. For
obtaining the Raman spectra a quantity of about 15 ␮l of each sam-
ple has been placed on the glass slide and dried for about 2 h at
room temperature. All the healthy and infected samples have been
processed following the standard safety rules [10].
2.2. Acquiring Raman spectra
Raman Spectra from all the samples have been measured
using Raman spectrometer (␮Ramboss DONGWOO OPRTON, South
Korea) having a spectral resolution of 4 cm−1 . A continuous laser
beam at 532 nm from a laser diode has been used for the exci-
tation. An objective lens with 100× magnification has been used
both for light focusing and collection in backscattering configu-
ration. Raman spectra for the entire samples have been recorded
within the spectral range of 600 cm−1 to 1750 cm−1 . An acquisition
time of 10 s has been used for recording each spectral data.
3. Results and discussion
Raman spectroscopy is a label free technique that identifies dif-
ferent constituents of the biological sample based on its molecular
vibration. The beauty of Raman spectroscopy lies in its specificity
where each biomolecule give rise to its own characteristic spec-
tral peak distinguish them from other molecules. In case of overlap
of Raman peaks of different components, one can distinguished
them in term of principle components such as Protein, lipids and
nucleotides [11,12].
The Raman spectra contain strong fluorescence background
because of the presence of natural fluorophores present in the bio-
logical samples. In addition, it also contains noise that might add
from detector electronics. Therefore all the Raman spectra from
healthy as well as diseased samples have been processed in Matlab
environment for the removal of noise and fluorescence background
[13]. Fig. 1 part (a) shows the fluorescence background subtracted
normalized Raman spectra of blood sera of 25 normal healthy
samples and part (b) shows the normalized Raman spectra of 40
confirmed dengue virus infected sera. The spectra of normal as well
dengue infected samples have been smoothed by using average
filter, Savitzky-Golay, with five points and 3rd order polynomial
2087
fitting [13]. For illustration purpose, the spectra of normal sera
are shown in blue color, whereas the spectra of dengue infected
samples are shown in red color. Raman spectra of normal healthy
samples show major Raman-peaks at 1002, 1156, 1123, 1445, 1516,
and 1668 cm−1 , whereas in the dengue infected samples, additional
lines have also been appeared at 750, 850, 1032, 1306, 1333, 1355,
1580, 1603 and 1660 cm−1 .
Raman peaks at 1156 and 1516 cm−1 have been assigned to ␤-
carotene [13–15]. It has been observed that in the dengue infected
sera the intensities of Raman peaks assigned to ␤-carotene at
1156 and 1516 cm−1 get decreased, indicates the deficiency of ␤-
carotenoinds. Raman peak at 1002 cm−1 appeared both in healthy
and diseased samples spectra as shown in Fig. 1(a) and (b), is the
most dominant signature of protein that is assigned to symmetric
ring breathing mode of phenylalanine and carotene [13–16]. Rising
trend in the Raman line at 1002 cm−1 in dengue infected sera is due
to high concentration of immunoglobulin G (IgG). The Raman lines
at 1123 cm−1 and 1445 cm−1 are present both in normal and dengue
infected blood sera. The line at 1123 cm−1 arises from C C stretch-
ing mode of protein and lipid labeled for IgG [13,14,16], whereas the
Raman line at 1445 cm−1 arises from CH2 bending mode, assigned
for fatty acid, lipid and collagen [13,14,16]. Increase in the inten-
sity of 1123 cm−1 line in dengue infected blood sera is due to high
concentration of IgG anti-bodies produced in response to infection
[14,17]. The Raman line at 1668 cm−1 exists in normal sera samples,
assigned for Amid I [18,19].
There are various Raman lines labeled at 750, 850, 1032, 1306,
1333, 1355, 1580, 1603 and 1660 cm−1 , which appeared only in
the dengue infected sera. A nice comparison of the Raman peaks
observed both in the normal and dengue infected sera are shown in
Table 1. Among all the spectral signatures, Raman lines at 750 cm−1
and 850 cm−1 are the most important lines that appeared in the
dengue infected blood sera. The Raman line at 750 cm−1 is assigned
s for adenosine diphosphate (ADP) [20] as well as for hemoglobin
[14,18,19]. The signal intensity of Raman line at 750 cm−1 assigned
for hemoglobin is small in comparison with other Raman lines at
1585 and 1682 cm−1 reported earlier [19]. Therefore if the Raman
line at 750 cm−1 is responsible for hemoglobin then other reported
lines of the hemoglobin must also be present in the Raman spectra
of dengue infected sera, which are not visible in Fig. 1(b). It implies
that an intense Raman line at 750 cm−1 in the dengue infected sam-
ples must have possible contributions from some other biochemical
changes that have been induced in the body as a result of dengue
virus infection.
It is well known that apart from red blood cells and white blood
cells (WBC), the blood also contain platelets. Platelets in the human
body play an important role in homeostasis and thrombosis. It is
well established that in the dengue virus infected patients the num-
ber of platelets decreases to less than 50,000 per micro liter [21,22]
whereas the normal range in the healthy subjects varies from
150,000 to 400,000 per micro liter. The decrease in platelets counts
is most likely due to two reasons i.e. firstly decreased occurred due
to less production and secondly, destruction in blood stream as a
result of dengue virus infection.
Platelets are produced from the cytoplasmic fragmentation of
megakaryocytes as anucleate cells [23–26]. Bone marrow stromal
cells get infected as a result of dengue virus infection, affecting
normal function of bone marrow that leads to low production of
platelets [27–29]. Destruction of platelets within the blood stream
may probably be due to carotene deficiency causing dryness of a
cellular wall, resulting an ultimate cell death. In normal condition
there are varieties of cytoplasmic structures inside the platelet.
These structures includes lysosomes and two types of granules
i.e. electron dense granules and ␣-granules actin (protein) uni-
formly distributed throughout and an irregular internal network of
canaliculi through which the granules content comes to the platelet
2088 S. Khan et al. / Optik 127 (2016) 2086–2088

Table 1
A comparison of Raman peaks of normal and dengue infected blood sera.

Raman shift (cm−1 ) Tentative assignment Dengue virus Normal healthy

infected blood sera blood sera
√
750 CH2,6 out-of-plane bending, observed in the spectra of single human RBC [14], ADP [20] –
√
850 Proline, Tyrosine [14], Tryptophan-IgG [16] –
√ √
1002 ␤-Carotenoids [16], Phenylalanine-IgG [18]
√
1032 Phenylalanine-IgG [14,16] –
√ √
1123 Labeled for IgG [61], C C stretching mode lipid and protein [14]
√ √
1156 C C and C N stretching of Carotenoids (protein) [14]
√
1306 IgM [16] –
√
1333 Phenylalanine, Tryptophan-IgG [14,16], Hemoglobin [13] –
√
1355 Tryptophan-IgG [16], Hemoglobin [13] –
√ √
1445 CH2 deformation, bending, scissoring labeled for Fatty acid and Lipid [14] − IgG [16]
√ √
1516 Arise from ␤-Carotenoids [14]
√
1580 C C stretching [14] –
√
1603 C C in-plane bending mode of phenylalanine and tyrosine [14] –
√
1660 Amide I band (protein band) [14] –
√
1668 Amide I band (protein band) [14] –

surface. The electron dense granules contain adenosine diphos-
phate (ADP), adenosine triphosphate (ATP), calcium and serotonin,
whereas the clotting factors, growth factors, and other proteins are
stored in ␣-granules. As previously mentioned, carotene deficiency
in blood causes dryness of a plasma membrane as a result most of
the cytoplasmic structures such as ADP, ATP, etc. leaks out of the cell
into the blood plasma [15]. Therefore, the Raman line at 750 cm−1
appeared in the spectra of dengue virus infected samples is likely
due to ADP [22]. Similarly, the Raman peak at 850 cm−1 is due
to the release of cytoplasmic structures from the platelets which
most probably assigned for ADP. In case of dengue virus infection,
ADP gets leak into extra cellular fluid because of the dryness of cell
membrane as mentioned earlier and remains in the sera even after
centrifugation.
4. Conclusion
This article presents the optical diagnosis of dengue virus infec-
tion in human blood sera through Raman spectroscopy. Raman
spectral peak at 750, 850, 1032, 1306, 1333, 1355, 1580, 1603
and 1660 cm−1 appeared only in dengue infected samples. The
Raman line at 750 cm−1 is the most prominent peak labeled for ADP,
releases to extra-cellular media, as a result of cells rupturing due
to dengue virus infection. The results obtained are quite promis-
ing, regarding the investigation of biochemical changes associated
with the dengue virus infection. The research work in our labora-
tory is still in progress striving for the development of an efficient
tool that might help in the early disease detection, which is still a
desired and challenging task.
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