Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

SERS-based viral load quantification of hepatitis B virus from PCR

products
Fatima Batool a, Haq Nawaz a,⇑, Muhammad Irfan Majeed a,⇑, Nosheen Rashid b, Saba Bashir a, Saba Akbar a,
Muhammad Abubakar a, Shamsheer Ahmad a, Muhammad Naeem Ashraf a, Saqib Ali a, Muhammad Kashif a,
Imran Amin c
a
Department of Chemistry, University of Agriculture, Faisalabad 38040, Pakistan
b
Department of Chemistry, University of Central Punjab, Lahore, Faisalabad Campus, Pakistan
c
PCR Laboratory, PINUM Hospital, Faisalabad, Pakistan

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 SERS analysis of PCR product of viral

DNA extracted from various patients
of hepatitis B is conducted.
 SERS features increasing and
decreasing in intensity with change in
viral load are used for diagnostic
purpose.
 Data is analyzed using multivariate
analysis techniques including PCA,
PLS-DA and PLSR.
 PCA differentiates between healthy
and diseased samples with 89% and
98% of sensitivity and specificity
respectively.
 PLSR model depicts 0.9031of
goodness value with RMSE value of
0.2923 and proves to be valid for
prediction of blind sample.

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

Article history: Hepatitis B is a contagious liver disorder caused by hepatitis B virus and if not treated at an early stage, it
Received 22 January 2021 becomes chronic and results in liver cirrhosis and hepatocellular carcinoma which can even lead to death.
Received in revised form 5 March 2021 In present study, surface-enhanced Raman spectroscopy (SERS) is employed for the analysis of poly-
Accepted 15 March 2021
merase chain reaction (PCR) products of DNA extracted from hepatitis B virus (HBV) infected patients
Available online 19 March 2021
in comparison with healthy individuals. SERS spectral features are identified which are solely present
in the HBV positive samples and consistently increase in intensities with increase in viral load which
Keywords:
can be considered as a SERS spectral marker for HBV infection. For sake of understanding, these various
SERS
Hepatitis B, Viral DNA
levels of viral loads in this study are classified as low (1–1000 IU), medium (1000–10,000 IU), high (above
Viral load 10,000 IU) and negative control (>1). In order to explore the efficiency of SERS for discrimination of SERS
Silver nanoparticles spectral datasets of different samples of varying viral loads and healthy individuals, principal component
Principal component analysis analysis (PCA) is applied. PCA is used for comparison of these classes including low, medium and high
Partial least square regression analysis levels of viral loads with each other and with healthy class. Moreover, partial least square discriminant
analysis and partial least square regression analysis are employed for the classification of different levels
of viral loads in the HBV positive samples and prediction of viral loads in the unknown samples,

⇑ Corresponding authors.
E-mail addresses: haqchemist@yahoo.com (H. Nawaz), irfan.majeed@uaf.edu.pk
(M.I. Majeed).

https://doi.org/10.1016/j.saa.2021.119722
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al.
respectively. PLS-DA is applied for validity of classification and its sensitivity and specificity was found to
be 89% and 98% respectively. PLSR model was constructed for prediction of viral loads on the bases of
SERS spectral markers of HBV infection with goodness value of 0.9031 and value of root means square
error (RMSE) 0.2923. PLSR model also proved to be valid for prediction of blind sample.
1. Introduction
Hepatitis B virus infection is a contagious but remediable liver
disease which is now becoming one of the major cause of death
throughout worldwide. HBV belongs to hepadnaviridae family
and causes transient and chronic liver disorders. Transient infec-
tions persist for months while chronic infections are long lasting
which can often result in liver failure with cirrhosis and hepatocel-
lular carcinoma [1,2]. Replication of HBV DNA begins in hepato-
cytes cells and the relaxed circular HBV DNA (RC-DNA) invades
into hepatocytes nucleus, where it is converted into covalently
closed circular DNA (cccDNA), from which various genomic RNAs
are transcribed by RNA polymerase 2. The pregenomic RNA is
packed into capsids and further reverse transcribed by P proteins
to form new RC-DNA. This matured RC-DNA is further used for
cccDNA amplification, these are enveloped and liberated from cell
as virions [3]. For better treatment of this disease an efficient, easy
and reliable diagnostic tool is required. It is evident to monitor
every patient’s virologic profile during antiviral therapy, in order
to measure disease progression. Viral load is an important param-
eter to examine disease progression and patient’s immune
response to given therapy of disease.
As previously reported, PCR assays are commonly used for qual-
itative purposes and real time PCR is used for quantitation of
nucleic acids. Using PCR, quantity of target nucleic acid can be
determined by two ways including relative quantitation and abso-
lute quantitation. The earlier method reveals variation in concen-
tration of target sequence as compare to its level in related
matrix while absolute quantitation shows exact quantity of nucleic
acid sequence related to specific unit. Sufficient information can be
obtained from relative quantitation while monitoring the progress
of infection [4]. PCR is a reliable technique for quantitation of
cccDNA in infected liver cells which provides estimation of viral
load of each individual patient. PCR process is repeated many times
for making desired number of copies.
The PCR products are usually analyzed using Gel documenta-
tion system (GDS) which is used for imaging and separation of
nucleic acids [5]. Agarose gel strength offers excellent handling
of less percentage of gels for separation of huge number of DNA
fragments. These gels are commonly strained using ethidium bro-
mide or other strainers like GelGreen. For visualization purpose,
Gel doc includes UV transilluminator [6]. Gel doc method has some
limitations including continuous irradiation of UV light weakens
the fluorescence of bands and also the samples with less molecular
weight or less amount further limit the use of Gel doc. Moreover
Gel doc requires expensive instrumentation and materials [7]. In
order to overcome these limitations, development of new methods
is required to analyze the PCR products more accurately for the
qualitative and quantitative purposes.
Recently, surface-enhanced Raman spectroscopy (SERS) has been
employed for various applications including disease diagnosis such
as tuberculosis [8] hepatitis C [9] dengue [10] and almost all types of
cancers like breast [11] prostate [12] and cervical cancer [13] etc. As
SERS provides clear differentiation so it is also used to for discrimi-
nation among stages of diseases. SERS is inexpensive, reliable tech-
nique and gives straightforward identification of sample with no
interference of water and requires no sample preparation [14].
2
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722
Ó 2021 Elsevier B.V. All rights reserved.
In the present study, SERS-based method is developed to ana-
lyze the PCR products of HBV DNA extracted from HBV infected/-
suspected patients with different levels of viral loads. These HBV
diseased samples are analyzed by comparing them with negative
control samples and true healthy samples. There is no such study
published yet which combines two important techniques of PCR
and SERS for demonstration of method which has great potential
to be employed for the quantitative characterization and diagnosis
of HBV.
2. Materials and method
2.1. Preparation of silver nanoparticles (AgNPs)
AgNPs are prepared by using chemical reduction method.
Briefly, one molar solution of Silver nitrate was prepared by adding
33.72 ml of silver nitrate to 200 ml of deionized water until it starts
to boil then it is added to 8 ml of 1% trisodium citrate (Na3C6H5O7)
with stirring at 600 rpm for about one hour. Solution is set free to
cool down at room temperature. Volume of 200 ml was maintained
by adding deionized water. The fresh prepared 2 ml suspension of
AgNPs are centrifuged at 600rmp for 7 min to remove supernatant
from it [15]. The size and morphology of nanoparticles were deter-
mined with electron microscopy for which methodology has been
described in our previously published work [9]. The AgNPs were
characterized with TEM and were found to be the spherical in
shape with an average diameter of 53 nm as shown in the supple-
mentary material (Fig. S1).
2.2. Sample collection
Blood samples of clinically diagnosed HBV infected individuals
were collected from Punjab Institute of Nuclear Medicine (PINUM),
Faisalabad. Sample preparation and DNA purification was per-
formed using Cobalt X 480 Hamilton instrument. DNA extraction
and DNA amplification (PCR products) was carried out using Natch
S biotech sansure instrument in PCR Lab of PINUM, Faisalabad, Pak-
istan. In this regard, as shown in Table 1, total 53 samples were col-
lected and their viral loads were predetermined. Out of these, 37
samples are HBV positive with different viral loads (VL) which
are further classified as low VL (1–1000 IU), Medium VL (1000–
10,000 IU) and high VL (above 10,000 IU), 13 are negative con-
trolled samples (pseudo healthy) with viral load of less than one
(<1) and three of them are true healthy samples with zero viral
load.
2.3. Polymerase chain reaction (PCR) protocol
Reagents used for PCR amplification include Primers, template
DNA, Buffer, Taq polymerase enzyme and nucleotide. For quantifi-
cation of HBV DNA using Real-time PCR, amplification was carried
out using set of primers and probes according to the surface anti-
gen (s gene) of HBV. The primers were: HBV_S_F: 5́-CCTCTT
CATCCTGCTGCT-3́, HBV_S_R: 5́-AACTGAAAGC CAAACAGTG-3́.
TaqMan probe used for PCR was: 5́-TET-TCCCATCCCAT
CATCCCTGGGCTTT-TAMRA-3́. For PCR, 900 nM of both primers
and 250 nM of the probe was used in 20 ml volume and initiative
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722

Table 1
Sample details of HBV positive, low VL, medium VL, high VL, negative control (pseudo healthy) and true healthy samples.

Sr. No. Sample Name Viral Load (IU) Log VL Class

1 S17 17.5 1.243038 Low
2 S28 27.9 1.445604 Low
3 S15 40.76 1.610234 Low
4 S18 56.18 1.749582 Low
5 S8 63.14 1.800305 Low
6 S21 82.81 1.918083 Low
7 S27 89.03 1.949536 Low
8 S13 118.4 2.073352 Low
9 S26 151.4 2.180126 Low
10 S34 166.23 2.220709 Low
11 S19 241.7 2.383277 Low
12 S29 299.5 2.476397 Low
13 S16 572.9 2.758079 Low
14 S12 581.8 2.764774 Low
15 S3 899 2.953760 Low
16 S6 1157 3.063333 Medium
17 S11 1158 3.063709 Medium
18 S10 1511 3.179264 Medium
19 S2 1662 3.220631 Medium
20 S30 2243 3.350829 Medium
21 S7 2355 3.371991 Medium
22 S20 2371 3.374932 Medium
23 S9 2390 3.378398 Medium
24 S37 2419 3.383636 Medium
25 S14 3266 3.514016 Medium
26 S1 3615 3.558108 Medium
27 S36 4483 3.651569 Medium
28 S31 5981 3.776774 Medium
29 S33 17,860 4.251881 High
30 S5 29,310 4.467016 High
31 S4 35,730 4.553033 High
32 S24 42,820 4.631647 High
33 S23 584,000 5.766413 High
34 S22 4,747,000 6.676419 High
35 S32 133,500,000 8.125481 High
36 S25 335,400,000 8.525563 High
37 S35 389,400,000 8.590396 High
38 S38 Negative control <1 Healthy
39 S39 Negative control <1 Healthy
40 S40 Negative control <1 Healthy
41 S41 Negative control <1 Healthy
42 S42 Negative control <1 Healthy
43 S43 Negative control <1 Healthy
44 S44 Negative control <1 Healthy
45 S45 Negative control <1 Healthy
46 S46 Negative control <1 Healthy
47 S47 Negative control <1 Healthy
48 S48 Negative control <1 Healthy
49 S49 Negative control <1 Healthy
50 S50 Negative control <1 Healthy
51 S51 True healthy 0 Healthy
52 S52 True healthy 0 Healthy
53 S53 True healthy 0 Healthy

denaturing step of PCR cycle was carried out at 95 °C for ten min-
utes followed by 45 PCR cycles for 15 s at 95 °C [16]. The PCR prod-
ucts were further analyzed by Surface Enhanced Raman
Spectroscopy.
2.4. SERS data acquisition
SERS data acquisition was carried out using Raman Microscope
Spectrometer (ATR8300BS) Optosky China equipped with diode
laser of 785 nm with a lens of 40X/0.6 leading to spot size of
2 mm. For this purpose, 30 ml of each sample was mixed with
30 ml of silver nanoparticles and left for 30 min as incubation time
to develop good interaction between sample and nanoparticles.
From this incubation mixture, 40 ml of each sample was put on
the aluminum slide grove and fifteen spectra were acquired from
each sample by using 100 mW laser power and 2 s as integration
time.
3
2.5. Data preprocessing and analysis
Preprocessing of raw SERS spectral data is performed in
MATLAB software version 7.8.0.347. Data is preprocessed using
algorithms for smoothening, baseline correction and vector nor-
malization and substrate removal by taking data in a single matrix.
The Savitzky-Golay algorithm is used for smoothening while Rub-
ber band and polynomial methods are used for baseline correction
of the spectral data. All the samples were given exactly same pre-
processing treatment in one matrix. The detailed peak assignment
of SERS spectral data is given in Table 2. Data was further analyzed
by multivariate data analysis techniques. Principal component
analysis (PCA), a statistical tool, was applied to SERS spectral data
to determine variability and differentiation in the different data
sets and the reason of this differentiation. PCA operates by reduc-
ing the dimensionality of data while variability remains unaffected
[17]. For selection of discriminative features for prediction of clas-
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al.
Table 2
DNA based Peak Assignment for SERS spectral data obtained from literature. Citations given in the last column of Table.
1
Assignments Raman Bands (cm ) References
Adenine 733(vs) 816(w)
Guanine 655(vs) 852(m)
Thymine 816(w) 1214(m)
Cytosine 617(w) 776(m)
Phosphate 1098(w) – –
Deoxyribose 945(w) 957(s)
Aromatic ring 1111(w) – –
Abbreviations: w; weak; m; medium; s; strong; vs; very strong.
sification and checking the authenticity of classification, Partial
least square discriminant analysis (PLS-DA) is a swift and easy tool.
PLS-DA is a versatile algorithm, applied to SERS spectral data for
prediction and descriptive modelling and classification of data into
various sets in order to classify different levels/ stages of the dis-
ease [18].
Partial least square regression (PLSR) is a statistical tool applied
to datasets, which limits the predictors to small groups of uncorre-
lated components to bring about least square regression in these
components instead of applying on original dataset [19]. Here PLSR
model is constructed for predicting HBV viral loads which are
dependent variables for SERS spectral data. SERS dataset is
regressed to explore the difference between measured and pre-
dicted variables which elaborate efficiency of PLS model. In PLSR,
root mean square error of cross validation (RMSECV) is used to
explore the significance or goodness of technique. Leave K-out
cross validation (LKOCV) is used to construct a model with mini-
mum latent variables in order to avoid overfitting of dataset. Data
is randomly selected and divided into two parts, from which 60% of
data is for calibration and 40% is left as test dataset [20].
3. Results and discussion
Characterization of silver nanoparticles was carried out using
transmission electron microscopy (TEM) which revealed that Ag-
NPs possess oval shaped geometry with average size of
65  45 nm as displayed in the Fig. 1(S). Studies shows that parti-
cles having such size range shows excellent SERS activity [21].
For comparison of samples on the basis of their viral loads, all
53 samples are divided into five classes high VL, Medium VL,
Low VL, Negative control and true healthy. Each class contains dif-
ferent samples of same ranges of viral load while negative control
is the one having viral load of below detection limits of PCR tech-
nique and true healthy possess zero viral load.
3.1. Mean SERS spectra
Fig. 1 shows mean SERS spectra of PCR products of true healthy,
negative control and different HBV positive samples including low
VL, medium VL and high VL. The spectral peaks observed are
mainly categorized into four classes on the basis of their intensity
including very strong (vs), strong (s), medium (m) and weak (w).
The significant SERS spectral features are labeled using solid lines,
dotted lines and dashed lines. The SERS bands which increase in
their intensities with increasing viral load are indicated by solid
lines including, 733(vs), 957(m), 1032(vs), 1111(w), 1221(s),
1419(vs), 1566(m), 1655(w), 1788(m) cm 1. SERS bands that go
on decreasing with increasing viral load are indicated by dotted
lines, are observed at 506(w), 617(w), 776(m), 816(w), 945(m),
1010(m), 1098(w), 1214(m), 1373(w), 1511(w) and 1702(w)
cm 1. Moreover, the peaks which are observed with a shift in the
HBV positive samples as compared to true healthy and negative
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722
1337(m) 1419(vs) [29,22,30,23,24]
1032(vs) 1566(m) 1605(w) [24,23,31]
1373(w) 1655(w) 1702(w) [29,22,25–27]
1221(m) 1511(w) [32,33,30,24,25]
– – [34]
1010(m) 1455(s) – [24,35,36]
– – [37]
control ones are shown by dashed lines and labelled in a red box,
include 655(vs), 852(s) 1337(m), 1455(s) and 1605(w) cm 1. In
Fig. 1 SERS band of guanine appears at 655(vs) and 852(s) cm 1
and bands of adenine appears at 1337(m) and 1605(w) in diseased
classes only while it has very low intensity in healthy class, which
depicts structural alterations of DNA of HBV. Similarly, at
1455 cm 1 band of deoxyribose appears in diseased class and
absent in healthy class which also depicts changes that occur in
structure of DNA after HBV infection.
The prominent spectral features of adenine are observed at 733
(vs), 816(w), 1337(m), 1373(w), 1419(vs) and 1605(w) cm 1. The
most dominant peak of adenine is observed at 733 cm 1, and its
intensity increases with increase in viral load and becomes very
dominant in high viral load class [22]. The weak band at
816 cm 1 is due to poly nucleotide duplexes poly[d(A)]. poly[d
(T)], this band depicts unique characteristics for backbone confor-
mation of DNA. In the mean spectra of disease this band go on
decreasing as compared to healthy ones and eventually disappear
in high VL class as shown in Fig. 1. The SERS band at 1337 cm 1
is due to CAN stretching vibration of pyrimidine ring. At
1419 cm 1 Raman peak of guanine is observed. Because of defor-
mation vibration of NH2 in Adenine a weak band appears at
1605 cm 1 [23,24].
The SERS bands of guanine appears at 655(vs), 852(m), 1419(s),
1605(w), 1032(vs), 1566(m) cm 1. Different extent of enhance-
ment in the intensities of these peaks associated with guanine
takes place with increase in viral load and appear much diminished
in negative control and true healthy classes. The SERS spectral fea-
tures at 816(w), 1214(m), 1373(w), 1655(w) and 1702(w) cm 1 are
assigned to thymine. The SERS band at 1214 cm 1 is denoted by
dotted line is prominent in healthy samples but decreases in inten-
sity as the viral load increases. It clearly shows the presence of thy-
midine [25]. The band of 1373 cm 1 shows the ring breathing
vibration modes of DNA bases (thymine), shows significant reduc-
tion in intensity as the viral load increases [26]. Another SERS band
appearing at 1655 cm 1 of thymine shows stretching of C2 = O-
C2N3 bond. It slightly appears in diseased classes and eventually
disappear in negative control and healthy class, denoted by dashed
lines [25]. A weak band of thymidine appears to be at 1702 cm 1
which decreases as the viral load increases but it is absent in true
healthy class [27].
The bands of cytosine appear to be at 617(w), 776(m), 1221(m),
1511(w) and 1605(w) cm 1. The bending vibration of cytosine
shows a weak band at 617 cm 1 [25] and the peak at 776 cm 1
is due ring breathing in cytosine, which is found to decrease in
intensity with increase in viral load. A weak band appearing at
1098 cm 1 is associated to phosphate group used in linkage of
DNA bases making a long polymer of nucleotide [28]. A medium
intensity peak at 945 cm 1 shows the presence of deoxyribose
complex is found with enhanced intensity. Further deoxyribose
bands appear at 947(w), 957(s), 1010(m), 1455(s) cm 1. The peaks
labelled in red boxes are 655(vs), 852(s) 1337(m), 1455(s) and
1605(w) cm 1. These peaks are characteristics peaks as they
4
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722

Fig. 1. Mean plot of SERS dataset. Black color is for true healthy, mustard yellow is for negative control, magenta is low VL, blue is medium VL and red is high VL. Solid lines
represent the peaks of ascending order which are increasing by increasing viral load, dotted lines represent the peaks of descending order which are decreasing with
increasing viral load and dashed lines indicates the peaks that are present in diseased samples but absent in true healthy and negative control samples (characteristics peaks).

Fig. 2. PCA scatter plot of SERS data of diseased (red), negative control (green) and true healthy (magenta) samples indicating different classes.

appear in diseased class and absent in healthy class which shows
changes that occur in structure of DNA after HBV infection.
3.2. Principal component analysis (PCA)
In order to interpret SERS spectral datasets of different groups
of viral load along with true healthy and negative controls, princi-
pal component analysis (PCA) is applied to reduce dimensionality
5
of SERS spectral data in such a way that no information is lost.
PCA was carried out using all SERS spectral data, to identify and
confirm the SERS features associated with HBV and to compare dif-
ferent classes of samples having different levels of viral load. Fig. 3
shows PCA scatter plot of different classes of samples having differ-
ent levels of viral load along with healthy samples. The SERS spec-
tral data of different groups is clustered and separated by PC-1 on
X-axis and indicated with different colors.
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al.
In Fig. 2 39% variability of data is explained by PC-1 and 17% by
PC-2. It is evident from the PCA scatter plot that classes are sepa-
rated on the basis of principal component 1 (PC-1) indicating that
due to different levels of viral load, the SERS spectral features are
different which cause the differentiation of these data sets. It is
observed that SERS spectral data of HBV diseased samples (red)
are shown on the negative side of PC-1 and true healthy (magenta)
along with negative control (green) samples are clustered on the
positive side of PC-1. Fig. 2 shows PCA scatter plots of different
samples of different viral loads from same class showing differen-
tiation of spectral data differentiated on the basis of their viral
loads. SERS spectral data of different samples is shown in the form
of clusters (a) PCA scatter plot of spectral data of 15 different sam-
ples of ‘‘class low VL”, each having fifteen spectra (b) PCA scatter
plot of spectral data of 13 different samples of ‘‘class Medium
VL” (c) PCA scatter plot of spectral data of 9 different samples of
‘‘class High VL”.
To further elaborate the differentiation of different levels/-
classes of viral load of different samples, pairwise PCA analysis
was performed in order to show the comparison within the same
class of viral loads which are categorized as different classes/-
groups including low (1–1000 IU), medium (1000–10,000 IU), high
(above 10,000 IU) and negative control (>1), as mentioned in
Table 1.
Fig. 4 shows the pairwise comparison of SERS spectral data of
different samples of different classes having different viral loads
including low VL, medium VL and high VL as PCA scatter plots.
Fig. 4(a-i) shows PCA scatter plot of SERS spectra of class Low VL
(15 samples) versus high VL (9 samples) indicated by green color
and red color dots, respectively. It is observed that low VL and high
VL classes are differentiated on the basis of PC-1 as explained vari-
Fig. 3. (a) PCA scatter plot of class Low VL containing 15 samples (b) PCA scatter plot of class Medium VL containing 13 samples (c) PCA scatter plot of class High VL
containing 9 samples.
6
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722
ance of PC-1 is 36% and PC-2 is 18%. Class Low VL is clustered on
the negative side of PC-1 and class high VL on the positive side
of PC-1 and no spectra (dots) are showing mixing which indicate
that Low VL and high VL samples are nicely differentiated due to
different viral load. Notably, as mentioned in Table 1, ‘‘Class low”
has samples with viral load less than 1000 IU/ml and ‘‘class high”
viral load has samples with range of viral load greater than
5000 IU/ml. Fig. (a-ii) shows loadings of class low VL vs high VL.
Raman bands labeled by solid lines are found increasing in intensi-
ties by increasing viral load, while dotted lines include the features
that are decreasing in intensities by increase of viral load. And
dashed lines indicates those features which are present in diseased
classes of HBV and observed shifts in their wavenumbers as com-
pared to negative control and true healthy samples. Positive load-
ings are observed at 733(vs), 957(m), 1032(vs), 1111(w), 1221(s),
1419(vs), 1566(m), 1655(w), 1788(m) cm 1, these are the features
that are increasing by increasing viral load, these characteristics
features are SERS biomarkers of HBV samples clustered on the pos-
itive side of the PC-1 in scatter plot. It is also observed in mean plot
that these samples possess high content of virus. The negative
loadings can be associated with the features that are decreasing
by increasing viral load and clustered on the negative side of PC-
1 in scatter plot. These are observed at 506(w), 617(w), 776(m),
816(w), 945(m), 1010(m), 1098(w), 1214(m), 1373(w), 1511(w)
and 1705(w) cm 1.
Fig. 4(b-i) shows PCA scatter plot of SERS spectra of class Low VL
(15 samples) versus Medium VL (13 samples) which shows separa-
tion of both the spectral data sets on the basis of PC-2 showing
variance of 18%. Blue dots are on the positive side of zero line indi-
cating medium VL class while green dots are on the negative side
indicating low VL. Notably, the ‘‘class low VL” includes samples
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722

Fig. 4. PCA pair wise comparison plot of all diseased classes of viral loads and their loadings. a(i) PCA comparison plot of class low VL vs high VL, light green dots denote class
low VL and red color is for high VL a(ii) loadings of low VL vs high VL. b(i) PCA comparison plot of class low VL (green color) vs medium VL (blue color) b(ii) loadings of low VL
vs medium VL c(i) PCA comparison plot of medium VL (blue color) vs high VL (high VL) c(ii) loadings of medium VL vs high VL.

with viral load of less than 1000 and class medium has viral load
within range of above 1000 and less than 10,000 IU/ml. Few sam-
ples are intermixing which is due to very minute difference of viral
load between two classes. Fig. 4(b-ii) shows PCA loadings of class
low VL versus class medium VL. The SERS spectral features that
are increasing are considered as positive loadings and lie on the
positive side of PC-1. These features contained high virus concen-
tration as observed in mean plot because it increases linearly.
These are noted at 733(vs), 957(m), 1032(vs), 1111(w), 1221(s),
1419(vs), 1566(m), 1655(w), 1788(m) cm 1. While negative load-
ings are clustered on the negative side of principle component 1
and decreases gradually as the viral load increases. Their virus con-
centration is decreased down the class. These features are observed
at 506(w), 617(w), 776(m), 816(w), 945(m), 1010(m), 1098(w),
1214(m), 1373(w), 1511(w) and 1705(w) cm 1 as observed in
mean plot.
Fig. 4(c-i) shows PCA scatter plot of SERS spectral data of med-
ium VL (13 samples) versus high VL (9 samples). These two classes
are separated on the bases of PC-2 by percentage variance of 17%,
class high VL (red colored dots) are on the positive side of a line
intersecting y axis and class medium VL (blue colored dots) are
on the negative side. Range of viral load for medium class is from
1000 to 10,000 IU/ml for ‘‘class high” is above 10,000 IU/ml. As
viral load between these two classes have less difference so it is
also showing a little mixing of spectral data sets of few samples
The SERS spectral datasets are found clustered with clear differen-
tiation between various classes of different viral loads. Fig. 4(c-ii)
shows loadings of class medium VL versus class high VL. Each SERS
feature is labelled by solid, dotted and dashed lines respectively.
Most prominent SERS features/loadings include 733 cm 1 and
1418 cm 1, which are the highest peaks of SERS dataset indicating,
among others, the reason of differentiation between two data sets.
Positive loadings are observed at 733(vs), 957(m), 1032(vs), 1111
(w), 1221(s), 1419(vs), 1566(m), 1655(w), 1788(m) cm 1 and neg-
ative loadings are observed at 506(w), 617(w), 776(m), 816(w),
945(m), 1010(m), 1098(w), 1214(m), 1373(w), 1511(w) and 1705
(w) cm 1.
Fig. 5 shows pairwise PCA scatter plot and loadings for compar-
ison of all diseased classes versus negative control class. Negative
control class is indicated by black color and other classes are indi-
cated by different colors. Fig. 5(a) Shows comparison of low VL
class versus negative control class, magenta dots are for spectral
data of low VL (15 samples) which lie on positive side of PC-1 line
and black dots are indicating negative control class (13 samples)

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F. Batool, H. Nawaz, Muhammad Irfan Majeed et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722

Fig.5. Pairwise PCA scatter plots of HBV positive samples including comparison of different classes of low VL, medium VL, high VL with negative control samples (a) Low VL vs
negative control (b) medium VL vs negative control (c) high VL vs negative control (d) loadings of low, medium and high VL class vs negative control.

that lie on the negative side of PC-1. Fig. 5(b) Shows comparison of
medium VL versus negative control where blue dots are for spec-
tral data of medium VL class (13 samples) which lie on the positive
side of PC-1 line and black dots are indicating negative control
class (13 samples) that lie on the negative side of PC-1. Fig. 5(c)
Shows comparison of high VL class versus negative control class
and spectral data of high VL (8 samples) class is clustered one
the positive side of PC-1 line and black dots are indicating negative
control class (13 samples) that lie on the negative side of PC-1.
Fig. 5(d) Shows loadings of all diseased classes in comparison with
negative control class. The positive and negative loadings indicate
the SERS spectral features associated with development of the
severity of the disease with the increase of viral load of HBV and
can be considered as SERS spectral markers of HBV infection. Pos-
itive loading are the features that are increasing by increasing viral
load and these are the SERS spectral features that is clustered on
the positive side of PC-1 in scatter plot. Negative loadings are the
features that are decreasing by increasing viral load and these fea-
tures are clustered on the negative side of PC-1 line in scatter plot.
The positive loadings are observed at 658, 733, 850, 1031, 1111,
1337, 1418, 1456, 1566, 1604 and 1788 cm 1, and negative load-
ings are observed at 506, 617, 776, 816, 945, 1010, 1098, 1211,
1373, 1511 and 1705 cm 1.
3.3. Partial least square discriminant analysis (PLS-DA)
Partial least square discernment analysis (PLS-DA) is a data
modelling technique that is applied for predictive and descriptive
modelling in addition to the selection of variables. PLS-DA is a ver-
satile algorithm which is recently used in many aspects in forensic
sciences for evidence analysis. It is widely used for disease classifi-
cation in medical sciences. Here PLS-DA is used for checking the
validity of classification system of True healthy, pseudo healthy
and diseased class. PLS-DA is applied to all five classes of SERS
spectral Dataset (low VL, medium VL, high VL, negative control
and true healthy). For this purpose, 60% of the SERS data was
selected was for validation and for selection of optimal number
of latent variables and the remaining 40% data was used for calcu-
lation of number of latent variables (NLV). The selected number of
latent variables for SERS data was 17 as shown in the scores plot of
LV-1 and LV-2 in Fig. 6(a), the score plot gave out clear differenti-
ation between HBV diseased, true healthy and pseudo healthy
class. PLS-DA was developed in order to check the authenticity of
constructed classes. Negative control is indicated by black color
and true healthy is indicated by blue color and both of their SERS
spectral data sets are clustered on the positive side of the line
intersecting y axis and all HBV positive classes are clustered on

8
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al.
Fig. 6. (a) PLS-DA Scatter diagram of HBV diseased class (red) vs negative control (Black) and true healthy (Blue) class. (b) Receiver operating characteristic curve (ROC)
showing area under curve (AUC).
the negative side. For PLS-DA model, 19 latent variables are
selected which are used to check diagnostic ability of this tech-
nique. Fig. 6(a) indicates the score plot of PLS-DA for SERS dataset
of all the different classes/groups of samples (low, medium and
high VL) viral load and shows that healthy and diseased classes
are well differentiated from each other on the bases of presence
of different levels of viral load. The SERS spectral data of the three
classes of disease show some mixing because of less difference of
viral load between them but they show good differentiation from
healthy samples. Calculated parameters of PLS-DA are accuracy
of 95%, precision of 74% sensitivity of 89% and specificity of 98%
which reveal that constructed model is valid and can be useful
for the classification of different HBV positive samples and discrim-
ination of healthy samples from HBV positive samples. It is
observed that PLS-DA plot explains the difference more clearly
between the classes then PCA scatter plot.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722
The extent of classification is further explained by generating
ROC curve which is used for cross validation of classification
model. Fig. 6(b) shows that the obtained value of area under curve
(AUC) for SERS dataset is 0.78. The maximum value for AUC is 1
which shows the 100% accuracy of model and zero value indicates
that model is not valid. As AUC value of SERS dataset is near about
1 so it can be stated that constructed model is valid and shows
excellent performance for classification.
3.4. Partial least square regression
PLSR is applied to further extend the capability of SERS method
for prediction of VLs. For construction of PLSR model, and to avoid
biasedness of dataset, SERS spectral data (555 spectra) are taken in
a single matrix and randomly divided into two halves 60% (333
spectra) and 40% (222 spectra), used as calibration dataset and val-
F. Batool, H. Nawaz, Muhammad Irfan Majeed et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722

idation dataset/ test dataset, respectively. Thirteen latent variables Table 3

are used for constructing PLSR model of 37 samples shown in Fig- Evolution Parameters of PLS-DA plot.

ure (S2). Fig. 7(a) shows Calibration PLSR model and (b) Predicted Parameters Values
PLSR model. Different evolution parameters, which predict the per- Area under curve 0.78
formance of PLSR are given in Table 3. The calculated value of Accuracy 95%
goodness (R2 calculated) for calibration is 0.9031 and for prediction Precision 74%
is 0.8376 which reveal the authenticity of regression plot. The Sensitivity 89%
Specificity 98%
adjusted R2 value for calibration is 0.87 and for prediction is 0.69
respectively. The errors recorded for regression model were mini-
mum in value which further confirm the validity of PLSR model. To further elaborate the validity of our constructed PLSR model,
Errors calculated for calibration are (MSE = 0.13, MAE = 0.34, one sample was considered as blind sample. PLS regression tool was
MRE = 0.29 and RMSE = 0.29) and for prediction/validation are applied for unknown blind sample S34. The predicted value of blind
(MSE = 0.08, MAE = 0.31, MRE = 0.23 and RMSE = 0.37), respectively sample S34 was found to be 2.558 as shown in Fig. 7(b) which is
(see Table 4). very close to 2.220 indicating that constructed model is valid.

Fig. 7. (a) PLSR model of calibration for SERS dataset of all diseased, 37 samples. (b) PLSR model of prediction (validation model) for SERS dataset of all diseased, 37 samples.
Red squared dot indicates blind/unknown sample (S37).

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F. Batool, H. Nawaz, Muhammad Irfan Majeed et al.
Table 4
Details of evolution parameters of PLSR model including both calibration and
validation dataset.
Evolution Parameters Calibration Dataset Validation Dataset
RMSE 0.29 0.37
R2 0.90 0.84
Adjusted R2 0.87 0.69
Mean absolute error 0.34 0.31
Mean relative error 0.29 0.23
Mean square error 0.13 0.08
4. Conclusions
Surface enhanced Raman spectroscopy is found to be an excel-
lent technique for analyzing PCR products of viral DNA of HBV as it
gave out clear identification of biochemical features associated to
HBV in blood samples. These SERS features related to DNA can be
used for differentiation of increasing viral load of the Hepatitis B.
PCA of SERS datasets of HBV positive samples in comparison with
healthy samples was performed to further elaborate the differenti-
ation of samples of diseased and healthy class. Moreover, pair-wise
PCA analysis was also found helpful for the differentiation of SERS
data sets of various classes such as low VL, medium VL, High VL,
Negative control (pseudo healthy) and True healthy classes. PLS-
DA was performed to further check the validity of classification
and its sensitivity and specificity was found to be 89% and 98%
respectively. PLSR model was constructed for prediction of viral
loads in HBV positive samples by keeping SERS spectral data of
an unknown viral load as a blind sample. The RMSEC value of PLSR
model was 0.2923 and value of goodness (R2) was found to be
0.9031, which ensure the validity of model.
CRediT authorship contribution statement
Fatima Batool: Writing - original draft. Haq Nawaz: Supervi-
sion, Project administration, Validation. Muhammad Irfan
Majeed: Conceptualization, Writing - review & editing, Validation.
Nosheen Rashid: Conceptualization, Writing - review & editing,
Validation. Saba Bashir: Conceptualization, Resources. Saba
Akbar: Writing - review & editing. Muhammad Abubakar: Data
curation. Shamsheer Ahmad: Software. Muhammad Naeem Ash-
raf: Software. Saqib Ali: Formal analysis. Muhammad Kashif: For-
mal analysis. Imran Amin: Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The authors are obliged to Higher Education Commission (HEC)
of Pakistan for providing financial funding for this research work
[Grant # 6400/NRPU and 8494/NRPU].
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.119722.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119722
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