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Biosensors and Bioelectronics 33 (2012) 190–195
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics
journal homepage:
Immobilization of enzyme on long period grating fibers for sensitive glucose
Akash Deep, Umesh Tiwari ∗ , Parveen Kumar, Vandana Mishra, Subhash C. Jain, Nahar Singh,
Pawan Kapur, Lalit M. Bharadwaj
Central Scientific Instruments Organisation, Sector (CSIR-CSIO) 30 C, Chandigarh, India
a r t i c l e
i n f o
Article history:
Received 15 September 2011
Received in revised form
14 December 2011
Accepted 28 December 2011
Available online 6 January 2012
Long period grating
Glucose oxidase
FTIR spectroscopy
Raman spectroscopy
a b s t r a c t
Glucose oxidase (GOD) immobilized long period grating (LPG) fibers have been proposed for the specific and sensitive detection of glucose. The treatment of LPG fibers with aminopropyl triethoxysilane
has induced biding sites for the subsequent GOD immobilization. Field emission scanning electron
microscopy, confocal laser scanning microscopy, infrared spectroscopy and Raman spectroscopy have
provided detailed evidences about the effectiveness of the adopted biofunctionalization methodology.
The enzyme activity is conserved during the immobilization step. Fabricated LPG sensor was tested on
different glucose solutions to record the transmission spectra on an optical spectrum analyzer. The wavelength shifts in the transmission spectra are linearly correlated with the glucose concentration in the
range of 10–300 mg dL−1 . The fabricated sensor gives fast response and is demonstrated to be of practical
utility by determining glucose contents in blood samples. Proposed technique can further be extended
to develop LPG fiber based novel, sensitive and label free nanosensors for disease diagnosis and clinical
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Fiber-optic based biosensors have been reported useful for medical and environmental applications (Brogan and Walt, 2005; Bosch
et al., 2007; Atias et al., 2009; Wolfbeis, 2006). Long period grating (LPG) fibers form a special class of transducers, and have been
proposed for some biosensing applications (Kuhlmey et al., 2008;
Barnes et al., 2008, 2010). The LPG fiber sensors produce highly sensitive resonance wavelength shift signals upon induced changes in
the ambient refractive index to offer direct and label free detections. The ability of LPGs to couple light from the fiber core to the
fiber cladding helps in direct probing of the surrounding media. Any
change in the refractive index of the surrounding media results into
spectral shifts, whose demodulation and proper correlations leads
to highly sensitive quantitative chemical analyses.
Apart from the already cited references, some other important
citations on the applications of LPG fibers report the detection
of antigen (DeLisa et al., 2000), pH (Goicoechea et al., 2008) and
medically relevant parameters (Mishra et al., 2011). Immunosensing with LPG requires their surface modification with molecular
recognition elements, such as antibodies, enzyme, etc. Main methods for the immobilization of bioreceptors on the LPG surface
∗ Corresponding author. Tel.: +91 172 2659951; fax: +91 172 2659951.
E-mail address: [email protected] (U. Tiwari).
0956-5663/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
include adsorption (Liu et al., 2000), ionic bonding by electrostatic
self-assembly technique (Elosúa et al., 2006; Wang et al., 2009),
cross-linking by means of multifunctional reagent (Lee and Walt,
2000), covalent bonding (Disley et al., 1998; Stanford et al., 2009),
and avidin–biotin interaction (Marks et al., 2002). Covalent binding
is considered as the most effective of all the above quoted attachment strategies. This approach allows the active sites to remain
unobstructed and chemically reactive.
Some of the extremely useful inherent properties of the LPG
fibers, such as their high sensitivity and smart transduction instrumentation, have motivated us to exploit them for enzyme based
immunosensing of glucose. Presently, only electrochemical methods are more popular for the glucose analysis. Proposed optical
based biosensing of glucose with LPG fibers may contribute to
the much required developments of alternative detection devices,
which can also compete with generally trusted laboratory tests.
The work described herein first time demonstrates the feasibility
of enzyme coated LPG fibers for the photonics based glucose sensing. Since the successful and precise working of the enzyme coated
LPG sensor depends entirely upon the quality of the fiber’s coverage with the protein molecule, the detailed spectroscopic and
microscopic investigations have been undertaken to confirm the
effectiveness of the adopted covalent immobilization methodology. In some published reports, unmodified LPG fibers have been
proposed for the non-selective analysis of very highly concentrated
glucose solutions, e.g. 300 mg mL−1 (Libish et al., 2011; Kim et al.,
A. Deep et al. / Biosensors and Bioelectronics 33 (2012) 190–195
2005). Dilute analyte solution tendered insignificant changes in
the refractive index and no wavelength shifts could be recorded to
deliver usable data. In our approach, the immobilization of a specific enzyme has caused the conversion of glucose to gluconic acid
during the analysis. This reaction props up change in the media’s
refractive index even with dilute (e.g. 0.1–3 mg mL−1 ) glucose solutions, which, in turn, changes the position of the transmission peak
in the associated optical spectrum. Wavelength shifts are agreeably
correlated within certain range of analyte concentration.
2. Materials and methods
Analytical grade reagents and sterile deionized water were used
for preparing all the working solutions. d-glucose, glucose oxidase (GOD, biological source: fungus Aspergillus niger), peroxidase
(POD), o-dianisidine and 3-aminopropyl-triethoxysilane (APTES)
were purchased from Sigma–Aldrich, India.
Long Period Grating fibers (periodicity 550 ␮m) were fabricated
by exposing hydrogenated standard single mode fiber core to KrF
laser using point-by-point method. Prior to further use, the LPG
fibers were cleaned by immersion in 100 mL of 5% nitric acid for
2 h at 90 ◦ C followed by thorough rinsing with deionized water and
ethanol. The hydroxyl-groups of the cleaned fibers were activated
by exposing them with 95% (v/v) sulphuric acid for 1 h at room
temperature. After drying in a convection oven for 4 h at 115 ◦ C,
the LPG fibers were left in contact with 10% (v/v) ethanolic solution of aminopropyl triethoxysilane (APTES) for 30 min at room
temperature. It was followed by rinsing with water and ethanol
to remove non-covalently adsorbed silane compounds. The above
reaction produced free amine groups on the fiber surface.
All the used buffers (acetic acid/sodium acetate and sodium
dihydrogen phosphate/disodium hydrogen phosphate) were prepared in compositions as calculated with the help of the software
‘Buffer Maker Version’. The ionic strengths of different
buffers were forced to a constant value of 50 mM.
Immobilization of GOD on the NH2 functionalized optical
fibers was achieved by immersing the fibers in 5 mg ml−1 buffered
solution (sodium acetate) of glucose oxidase (GOD) for 100 min.
Enzyme immobilized fibers were washed with sodium acetate
buffer (SA) and then dried in the air.
To investigate any change in the GOD activity after immobilization on the optical fibers, a reaction mixture (S1) of 0.1 mL glucose
solution (5 mM), 20 ␮L peroxidase and 0.05 mL of o-dianisidine
(1%, w/w) was prepared in 3 mL of phosphate buffer medium
(50 mM, pH 7.5). Immobilized fibers were cut in to number of pieces
and then their weighed (15 mg–40 mg) amounts were added into
500 ␮L of the prepared reaction mixture‘S1’. These contents (in
micro centrifuge vials) were gently mixed in a temperature controlled (35 ± 1 ◦ C) shaker for 1 h. After the reaction, 50 ␮L of the
liquid was pipetted out and tested for absorbance (at 500 nm) on a
spectrophotometer (UV–Vis–NIR, Varian, Cary 5000).
Consistency in the coating of APTES and GOD on the optical
fibers was investigated by Field Emission Scanning Electron Microscope (FESEM, Hitachi S4300 SE/N) and Confocal Laser Scanning
Microscope (CLSM, Zeiss, LSM 510). Chemical modification of the
fibers with NH2 functional groups was characterized with Infrared
(FTIR, Nicolet iS10) and Raman (Invia Raman, Renishaw) spectrometers.
For glucose analysis, the enzyme immobilized LPG sensor was
fixed between the two micropositioner stages to maintain the
conditions of constant temperature, pressure and strain throughout the experiment. One end of the fiber was connected to white
light source while the other was connected to an optical spectrum
analyzer (OSA, Yokogawa AQ6319, Ando electric Co. Ltd., Japan)
for constant monitoring of the transmission spectrum. A vertical
moving stage was used to dip the LPG in different test solutions. The
sensor was exposed to the different analyte concentrations (0.1, 0.5,
1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mg ml−1 glucose in sodium acetate buffer
of pH 5.6) and the resulting transmission spectra were collected.
Simple refractive index measurements were done on Bellingham
Stanley (RFM 840) system.
3. Results and discussion
3.1. Optimization of the fiber silanization and enzyme
immobilization steps
The silanization of LPG surface is a crucial step to ensure the
efficiency and reproducibility of the subsequent enzyme immobilization step. Some preliminary experiments were carried out to
work out best silanization conditions and to avoid excess aggregation. A fix concentration (10%) of the ethanolic APTES solution
was used to treat the fibers for different durations. After every
experiment, the refractive index of the residual APTES solution was
checked and its value was associated with the unreacted amount of
the reagent. The values of refractive index of residual APTES solution became constant (1.322 ± 0.001) after 25 min of the incubation
of fibers in the reagent solution. For all further studies, the silanization of the fibers was carried out by keeping the incubation time to
30 min. Similarly, the time required for the immobilization of GOD
on the silanized LPG was optimized by immersing the functionalized fibers in 5 mg ml−1 solution of GOD in sodium acetate buffer for
different time durations. The medium was intermittently disturbed
and manually stirred to maintain homogenous enzyme dispersion.
Absorption data of the supernatant liquor collected after each set of
the experiment (Fig. S1) revealed that the desired enzyme immobilization on the silanized fibers may need approximately 75 min
of their incubation with GOD solution. For all further studies, the
enzyme immobilization on the silanized LPG fibers was achieved
by 100 min of incubation.
Some experiments were carried out to test the conservation of
enzyme activity after immobilization onto the optical fibers. In general, the glucose oxidase catalyses the oxidation of ␤-d-glucose to
d-glucono-␦-lactone with the concurrent release of hydrogen peroxide (H2 O2 ) (Eq. (1)). During the enzyme activity test, the presence
of peroxidase (POD) causes the evolved H2 O2 to enter into a second
reaction involving o-dianisidine (reduced) with the quantitative
formation of a o-dianisidine (oxidized) dye complex (Eq. (2)) which
is measured at 500 nm.
D-Glucose + O2 + H2 O−→D-Glucono-1,5-lactone + H2 O2
H2 O2 + o-Dianisidine (reduced)−→o-Dianisidine (oxidized)
Fig. S1 (inset) also gives the absorption data for enzyme assay
test carried out on the GOD immobilized fibers. Linearity of the
reaction curve for various concentrations of glucose oxidase in the
assay mixture is an evidence of the conservation of GOD activity
after its immobilization on the optical fibers.
3.2. Surface characteristics of the silanized and enzyme
immobilized fibers
Fig. 1 shows the FESEM micrographs of the original, silanized
and GOD immobilized optical fibers. Surface modification is evident. It is also clear that the protocols used for the silanization
and the subsequent enzyme immobilization produce uniform and
nicely adhered biomolecule layers. Some charging was observed
during the scanning of GOD coated fiber, which may be due to
the interaction of electron beam with some of the non-conducting
organic groups present in the enzyme structure.
A. Deep et al. / Biosensors and Bioelectronics 33 (2012) 190–195
Fig. 1. (a) FESEM micrograph of the optical fiber, (b) FESEM micrograph of the silanized optical fiber, (c) FESEM micrograph of the enzyme immobilized optical fiber and (d)
CLSM images of the GOD immobilized fluorescent optical fibers.
Intrinsic fluorescent property of the GOD was utilized to optically scan the enzyme immobilized fibers under a confocal laser
scanning microscope. A laser wavelength of 458 nm with (10%
transmission) was used for the excitation. Laser scanning at 200×
optical magnification yielded fluorescent images (Fig. 2d) of the
optical fibers, giving fair evidence about the consistent enzyme
immobilization. As a control experiment, APTES treated fibers were
also monitored under the same conditions. No fluorescent signal
was identifiable. Hence, the observed fluorescence in the fibers can
be attributed to the presence of GOD molecules.
molecules. Thus available NH2 groups bind with the enzyme’s
COOH group through covalent interaction to yield the protein
immobilization according to Eq. (3).
3.3. FTIR and Raman spectroscopic investigations
FTIR and Raman data provide complementary information on
various vibration transitions and vibrational bands to verify the
efficiency of fiber functionalization and subsequent GOD grafting
processes. Fig. 2 shows the FTIR spectra of the silanized (solid lines)
and GOD immobilized (dotted lines) optical fibers. The appearance
of peaks at 1538 and 1634 cm−1 signifies the vibration of primary
amine NH2 and N H scissoring bending. This is further supported
by strong stretching at 3428 cm−1 . Attachment of aliphatic groups
is highlighted by C H asymmetric and symmetric stretching modes
at 2854 and 2925 cm−1 . Furthermore, absence of free Si OH functional groups (no peaks at 795 or 1177 cm−1 ) indicate that APTES
molecules almost smoothly cover the fiber surface and provide
NH2 functional groups to support further immobilization of GOD.
The attachment of enzyme is characterized with the appearance of
a peak at 1637 cm−1 depicting the N C vibrations. A weak band
at 1559 cm−1 also suggests the interaction between the NH2
group of the APTES treated optical fiber and the carboxyl group of
GOD. A broad and intense band at 3448 cm−1 confirms the N H
stretching mode. The presence of two weak bands at 2825 and
2930 cm−1 is also suggesting the successful grafting of the protein
Eq. (1) depicts the scheme of GOD immobilization on the fiber
surface. Sulphuric acid treatment of the cleaned fibers activates
OH groups that have further been used for the assembly of APTES
Fig. 2. FTIR spectra of the silanized (solid line) and enzyme immobilized (dotted
line) optical fibers.
A. Deep et al. / Biosensors and Bioelectronics 33 (2012) 190–195
Fig. 3. Raman spectral studies of (a) silanized optical fibers, (b) native GOD and (c)
GOD immobilized optical fibers.
components on to the optical fiber. The above FTIR studies prove
that GOD molecules are well bonded on the silanized fiber surface.
Raman spectra of the silanized and enzyme coated fibers were
collected at a laser excitation wavelength of 785 nm. Fig. 3a
shows the recorded data for the silanized fiber in two different
ranges, 1200–1700 cm−1 and 2700–3000 cm−1 , for the sake of better peak presentations. Bands observed at 1639 and 1336 cm−1
are attributed to the NH2 group and NH with CH stretch modes,
respectively. Bands at 1459 and 1414 cm−1 suggest the presence
of CH2 scissoring mode and CN, respectively. It indicates the
involvement of silane molecules belonging to APTES attached at the
surface of the fiber. A distinct Raman band at 1314 cm−1 accounts
for the C C stretch mode. The broad Raman bands at 2906, 2921 and
2967 cm−1 are ascribed to CH stretching modes due to the attachment of APTES molecules. Other weak and broad Raman bands
at 3300–3500 cm−1 are assigned to the asymmetric and symmetric modes of NH2 stretching. Raman studies have verified that
the APTES treatment to the optical fibers yields in the generation of free amine groups, which can further be utilized for the
GOD attachment. Fig. 3b and c show the Raman spectra of the
native GOD and the GOD immobilized optical fiber, respectively.
Spectrum 3b reveals characteristic Raman bands for the native
GOD molecule at 409, 632, 682, 863, 999, 1262, 1341, 1535 and
1775 cm−1 . Observed bands at 682, 409 and 632 cm−1 indicate the
presence of S atom, ı (C S) deformations and (C S) stretching, respectively. CNC symmetric stretching of amine groups is
highlighted by a band at 863 cm−1 . C O, C C stretching, p(NH2 )
rocking and NH2 twisting vibrations lead to the appearance of
Raman bands at 1262 and 1535 cm−1 . Significant bands at 1535
and 1775 cm−1 may be assigned to the C C stretching and C O
stretching vibrations, respectively. Raman spectrum of the enzyme
immobilized fiber (Fig. 3c) reveals almost all the peaks of the native
GOD molecule. This observation suggests that the main features of
the native glucose oxidase are conserved after being immobilized
Fig. 4. Transmission spectra and detailed graphical illustration showing variation of wavelength shift as a function of varying glucose concentrations
(0.1–5 mg mL−1 ). Relative standard deviation (RSD) for 3 different sample measurements: 0.015 < RSD < 0.030.
on the fiber surface. Generally amide II band remain inactive or
very weak in the Raman spectrum, however, notable emergence of
a weak band at 1670 cm−1 (amide I) is suggestive of APTES-GOD
covalent interaction.
FTIR and Raman spectral studies have successfully revealed that
the LPG fibers can be conveniently modified by APTES to generate
free NH2 groups, which effectively function as enzyme binding
sites. These modified optical fibers have further been coupled with
measurement system to test their usefulness for glucose detection.
3.4. Glucose detection with GOD immobilized LPG fibers
Immobilization of the enzyme on the LPG fibers has rendered
a sensing platform, which not only provides specificity towards
glucose detection but also protect the fiber from detecting minor
refractive index changes in the presence of possible interfering
species. Fig. S2 schematically represents the experimental setup
with GOD immobilized LPG fiber (exposed length = 2 cm). A blank
solution (2 mL) of sodium acetate buffer was first transferred in to
the sample cell and the stable optical spectrum collected (Fig. S3).
The observed peak position was referred as reference. The buffer
was then carefully removed from the sample cell, and a glucose
solution (in sodium acetate buffer) was introduced for analysis. The
conversion of ␤-d-glucose to d-glucono-␦-lactone (which is finally
hydrolyzed to gluconic acid) caused the change in the refractive
index of the medium, thus inducing wavelength shift in the optical spectrum. A stable signal (Fig. S3) was recordable after 30 s of
the reaction. Subsequently, glucose solutions with different physiologically important concentrations (0.1–5 mg mL−1 ) were analyzed
in the above manner. The obtained optical spectra are presented
in Fig. 4. Data for only 3 representative concentrations have been
plotted for the sake of clear graphical illustration, which shows
A. Deep et al. / Biosensors and Bioelectronics 33 (2012) 190–195
Table 1
Analysis of glucose from serum samples with glucose oxidase immobilized LPG fiber.
Glucose content as per
hospital test (in mg mL−1 )
Wavelength shift on GOD-LPG sensor w.r.t.
serum plasma sample having known
1.05 mg mL−1 glucose content (nm)
experimental glucose
content (mg mL−1 )
Estimated glucose content after
incorporating reference data
(column 4 + 1.05 mg mL−1 )
% Variation w.r.t.
hospital data
1.2 ± 0.02
1.8 ± 0.02
2.2 ± 0.03
+4.06 to 2.57
transmission peaks with wavelength shifts at two positions, near
1620 and 1550 nm. The signals obtained near 1620 nm provided
the desired pattern of wavelength shifts with respect to the changing glucose concentration. Detailed information on the wavelength
shifts (with respect to control transmission peak at 1620 nm) as a
function of glucose concentrations is also plotted in Fig. 4. It is clear
that the GOD immobilized LPG fiber give fairly linear response in the
glucose concentration range of 0.1–3 mg mL−1 . The assay test with
higher concentrations of glucose did not give proper response possibly due to the fact that the selected length (2 cm) of the LPG fiber
did not have enough enzyme loading to support the complete oxidation of the test solution. However, it must be noted that the above
sensor in the said conditions is suitable for the analysis of physiologically important glucose concentrations (10–300 mg dL−1 ). All
the above described experiments were conducted at least three
times with different sets of enzyme immobilized optical fibers, and
the observed average variation in the wavelength shifts was limited
to ±0.1 nm.
The glucose detection with GOD immobilized LPG fibers has
been proved to be very sensitive and the technique’s usability
is demonstrated for the analysis of dilute (10–300 mg dL−1 ) glucose concentrations. Though it has been shown earlier that the
unmodified LPG fibers may also be used for detecting glucose concentrations (Libish et al., 2011; Kim et al., 2005), those reported
studies dealt with much higher (e.g. 30 g mL−1 ) levels of the analyte. Our investigations (Fig. S4) on a simple LPG fiber have shown
insignificant optical response with respect to the analysis of dilute
glucose solutions (0.1–5 mg mL−1 ). It can be correlated with minuscule changes in the medium’s refractive index when working with
dilute solutions (Fig. S5). Glucose analysis on GOD modified LPG
fiber is associated with the conversion of glucose to the gluconic
acid; the evolution of which changes the medium’s refractive index.
Study of solutions after the reaction indicated wider changes in the
refractive index (Fig. S5) accounting for the appearance of significant transmission shifts even at low glucose concentrations.
Solution pH is known to be one of the most important factors
affecting the enzyme activity and stability, and hence the sensor’s performance. Effect of pH on the LPG sensor performance was
determined in the range of 5.0–8.0 against a constant glucose concentration of 2.0 mg mL−1 . Acetic acid/sodium acetate buffer was
used for lower pH value experiments while sodium dihydrogen
phosphate/disodium hydrogen phosphate buffer was employed for
making analyte solutions of higher pH values. Fig. 5 shows that
the performance of the LPG sensor was better in the pH range of
5.0–7.0. Higher pH values inhibit the enzyme activity, and therefore
the wavelength shifts were comparatively less resolved. Response
time of the sensor was assessed by recording several optical spectra
during the analysis of 1.0 mg mL−1 glucose solution. The data of the
study are also given in Fig. 5. Stable optical spectrum was recorded
within 30 s, thereby demonstrating that the enzyme–substrate
reaction took little time to complete and yield the stable result.
The different detailed investigations have identified the optimized experimental parameters for the analysis of glucose
(10–300 mg dL−1 ) under certain test conditions (exposed length
of LPG = 2 cm, GOD for immobilization = 5 mg mL−1 , phase volume = 2 mL). However, these parameters are selectable according
to the specific requirements. In such a case, the calibration of the
Fig. 5. Effects of pH and reaction time on the optical signal during the analysis of
2.0 mg mL−1 glucose on GOD immobilized optical fibers. Relative standard deviation
(RSD) for 3 different sample measurements: 0.020 < RSD < 0.035.
system will need to be redrawn. The practical utility of the proposed LPG sensor was evaluated by analyzing the glucose content
in serum samples. Fresh and clinically analyzed plasma samples
were collected from the local hospital. These were diluted before
introducing into the LPG setup. A plasma sample with known
(1.05 mg ml−1 ) glucose content was first analyzed to record a reference optical spectrum. Subsequent analyses of the samples of the
diabetic patients were analyzed and the results are summarized in
Table 1. The glucose content of the blood was calculated from the
calibration curve. The results were satisfactory and agreed closely
with those measured by the biochemical analyzer done by local
4. Conclusions
The presented research data demonstrate the successful immobilization of GOD on to the silanized LPG fibers for the development
of a new glucose sensing technique. The enzyme immobilization
has been achieved by modifying the fiber surface with APTES. Free
amine groups of the APTES bind with carboxyl groups of GOD to
render a sensing probe. Microscopic studies prove the uniform
and effective coating of the optical fiber with the protein. FTIR
and Raman spectroscopic studies have further revealed the efficacy of the undertaken strategy to develop a LPG based sensor
by giving detailed information on various vibrations transitions
and bands after each step. FTIR, Raman spectroscopy and Confocal microscopy investigations provide new dimensions to the LPG
based sensor fabrication, which may also be usefully referred for
other similar purposes. During the immobilization process, intermittent washing and rinsing steps ensured the removal of extra
materials attached through non-specific interactions. The contact
of different glucose solutions with the enzyme immobilized LPG
fiber leads to patterned wavelength shifts. A practical range of
signal linearity implies the usefulness of the proposed sensor for
the detection of physiologically significant glucose concentrations.
A. Deep et al. / Biosensors and Bioelectronics 33 (2012) 190–195
Development of simple, inexpensive, direct and real-time monitoring glucose sensors is of continuous interest as the traditional
methods for glucose concentration measurement require different
laboratorial analyses. LPG fiber based sensitive photonic biosensing may offer an attractive solution in this respect due to some
intrinsic merits, such as high sensitivity, immunity to electromagnetic interference, small size, fast response etc. Some other
important advantages of the proposed optical technique over
other label free amperometric, potentiometric and conductometric metal/dielectric platforms include the use of much efficient and
convenient signal transduction protocol and low signal to noise
ratio. The proposed technique of protein immobilization could also
be useful to develop optical fiber based sensors for applications in
various other fields like disease diagnosis, pathogen testing, study
of environmental pollutants, etc.
Thanks are due to the Council of Scientific and Industrial
Research, India (NWP 035 & NWP 026) for the financial support
to the work.
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