Sensors and Actuators B 178 (2013) 458–464

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Sensors and Actuators B: Chemical

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Enhancement in sensitivity of fluorescence based assay for organophosphates

detection by silica coated silver nanoparticles using organophosphate hydrolase
Shaveena Thakur a , Pardeep Kumar b , M. Venkateswar Reddy c , D. Siddavattam c , A.K. Paul a,∗
a
CSIR-Central Scientific Instruments Organisation, Sector-30, Chandigarh 160030, India
b
National Institute of Technology (NIT), Kurukshetra 136119, India
c
Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

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

Article history: The aim of the present work is to enhance the sensitivity of the fluorescence based developed detection
Received 27 October 2012 assay for the estimation of organophosphates by exploiting the spectral property of metal enhanced
Received in revised form 2 January 2013 fluorescence shown by silver nanoparticles. The receptor entity consisted of organophosphorus hydrolase
Accepted 4 January 2013
with a histidine tail (OPH6His ) at its C terminus which has been conjugated to pH sensitive high quantum
Available online 11 January 2013
yield fluorophore i-e pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt). The introduction
of silica nanoshell (∼30 nm) on silver nanoparticles (∼35 nm) ameliorated tuning of surface plasmon
Keywords:
resonance wavelength of silver nanoparticles to the excitation wavelength of pyranine which ultimately
Silica coated silver nanoparticles
Organophosphates
led to a ten-fold increase in the fluorescence signal to that given by OPH6His –pyranine bioconjugate. This
Metal enhanced fluorescence fluorescence enhancement resulted in lowering of detection limit from 20 ppb to 2 ppb for paraoxon and
Organophosphorous hydrolase 50 ppb to 10 ppb for methyl parathion.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction the spectral properties of fluorophores [4]. The phenomenon aris-

The environmental pollution caused by the organophosphates
pesticides is a global concern. These nitroaromatic pesticides being
highly active and less persistent in environment have replaced
organochlorines which bioaccumulate along the food chain [1].
Organophosphates are widely used to control pests in a vari-
ety of agricultural applications. Although these compounds are
degraded under many environmental conditions, potential expo-
sure from sources such as fruits, vegetables and processed food
cannot be ruled out. Recently, there have been an intense research
efforts to develop bioanalytical devices for the determination of
organophosphates. The enzyme based biosensors either employ
acetylcholinesterase (AChE), detecting organophosphates by AChE
inhibition or organophosphorus hydrolase (OPH) which hydrolyze
the organophosphate into its metabolites [2,3]. In the present study
the release of the protons in the hydrolysis reaction of organophos-
phates by OPH has been estimated by pH sensitive fluorophore –
pyranine using fluorescence based optical set up.
Fluorescence due to its high sensitivity and ability to detect sin-
gle molecule interactions has dominated the sensing technology in
diagnostics and biotechnology. The photophysical limitations such
as photostability and quantum yield posed by fluorophore had been
alleviated by employing metallic nanoparticles to favorably modify
∗ Corresponding author. Tel.: +91 172 2642545; fax: +91 172 2657267.
E-mail address: paul ak@rediffmail.com (A.K. Paul).
ing due to interaction of fluorophore with localized surface
plasmons of the metal is known as metal enhanced fluorescence
(MEF) or surface-enhanced fluorescence. Metallic nanostructures
alter the fluorescence either by enhancing the excitation rate or by
altering the radiative and non-radiative decay rate of the nearby
fluorophore resulting a hike in the quantum yield and lifetime of
the fluorophore. The spectral properties of metallic nanoparticles
originate from the collective oscillations of conduction electrons on
excitation by electromagnetic (EM) radiation, which is termed as
surface plasmon resonance (SPR). There are number of factors such
as acceleration of the conduction electrons by the electric field of
incident radiation, presence of restoring forces that result from the
induced polarization in both the particle and surrounding medium
and confinement of the electrons to dimensions smaller than the
wavelength of light that collectively lead to these oscillations [5].
The electric field of the incident EM radiation displaces the parti-
cle’s electrons from equilibrium and, in turn, produces a restoring
force that results in oscillatory motion of the electrons with a
characteristic frequency, that is, the SPR frequency. At the same
time, the oscillating electrons induce polarization of the oppo-
site direction in the surrounding medium, and this polarization
reduces the restoring force for the electrons thereby shifting the
SPR frequency. When surface plasmons are excited, the oscillating
electrons generate an EM field consisting of two components, a
local non-radiative field around the particles that is enhanced
and a radiative EM field due to resonant scattering. The distance
to which the local field extends from the particle surface plays a

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http://dx.doi.org/10.1016/j.snb.2013.01.010
 S. Thakur et al. / Sensors and Actuators B 178 (2013) 458–464
Fig. 1. Schematic representation of organophosphates detection by pyranine–OPH6His bioconjugate with the aid of silica coated silver nanoparticles.
major role in various surface-enhanced phenomena as molecules
placed within this field experience increased induced polarization.
Plasmons excited in metallic nanoparticles have the characteristic
frequencies dependent on sizes, shapes, and dielectric functions of
these nanoparticles and surrounding medium [6].
Silver exhibits highest efficiency of plasmon excitation among
the other metals (Ag, Au, Cu) which display surface plasmon
resonance peak in the visible spectrum. The light-interaction cross-
section for Ag can be ten times that of the geometric cross-section,
which indicates that the particles capture much more light than
is physically incident on them [7]. The coating of the metallic
nanoparticles with a thin layer of another material with desired
properties in terms of stability and functionality results in concen-
tric particles which are called nanoshell particles. Nanoparticles are
susceptible to coalescence and oxidation as their surface is unsta-
ble because of the presence of dangling bonds and surface strains.
Stability of colloids is greatly enhanced by coating them with stable
material such as silica. Silica coated nanoparticles have advantage
because of their extraordinary stability against coagulation, more-
over high dielectric constant and refractive index of silica shell
brings shift in SPR of silver nanoparticles (AgNPs) to the desired
range [8].
The fluorescence based detection assay with OPH6His –pyranine
bioconjugate has been reported earlier for the estimation of
organophosphate pesticides. In the present work, which is an
extended study of the previous reported work [20], MEF exhibited
by silica coated AgNPs has been utilized to increase the fluores-
cence signal of OPH6His –pyranine bioconjugate so as to enhance the
detection limit of organophosphorus pesticides as shown in (Fig. 1).
The enzyme OPH6His catalyses the hydrolysis of organophosphates
releasing H+ as a byproduct in its micro-environment and the
released protons turn the medium acidic in the vicinity of the
459
enzyme. The change in the pH is being detected by the pH reporter
– pyranine which had been electrostatically labeled to the OPH6His.
The fluorescence sensitivity of pyranine in the pH range of 5–8
is due to presence the of 8-hydroxyl group and fast equilibration
between protonated and deprotonated species in the excited state
which results in the change of relative fluorescence intensity. This
change in the fluorescence signal makes the development of fluo-
rescence based optical detection of organophosphates by OPH6His
feasible. The excitation and therefore emission intensity of the
pyranine increased ten folds in the presence of AgNPs with sil-
ica nanoshell due to the metal enhanced fluorescence. The present
study was carried out to lower the detection limit of the synthesized
bioconjugate by introducing silica shell AgNPs.
2. Methodology
2.1. Materials and reagents
All the chemicals such as silver nitrate (AgNO3 ), trisodium
citrate (Na3 C6 H5 O7 ), tetraethoxysilane (TEOS), isopropanol
(C3 H8 O), ammonium hydroxide (NH4 OH), 8-hydroxypyrene-
1,3,6-trisulfonic acid trisodium salt (HPTS or pyranine),
2-[N-cyclohexylamino] ethanesulfonic acid (CHES), paraoxon
and methyl parathion were obtained from Sigma Chemical Co. All
other compounds and solvents used were of analytical grade from
Merck.
2.2. Preparation of silver colloids with silica shell by chemical
method
The silver nanoparticles used in these experiments were pre-
pared by chemical reduction of silver nitrate with trisodium citrate
460 S. Thakur et al. / Sensors and Actuators B 178 (2013) 458–464
Fig. 2. Absorbance spectra of (a) silver nanoparticles (∼35 nm) at 437 nm and (b) silica coated AgNPs (∼65 nm) at 462 nm.
using a modified Lee and Meisel method [9,10]. Silver colloids
were prepared by dropwise adding 0.5 ml of 38.8 mM trisodium
citrate aqueous solution within 2 min into 24.5 ml of boiling aque-
ous solution containing 4.5 mg of AgNO3 under vigorous stirring.
After continuous stirring for 1 h at 80 ◦ C, the reaction solution was
cooled to room temperature. The prepared silver colloid solution
was centrifuged at 5000 rpm for 20 min to remove smaller sized
particles. According to this method the trisodium citrate acted as a
reducing agent, and later the negative citrate ions were adsorbed
on silver nanoparticles which prevented aggregation of nanoparti-
cles [11,12]. The collected pellet was dispensed in deionized water
and was characterized using UV–vis spectrophotometer (Lab-India
UV-3200).
The capping of synthesized AgNPs by silica was done following
Stöber method [13]. Under vigorous stirring, 0.4 ml of silver col-
loids solution in 10 ml of deionized water was mixed with 100 ml of
isopropanol. After the addition of 1.6 ml of 30% ammonium hydrox-
ide, 100 ␮l TEOS solution having 100% concentration was added
immediately to the reaction mixture which was stirred at room
temperature for 30 min and then allowed to age without agita-
tion at 4 ◦ C overnight. Each suspension of silica-coated AgNPs was
washed and centrifuged three times with water and ethanol mix-
ture (5:4) at 3500 rpm for 30 min. The increase in size of AgNPs
in terms of shift in SPR was monitored regularly by UV–vis spec-
trophotometer and thickness of the silica layers was determined
by electron microscopy (SEM & TEM).
2.3. Fluorescence assays of pyranine with synthesized silica
coated AgNPs
1 mg/ml stock solution of a fluorophore pyranine was prepared
in double distilled water. 2 ␮M solution of pyranine was prepared
in 50 mM CHES buffer (pH 8). The dilutions of the synthesized
silica coated silver nanoparticles of concentration 0.02 mg/ml ran-
ging from 1:10 to 1:50 were added to the fluorophore solutions
to optimize the enhancement in fluorescence signal. The same
experiment regarding the optimization of fluorescence enhance-
ment of OPH6His –pyranine bioconjugate with silica coated AgNPs
was performed. All fluorescence measurements were done in
quartz cuvettes by fluorescence spectrophotometer of Varian-Cary
Eclipse.
2.4. Detection of organophosphate in the presence of silica coated
AgNPs by bioconjugate of organophosphorus hydrolase and
pyranine
The 1 mg/ml stock of paraoxon and methyl parathion in
methanol was prepared and further dilutions were made with the
reaction buffer i.e. 1 mM CHES buffer (pH 8). The detection assay
for organophosphate reaction mixture comprised of bioconjugate
of histidine tagged organophosphorus hydrolase (OPH6His ) electro-
statically labeled with pyranine. The optimized concentration of
silica coated AgNPs was added to the reaction mixture and change
in fluorescence intensity was monitored. Calibration plots were
prepared by varying the concentrations of methyl parathion and
paraoxon versus relative fluorescence intensity.
3. Results and discussion
3.1. Characterization of nanoparticles
A great deal of information about the physical state of the
nanoparticles can be obtained by analyzing the unique spectral
properties of silver nanoparticles in solution utilizing FTIR and
UV–visible spectroscopic techniques. The size and shape of metal
nanoparticles was measured by employing different analytical
techniques such as X-ray diffraction, electron microscopy (SEM &
TEM). The size characterization during the synthesis was monitored
at regular intervals by employing UV–vis spectrophotometer as dif-
ferent sized nanoparticles showed SPR between 400 and 500 nm.
When the frequency of the electromagnetic field becomes resonant
with the coherent electron motion, a strong absorption takes place
at that particular wavelength. The strongest absorption peak of the
silver nanoparticles was observed at 437 ± 5 nm and silica coated
AgNPs showed broader peak at 462 ± 5 nm as shown in Fig. 2a and
b respectively. These results suggested a red shift in SPR which
was found to be dependent on increase in particle size, dielectric
medium and surface adsorbed species [14,15]. The results also sug-
gested that extinction coefficient of core–shell particles was much
larger than nanoparticles. The formation of silica shell of ∼30 nm
thick around silver particles was observed in TEM images as shown
in Figs. 3a and b which were spherical in shape with a smooth
surface morphology. Since the electron density of the metal is sig-
nificantly higher than that of amorphous silica, darker part in the
 S. Thakur et al. / Sensors and Actuators B 178 (2013) 458–464 461

Fig. 3. TEM images of synthesized (a) AgNPs and (b) AgNPs with silica shell.

center is associated with silver [16]. The size of the synthesized

AgNPs was estimated to be 65 nm and 35 nm with and without silica
shell respectively by SEM images as shown in Fig. 4a and b. In XRD
Fig. 4. SEM images of (a) AgNPs and (b) AgNPs with silica shell.
pattern a number of strong Bragg reflections were obtained corre-
sponding to h k l values of (1 1 1), (2 0 0), (2 2 0), (3 1 1) as shown in
(Fig. 5) which matched with the JCPDS database (PDF2-Numbers AgNPs thereby shifting the surface plasmon peak toward the higher
40783 and 411402) confirming fcc structure of AgNPs. The size wavelength.
of silver nanoparticles as calculated from the XRD pattern using
the Debye–Scherrer formula was found to be 33 nm which is also 3.3. Detection of organophosphates by silica coated AgNPs
in close approximation to the size as obtained by TEM and SEM
analysis [17]. The detection of organophosphate pesticides is based on the
The synthesized silver colloids with and without silica nanoshell change in relative fluorescence intensity of the pyranine attached
were characterized by FTIR to monitor the formation of silica shell
on AgNPs. The presence of spectral signatures of silver nanoparti-
cles at 1650–1600 cm−1 as shown in (Fig. 6a) are due to the presence
of citrate layer on silver which confirms the reduction of silver ions
which subsequently resulted in the formation of AgNPs. The pres-
ence of peaks in the region 1400–1100 cm−1 as shown in (Fig. 6b)
are due to the formation of Si O Si bonds which is considered to
be the fingerprint of metal oxides. These bands are attributed to ␯
(Si O), ␦ (Si OH), ␯ (Si O Si), and ␦ (CH2 ) from residual TEOS or
Si-ethoxy residuals due to incomplete TEOS hydrolysis [18,19].

3.2. Fluorescence signal enhancement

The 1:20 molar ratio of AgNPs to OPH6His –pyranine complex

enhanced the fluorescence signal by three folds as compared to
tenfolds in the presence of silica coated silver colloids as shown
in (Fig. 7) which is the maximum limit for pyranine due to
its high quantum yield. The main reason for enhancement of
fluorescence signal is attributed to the proximity of pyranine exci-
tation wavelength (ex = 460 nm) to the wavelength at which silica
coated AgNPs (65 nm) exhibit maximum cross sectional scatter-
ing at 462 nm as compared to 437 nm by AgNPs. The silica coating Fig. 5. X-ray diffraction pattern of AgNPs showing distinct three peaks correspond-
increased the dielectric constant of the surrounding medium of ing to (1 1 1), (2 0 0) and (2 2 0) planes of a face-centered cubic lattice.
462 S. Thakur et al. / Sensors and Actuators B 178 (2013) 458–464

Fig. 6. FTIR of (a) synthesized AgNP and (b) AgNPs with silica shell.
Fig. 8. Calibration plot for paraoxon with (a) bioconjugate of OPH6His –pyranine in
the presence of synthesized AgNPs coated with silica shell and (b) bioconjugate of
OPH6His –pyranine.

to hydrolyzing enzyme OPH6His (bioconjugate) due to the change

in the pH of bioconjugate micro environment on the hydrolysis
of organophoshates in the presence of silica coated AgNPs. The
1:20 molar ratio of the bioconjugate with silica coated AgNPs in
1 mM CHES (pH 8) was selected for the detection assay which
are the optimized conditions for maximum enhancement of flu-
orescence intensity. Lower detection limit has been achieved upto
∼2 ppb for paraoxon qualitatively as shown in (Fig. 8a), compared to
∼20 ppb which was prior to the introduction of AgNPs to the assay
as shown in (Fig. 8b). The dynamic concentration range was from 5
to 100 ppb with a standard deviation of 1.1% (n = 10) for paraoxon.
In case of methyl parathion, lower detection limit of ∼10 ppb has
been obtained qualitatively as compared to ∼50 ppb without sil-
ica coated AgNPs as shown in Figs. 9a and b respectively [20]. The
standard deviation in this case was 1.51% (n = 10) with a dynamic
concentration range from 20 to 100 ppb. These lower detection lim-
its of paraoxon and methyl parathion are attributed to the tenfold
increase in the fluorescence intensity of the bioconjugate which
Fig. 7. Enhancement in fluorescence signal of bioconjugate (pyranine–OPH6His ) in
is exhibited in the presence of silica coated AgNPs of the existing
the presence of AgNPs with and without silica shell.
biosensing assay.
 S. Thakur et al. / Sensors and Actuators B 178 (2013) 458–464
Fig. 9. Calibration plot for methyl parathion with (a) bioconjugate of
OPH6His –pyranine in the presence of synthesized AgNPs coated with silica
shell and (b) bioconjugate of OPH6His –pyranine.
4. Conclusions
The studies have demonstrated the detection of organophos-
phate pesticides with higher sensitivity due to the tenfold
enhancement of fluorescence signal by employing silica coated
silver nanoparticles to the OPH6His –pyranine bioconjugate. The
qualitative detection limits as obtained by this biosensing assay
are ∼2 ppb and ∼10 ppb for paraoxon and methyl parathion respec-
tively which are lower as compared to the enzyme based biosensor
for organophosphate pesticides. The presence of silica nanoshell
on AgNPs increased its stability and made the tagging possi-
ble with bioconjugate due to the presence of hydroxyl groups
on silica shell. Therefore, we conclude that AgNPs with silica
shell have capability to significantly enhance the optical sensing
signal through amplifying fluorescence intensity leading to the
enhancement in sensitivity of the developed assay which fur-
ther widens its applicability for detecting organophosphate nerve
agents.
463
Acknowledgements
The authors gratefully acknowledge the support of the Director,
CSIR-CSIO, Chandigarh. The financial assistance provided to CSIO by
CSIR, New Delhi is highly acknowledged. Research in the laboratory
of Dr. D. Siddavattam is supported by DRDO, New Delhi.
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Biographies
Shaveena Thakur completed her Master’s degree in Biotechnology in 2008 from
Panjab University Chandigarh, India. She is presently working on the project to
develop biosensor for environmental monitoring.
Pardeep Kumar completed his Master of Technology in Nanotechnology from
National Institute of Technology, Kurukshetra in 2012. The present work has been
carried out by him at Central Scientific Instruments Organization during his project
training.
464 S. Thakur et al. / Sensors and Actuators B 178 (2013) 458–464

M. Venkateswar Reddy completed his Master’s degree in Bio-technology from Ban- found in structurally diverse group of OP-compounds, targets to membrane through
galore University in 2003. Currently, he is a Senior Research Fellow in a DRDO an alternate protein transport pathway known as twin arginine transport (TAT)
sponsored research project involved in developing nano biosensors. pathway.

D. Siddavattam did his graduation from S.V. University, Tirupati, India in Envi-
ronmental Biology and Ph.D. in 1985. Currently he is a senior professor in the
University of Hyderabad, Hyderabad and specializes in optimizing expression and
large scale purification of recombinant OPH. His laboratory has shown that the
organophosphate hydrolase (OPH), the key enzyme in inactivating triester bond
A.K. Paul received his Master of Science in Physics from Punjabi University, Patiala,
India; Master of Technology in Applied Optics from Indian Institute of Technology,
Delhi and PhD degree in Physics from Panjab University, Chandigarh, India. His cur-
rent activities include the development of bio-sensors for pesticides estimation in
water and soil, explosive detection and analytical instrumentation.