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Sensors and Actuators B 178 (2013) 458– 464

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o me pa ge: www.elsev ier .com/ locate /snb

nhancement in sensitivity of fluorescence based assay for organophosphatesetection by silica coated silver nanoparticles using organophosphate hydrolase

haveena Thakura, Pardeep Kumarb, M. Venkateswar Reddyc, D. Siddavattamc, A.K. Paula,∗

CSIR-Central Scientific Instruments Organisation, Sector-30, Chandigarh 160030, IndiaNational Institute of Technology (NIT), Kurukshetra 136119, IndiaDepartment of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

r t i c l e i n f o

rticle history:eceived 27 October 2012eceived in revised form 2 January 2013ccepted 4 January 2013vailable online xxx

a b s t r a c t

The aim of the present work is to enhance the sensitivity of the fluorescence based developed detectionassay for the estimation of organophosphates by exploiting the spectral property of metal enhancedfluorescence shown by silver nanoparticles. The receptor entity consisted of organophosphorus hydrolasewith a histidine tail (OPH6His) at its C terminus which has been conjugated to pH sensitive high quantum

eywords:ilica coated silver nanoparticlesrganophosphatesetal enhanced fluorescence

yield fluorophore i-e pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt). The introductionof silica nanoshell (∼30 nm) on silver nanoparticles (∼35 nm) ameliorated tuning of surface plasmonresonance wavelength of silver nanoparticles to the excitation wavelength of pyranine which ultimatelyled to a ten-fold increase in the fluorescence signal to that given by OPH6His–pyranine bioconjugate. Thisfluorescence enhancement resulted in lowering of detection limit from 20 ppb to 2 ppb for paraoxon and

yl pa

rganophosphorous hydrolase 50 ppb to 10 ppb for meth

. Introduction

The environmental pollution caused by the organophosphatesesticides is a global concern. These nitroaromatic pesticides beingighly active and less persistent in environment have replacedrganochlorines which bioaccumulate along the food chain [1].rganophosphates are widely used to control pests in a vari-ty of agricultural applications. Although these compounds areegraded under many environmental conditions, potential expo-ure from sources such as fruits, vegetables and processed foodannot be ruled out. Recently, there have been an intense researchfforts to develop bioanalytical devices for the determination ofrganophosphates. The enzyme based biosensors either employcetylcholinesterase (AChE), detecting organophosphates by AChEnhibition or organophosphorus hydrolase (OPH) which hydrolyzehe organophosphate into its metabolites [2,3]. In the present studyhe release of the protons in the hydrolysis reaction of organophos-hates by OPH has been estimated by pH sensitive fluorophore –yranine using fluorescence based optical set up.

Fluorescence due to its high sensitivity and ability to detect sin-le molecule interactions has dominated the sensing technology in

iagnostics and biotechnology. The photophysical limitations suchs photostability and quantum yield posed by fluorophore had beenlleviated 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).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.01.010

rathion.© 2013 Elsevier B.V. All rights reserved.

the spectral properties of fluorophores [4]. The phenomenon aris-ing due to interaction of fluorophore with localized surfaceplasmons of the metal is known as metal enhanced fluorescence(MEF) or surface-enhanced fluorescence. Metallic nanostructuresalter the fluorescence either by enhancing the excitation rate or byaltering the radiative and non-radiative decay rate of the nearbyfluorophore resulting a hike in the quantum yield and lifetime ofthe fluorophore. The spectral properties of metallic nanoparticlesoriginate from the collective oscillations of conduction electrons onexcitation by electromagnetic (EM) radiation, which is termed assurface plasmon resonance (SPR). There are number of factors suchas acceleration of the conduction electrons by the electric field ofincident radiation, presence of restoring forces that result from theinduced polarization in both the particle and surrounding mediumand confinement of the electrons to dimensions smaller than thewavelength 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 restoringforce that results in oscillatory motion of the electrons with acharacteristic frequency, that is, the SPR frequency. At the sametime, the oscillating electrons induce polarization of the oppo-site direction in the surrounding medium, and this polarizationreduces the restoring force for the electrons thereby shifting theSPR frequency. When surface plasmons are excited, the oscillating

electrons generate an EM field consisting of two components, alocal non-radiative field around the particles that is enhancedand a radiative EM field due to resonant scattering. The distanceto which the local field extends from the particle surface plays a

S. Thakur et al. / Sensors and Actuators B 178 (2013) 458– 464 459

anine–

mpPft

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boebcdTrr

Fig. 1. Schematic representation of organophosphates detection by pyr

ajor role in various surface-enhanced phenomena as moleculeslaced within this field experience increased induced polarization.lasmons excited in metallic nanoparticles have the characteristicrequencies dependent on sizes, shapes, and dielectric functions ofhese nanoparticles and surrounding medium [6].

Silver exhibits highest efficiency of plasmon excitation amonghe other metals (Ag, Au, Cu) which display surface plasmonesonance peak in the visible spectrum. The light-interaction cross-ection for Ag can be ten times that of the geometric cross-section,hich indicates that the particles capture much more light than

s physically incident on them [7]. The coating of the metallicanoparticles with a thin layer of another material with desiredroperties in terms of stability and functionality results in concen-ric particles which are called nanoshell particles. Nanoparticles areusceptible to coalescence and oxidation as their surface is unsta-le because of the presence of dangling bonds and surface strains.tability of colloids is greatly enhanced by coating them with stableaterial such as silica. Silica coated nanoparticles have advantage

ecause of their extraordinary stability against coagulation, more-ver high dielectric constant and refractive index of silica shellrings shift in SPR of silver nanoparticles (AgNPs) to the desiredange [8].

The fluorescence based detection assay with OPH6His–pyranineioconjugate has been reported earlier for the estimation ofrganophosphate pesticides. In the present work, which is anxtended study of the previous reported work [20], MEF exhibitedy silica coated AgNPs has been utilized to increase the fluores-ence signal of OPH6His–pyranine bioconjugate so as to enhance the

etection limit of organophosphorus pesticides as shown in (Fig. 1).he enzyme OPH6His catalyses the hydrolysis of organophosphateseleasing H+ as a byproduct in its micro-environment and theeleased protons turn the medium acidic in the vicinity of the

OPH6His bioconjugate with the aid of silica coated silver nanoparticles.

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 5–8is due to presence the of 8-hydroxyl group and fast equilibrationbetween protonated and deprotonated species in the excited statewhich results in the change of relative fluorescence intensity. Thischange in the fluorescence signal makes the development of fluo-rescence based optical detection of organophosphates by OPH6Hisfeasible. The excitation and therefore emission intensity of thepyranine increased ten folds in the presence of AgNPs with sil-ica nanoshell due to the metal enhanced fluorescence. The presentstudy was carried out to lower the detection limit of the synthesizedbioconjugate by introducing silica shell AgNPs.

2. Methodology

2.1. Materials and reagents

All the chemicals such as silver nitrate (AgNO3), trisodiumcitrate (Na3C6H5O7), tetraethoxysilane (TEOS), isopropanol(C3H8O), ammonium hydroxide (NH4OH), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS or pyranine),2-[N-cyclohexylamino] ethanesulfonic acid (CHES), paraoxonand methyl parathion were obtained from Sigma Chemical Co. Allother compounds and solvents used were of analytical grade fromMerck.

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

nm) a

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Fig. 2. Absorbance spectra of (a) silver nanoparticles (∼35

sing a modified Lee and Meisel method [9,10]. Silver colloidsere prepared by dropwise adding 0.5 ml of 38.8 mM trisodium

itrate aqueous solution within 2 min into 24.5 ml of boiling aque-us solution containing 4.5 mg of AgNO3 under vigorous stirring.fter continuous stirring for 1 h at 80 ◦C, the reaction solution wasooled to room temperature. The prepared silver colloid solutionas centrifuged at 5000 rpm for 20 min to remove smaller sizedarticles. According to this method the trisodium citrate acted as aeducing agent, and later the negative citrate ions were adsorbedn silver nanoparticles which prevented aggregation of nanoparti-les [11,12]. The collected pellet was dispensed in deionized waternd was characterized using UV–vis spectrophotometer (Lab-IndiaV-3200).

The capping of synthesized AgNPs by silica was done followingtöber method [13]. Under vigorous stirring, 0.4 ml of silver col-oids solution in 10 ml of deionized water was mixed with 100 ml ofsopropanol. After the addition of 1.6 ml of 30% ammonium hydrox-de, 100 �l TEOS solution having 100% concentration was addedmmediately to the reaction mixture which was stirred at roomemperature for 30 min and then allowed to age without agita-ion at 4 ◦C overnight. Each suspension of silica-coated AgNPs wasashed and centrifuged three times with water and ethanol mix-

ure (5:4) at 3500 rpm for 30 min. The increase in size of AgNPsn terms of shift in SPR was monitored regularly by UV–vis spec-rophotometer and thickness of the silica layers was determinedy electron microscopy (SEM & TEM).

.3. Fluorescence assays of pyranine with synthesized silicaoated AgNPs

1 mg/ml stock solution of a fluorophore pyranine was preparedn double distilled water. 2 �M solution of pyranine was preparedn 50 mM CHES buffer (pH 8). The dilutions of the synthesizedilica coated silver nanoparticles of concentration 0.02 mg/ml ran-ing from 1:10 to 1:50 were added to the fluorophore solutionso optimize the enhancement in fluorescence signal. The same

xperiment regarding the optimization of fluorescence enhance-ent of OPH6His–pyranine bioconjugate with silica coated AgNPsas performed. All fluorescence measurements were done in

uartz cuvettes by fluorescence spectrophotometer of Varian-Caryclipse.

t 437 nm and (b) silica coated AgNPs (∼65 nm) at 462 nm.

2.4. Detection of organophosphate in the presence of silica coatedAgNPs by bioconjugate of organophosphorus hydrolase andpyranine

The 1 mg/ml stock of paraoxon and methyl parathion inmethanol was prepared and further dilutions were made with thereaction buffer i.e. 1 mM CHES buffer (pH 8). The detection assayfor organophosphate reaction mixture comprised of bioconjugateof histidine tagged organophosphorus hydrolase (OPH6His) electro-statically labeled with pyranine. The optimized concentration ofsilica coated AgNPs was added to the reaction mixture and changein fluorescence intensity was monitored. Calibration plots wereprepared by varying the concentrations of methyl parathion andparaoxon versus relative fluorescence intensity.

3. Results and discussion

3.1. Characterization of nanoparticles

A great deal of information about the physical state of thenanoparticles can be obtained by analyzing the unique spectralproperties of silver nanoparticles in solution utilizing FTIR andUV–visible spectroscopic techniques. The size and shape of metalnanoparticles was measured by employing different analyticaltechniques such as X-ray diffraction, electron microscopy (SEM &TEM). The size characterization during the synthesis was monitoredat 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 resonantwith the coherent electron motion, a strong absorption takes placeat that particular wavelength. The strongest absorption peak of thesilver nanoparticles was observed at 437 ± 5 nm and silica coatedAgNPs showed broader peak at 462 ± 5 nm as shown in Fig. 2a andb respectively. These results suggested a red shift in SPR whichwas found to be dependent on increase in particle size, dielectricmedium and surface adsorbed species [14,15]. The results also sug-gested that extinction coefficient of core–shell particles was muchlarger than nanoparticles. The formation of silica shell of ∼30 nm

thick around silver particles was observed in TEM images as shownin Figs. 3a and b which were spherical in shape with a smoothsurface 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

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The detection of organophosphate pesticides is based on thechange in relative fluorescence intensity of the pyranine attached

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

enter is associated with silver [16]. The size of the synthesizedgNPs was estimated to be 65 nm and 35 nm with and without silicahell respectively by SEM images as shown in Fig. 4a and b. In XRDattern a number of strong Bragg reflections were obtained corre-ponding to h k l values of (1 1 1), (2 0 0), (2 2 0), (3 1 1) as shown inFig. 5) which matched with the JCPDS database (PDF2-Numbers0783 and 411402) confirming fcc structure of AgNPs. The sizef silver nanoparticles as calculated from the XRD pattern usinghe Debye–Scherrer formula was found to be 33 nm which is alson close approximation to the size as obtained by TEM and SEMnalysis [17].

The synthesized silver colloids with and without silica nanoshellere characterized by FTIR to monitor the formation of silica shell

n AgNPs. The presence of spectral signatures of silver nanoparti-les at 1650–1600 cm−1 as shown in (Fig. 6a) are due to the presencef citrate layer on silver which confirms the reduction of silver ionshich subsequently resulted in the formation of AgNPs. The pres-

nce of peaks in the region 1400–1100 cm−1 as shown in (Fig. 6b)re due to the formation of Si O Si bonds which is considered toe the fingerprint of metal oxides. These bands are attributed to �Si O), � (Si OH), � (Si O Si), and � (CH2) from residual TEOS ori-ethoxy residuals due to incomplete TEOS hydrolysis [18,19].

.2. Fluorescence signal enhancement

The 1:20 molar ratio of AgNPs to OPH6His–pyranine complexnhanced the fluorescence signal by three folds as compared toenfolds in the presence of silica coated silver colloids as shownn (Fig. 7) which is the maximum limit for pyranine due tots high quantum yield. The main reason for enhancement ofuorescence signal is attributed to the proximity of pyranine exci-ation wavelength (�ex = 460 nm) to the wavelength at which silica

oated AgNPs (65 nm) exhibit maximum cross sectional scatter-ng at 462 nm as compared to 437 nm by AgNPs. The silica coatingncreased the dielectric constant of the surrounding medium of

Fig. 4. SEM images of (a) AgNPs and (b) AgNPs with silica shell.

AgNPs thereby shifting the surface plasmon peak toward the higherwavelength.

3.3. Detection of organophosphates by silica coated AgNPs

Fig. 5. X-ray diffraction pattern of AgNPs showing distinct three peaks correspond-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. 7. Enhancement in fluorescence signal of bioconjugate (pyranine–OPH6His) inthe presence of AgNPs with and without 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 ofOPH6His–pyranine.

to hydrolyzing enzyme OPH6His (bioconjugate) due to the changein the pH of bioconjugate micro environment on the hydrolysisof organophoshates in the presence of silica coated AgNPs. The1:20 molar ratio of the bioconjugate with silica coated AgNPs in1 mM CHES (pH 8) was selected for the detection assay whichare 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 assayas shown in (Fig. 8b). The dynamic concentration range was from 5to 100 ppb with a standard deviation of 1.1% (n = 10) for paraoxon.In case of methyl parathion, lower detection limit of ∼10 ppb hasbeen obtained qualitatively as compared to ∼50 ppb without sil-ica coated AgNPs as shown in Figs. 9a and b respectively [20]. Thestandard deviation in this case was 1.51% (n = 10) with a dynamicconcentration 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 whichis exhibited in the presence of silica coated AgNPs of the existingbiosensing assay.

S. Thakur et al. / Sensors and Actua

Fig. 9. Calibration plot for methyl parathion with (a) bioconjugate ofOPH6His–pyranine in the presence of synthesized AgNPs coated with silicashell and (b) bioconjugate of OPH6His–pyranine.

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Pardeep Kumar completed his Master of Technology in Nanotechnology fromNational Institute of Technology, Kurukshetra in 2012. The present work has been

. Conclusions

The studies have demonstrated the detection of organophos-hate pesticides with higher sensitivity due to the tenfoldnhancement of fluorescence signal by employing silica coatedilver nanoparticles to the OPH6His–pyranine bioconjugate. Theualitative detection limits as obtained by this biosensing assayre ∼2 ppb and ∼10 ppb for paraoxon and methyl parathion respec-ively which are lower as compared to the enzyme based biosensoror organophosphate pesticides. The presence of silica nanoshelln AgNPs increased its stability and made the tagging possi-le with bioconjugate due to the presence of hydroxyl groupsn silica shell. Therefore, we conclude that AgNPs with silicahell have capability to significantly enhance the optical sensingignal through amplifying fluorescence intensity leading to thenhancement in sensitivity of the developed assay which fur-

her widens its applicability for detecting organophosphate nervegents.

tors B 178 (2013) 458– 464 463

Acknowledgements

The authors gratefully acknowledge the support of the Director,CSIR-CSIO, Chandigarh. The financial assistance provided to CSIO byCSIR, New Delhi is highly acknowledged. Research in the laboratoryof 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 fromPanjab University Chandigarh, India. She is presently working on the project to

carried out by him at Central Scientific Instruments Organization during his projecttraining.

4 Actua

Mgs

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. Venkateswar Reddy completed his Master’s degree in Bio-technology from Ban-alore University in 2003. Currently, he is a Senior Research Fellow in a DRDOponsored research project involved in developing nano biosensors.

. Siddavattam did his graduation from S.V. University, Tirupati, India in Envi-onmental Biology and Ph.D. in 1985. Currently he is a senior professor in theniversity of Hyderabad, Hyderabad and specializes in optimizing expression and

arge scale purification of recombinant OPH. His laboratory has shown that therganophosphate hydrolase (OPH), the key enzyme in inactivating triester bond

tors B 178 (2013) 458– 464

found in structurally diverse group of OP-compounds, targets to membrane throughan alternate protein transport pathway known as twin arginine transport (TAT)pathway.

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 inwater and soil, explosive detection and analytical instrumentation.