sensors and actuators b: chemical - iisc bangalore · melamine added during the biosynthesis....
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Sensors and Actuators B 238 (2017) 641–650
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical
jo ur nal home page: www.elsev ier .com/ locate /snb
nstantaneous detection of melamine by interference biosynthesis ofilver nanoparticles
.C.G. Kiruba Daniel, Lourdes Albina Nirupa Julius, Sai Siva Gorthi ∗
epartment of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore, India
r t i c l e i n f o
rticle history:eceived 5 April 2016eceived in revised form 27 June 2016ccepted 21 July 2016vailable online 25 July 2016
eywords:elamine
nterference biosynthesisilver nanoparticles
a b s t r a c t
Instantaneous detection of melamine, a potential milk adulterant has been demonstrated at room tem-perature by means of interference biosynthesis of silver nanoparticles. The sensing mechanism is basedon the colorimetric change observed during the synthesis of silver nanoparticles due to the presenceof melamine added during the biosynthesis. Presence and absence of melamine led to either inhibitionof nanoparticle formation or enable partial synthesis of nanoparticles which is detected spectrally. Alimit of detection (LOD) of 0.1 ppm in water and 0.5 ppm in raw milk was detected by the proposed tech-nique at room temperature. UV–vis spectroscopy and High Resolution Transmission Electron Microscopy(HR-TEM) have been used to detect the spectral Surface Plasmon Resonance (SPR) and morphologicalchanges of synthesized silver nanoparticle with and without the presence of the analyte melamine. Fur-
nstant detection ther, interference synthesis based sensing of melamine was done with caffeic acid as a reducing agentwhich confirms the role of caffeic acid a major constituent of Parthenium leaf extract for interferencebiosynthesis based sensing. Melamine is detected from raw milk by interference biosynthesis based sens-ing after a facile milk pre-processing step. Thus the method can be converted into a workable handheldprototype for detection of melamine for in-situ field applications.
© 2016 Elsevier B.V. All rights reserved.
. Introduction
Adulteration of food is one of the major problems in the foodndustry and their detection has become the need of the hour [1].t will be a boon to detect food adulteration at a rapid rate within
few minutes using a simple technology to prevent health haz-rds. Food materials/drinks when adulterated will lead to seriousealth issues based on the adulterant being used [1]. Melamine
s one such adulterant milk which when added to milk makes itook protein rich due to the high nitrogen content in melamine.dulteration of milk by melamine will lead to formation of kid-ey stones and other renal problems in infants as well as adults2]. Widespread melamine adulteration was reported in China in008 where about 54,000 children were hospitalized with feweported deaths [3]. Currently melamine is being detected com-ercially by means of Gas Chromatography–Mass Spectrometry
GC–MS), High Performance Liquid Chromatography (HPLC), Liquidhromatography–Mass Spectrometry (LC–MS) and other sophisti-ated techniques [4]. The above mentioned centralized laboratory
∗ Corresponding author.E-mail addresses: [email protected], [email protected]
S.S. Gorthi).
ttp://dx.doi.org/10.1016/j.snb.2016.07.112925-4005/© 2016 Elsevier B.V. All rights reserved.
testing techniques require skilled personnel for conducting the testand to interpret the results obtained. Hence there is a need for thedetection of melamine in a rapid, simple and affordable way.
With the advent of nanotechnology, different research groupshave demonstrated the usage of silver and gold nanoparticles forsensing of melamine [5]. Most of the literature employed previouslysynthesized metal nanoparticles for sensing applications. Noblemetal nanoparticles owing to their Localized Surface Plasmon Res-onance (LSPR) have been utilized for different sensing applications[6–14]. Similarly LSPR of metal nanoparticles − Gold and Silver havebeen put into use for the detection of melamine. In most of thepublications where melamine is detected by metal nanoparticles,two steps are followed viz − synthesis of nanoparticles followed bysensing of analytes [5]. Synthesis followed by sensing requires sometime before the sensing takes place. In the current strategy sensingof the analyte by the nanoparticles will be due to the interferencein the biosynthesis of nanoparticles by the analyte at room temper-ature. Recently biosynthesized silver nanoparticles have been usedto detect melamine where, previously synthesized nanoparticleswere utilized to sense melamine [15]. Interruption in synthesis of
nanoparticles (gold and silver) based sensing has been previouslyreported for the detection of melamine using reducing agents likehydrogen peroxide, dopamine, ellagic acid [16–18] while there areno reports of a leaf extract based synthesis interruption leading to642 S.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650
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ig. 1. A) Color photo of sensing of melamine at different ppm concentrations of A −ized silver nanoparticles by means of interference biosynthesis. B) UV–vis spectroith different ppm concentration of melamine.
ensing of any analyte. This single step synthesis cum sensing ofanoparticles is fast and lead to rapid detection in a few seconds<20 s). Previous interference based sensing methods use chemi-als as reducing agents and the sensing time are in the order ofinutes. The current strategy of using interference biosynthesis is
method of biocompatible sensing due to the involvement of leafxtracts as reducing agents instead of chemical reducing agents.lso the present technique is faster (in seconds) compared to exist-
ng interference sensing techniques for the detection of melamine.reviously reported sensing strategies employed like synthesis fol-owed by sensing consumes a minimum of 30 min of sensing time
ithout including the synthesis time, where as in the current inter-erence biosynthesis based sensing, sensing along with synthesisappens in seconds leading to faster detection of melamine.
. Materials and method
Silver nitrate (Merck) is used as such for preparation of 1 mMrecursor solution. Leaf extract of Parthenium histerophorus (nox-
m, 1 ppm, 10 ppm, 100 ppm, 1000 ppm using Parthenium sp. leaf extract biosynthe- graph exhibiting the spectral shift in SPR peak of silver nanoparticles synthesized
ious weed) has been prepared [19] by boiling 30 g of leaves of in100 ml deionized water for one hour. Caffeic acid (Merck) was dis-solved in water at appropriate concentration (4 mM) and was alsoused as a reducing agent for interference sensing. For interferencebased biosynthesis based sensing experiments, 200 �l of differentconcentrations of analyte (melamine) is added to 500 �l of theprecursor (silver nitrate) and 10 �l of the pH adjusted reducingagent (leaf extract) added subsequently. For synthesis and sensingexperiments, previously synthesized nanoparticle solutions addedto different concentrations of analytes (0.1 ppm to 1000 ppm ofmelamine). In all the sensing experiments, the microliter volumesof samples were taken in a 96-well plate and subjected to UV–visabsorption spectroscopy scan from 300 nm to 800 nm using theTECAN 200 INFINITE plate reader. High Resolution TransmissionElectron Microscopy analysis was done utilizing a F30 TECNAIHRTEM operating at 200 kV. Sample preparation for HR-TEM was
done by adding a drop of the aqueous nanoparticles on a 400 meshcarbon coated copper grid and subsequently dried at 50 ◦C. Milkspiking is done by adding melamine solutions to 1 ml of raw milkS.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650 643
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ig. 2. Different scales of High Resolution Transmission Electron Microscopy imageifferent scales.
o make up to the ppm concentration of melamine as 500 ppm,0 ppm, 5 ppm and 0.5 ppm. Control milk sample constitutes addi-ion of water to 1 ml of raw milk. A modified milk pre-processing
lver nanoparticles biosynthesized without (A) and with (B) melamine (100 ppm) at
[20] step was carried out by adding 30% Tri Chloro Acetic acid(TCA) and 3 M Sodium hydroxide (NaOH) in consecutive steps.Repeatability of each experiment was verified by conducting the
644 S.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650
Fig. 3. Absorbance (414 nm) of silver nanoparticles biosynthesized using leaf extract of Parthenium in the presence of melamine (different concentrations from 0.1 to 5 ppm).
Fig. 4. Comparison of 3D (A and B) and 2D (C and D) graphs of biosynthesis/sensing and interference sensing using silver nanoparticles synthesized with and withoutmelamine (1 ppm and 10 ppm respectively); Black lines in Fig. 4C and 4D denotes the increase in the absorbance of silver nanoparticles with respect to time (0 min to 50 min)which is not occurring for control and biosynthesis/sensing.
S.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650 645
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Fig. 5. UV–vis spectroscopy graph exhibiting biosynthesized silve
xperiments in triplicates. Conventional statistical methods likerror bars for selectivity experiments and standard deviation forecovery experiments were used.
. Results and discussion
Melamine sensing has been achieved based on the interactionf melamine with the reducing agent while forming nanoparticles.t is possible to sense melamine based on colour change leading toubsequent spectral change. Previous literature reported the sens-ng of melamine [5], based on aggregation among the synthesizedanoparticles whereas in the current study substantial change inhe size and shape of the nanoparticles is observed. Detection (dif-erent ppm from 0.1 to 1000) is determined by the colour changef partially formed silver nanoparticles (Fig. 1A) and its variation inPR is determined spectrophotometrically. There is an absorbanceecrease in the SPR peak which is due to the differential size ofhe nanoparticles formed due to interference of biosynthesis of sil-er nanoparticles by melamine (Fig. 1B). The peak observed near80 nm is for the melamine [21], hence it becomes more remarkableith excess of melamine.
In the earlier work that employed previously synthesizedanoparticle for sensing application as a result of the aggregationf nanoparticles in proportionate to the analyte concentration, ateady decrease in the SPR of silver nanoparticles at 395 nm and
steady increase in the absorption at 645 nm has been observed5]. However, in the present study there is a steady decrease atPR peak of 418 nm but there is no increase at any other wave-ength which may be due to the formation of silver nanoparticletself getting disrupted when subjected to different concentrationf melamine. High Resolution Transmission Electron Microscopymages (Fig. 2) of silver nanoparticles without melamine and silveranoparticles with melamine (100 ppm) by interference in biosyn-hesis taken at different scales of 2 nm, 20 nm, 50 nm, 100 nm. Theyeveal the differences in size and shape of the nanoparticles. Sil-er nanoparticles without melamine (control) exhibited uniform
haped particles of size between 5–20 nm while melamine inter-ered biosynthesis of silver nanoparticles led to formation of big,rregular shape and sized nanoparticles. This leads to change in SPRroperties and hence there is a change in colour of the solution.particles being used for detection of melamine at different hours.
A linear plot is obtained between absorbance (414 nm) anddifferent melamine concentration between 0.1–5 ppm (Fig. 3).Fig. 4 elucidates the three dimensional graph of comparisonbetween synthesis/sensing (biosynthesis followed by sensing)versus synthesis-interference based sensing for 1 and 10 ppm.There is no much change in the SPR between silver nanoparti-cles without melamine (control) and biosynthesis/sensing samples,whereas there is an appreciable SPR difference for 1 ppm, 10 ppmand control (without melamine) of interference biosynthesis basedsensing. Also in Fig. 4 the change in absorbance (SPR) with respectto time for 1 ppm and 10 ppm of melamine was recorded from0th minute to 50th minute for control, synthesis/sensing andsynthesis-interference sensing. In synthesis-interference basedsensing there is gradual increase in absorbance/SPR with respectto time but exhibited clear difference compared to control andsynthesis/sensing from the 0th minute onwards. Silver nanopar-ticles previously biosynthesized, when subjected with melamine(100 ppm) and it is found that there is not much change in theSPR peak for 12 h (Fig. 5). The main reason is due to the presenceof different biomolecules present in the Parthenium leaf extractcapping the silver nanoparticles efficiently blocking them fromsensing melamine after synthesis whereas melamine interferedwith biosynthesis- based sensing is efficient in sensing.
Selectivity test has been carried out using different chemicalspresent in milk that may simultaneously interfere like lactose,lysine, dextrose, glycine, leucine, magnesium, cysteine, citric acid,along with melamine at 100 ppm concentration, control (with-out melamine) and not much colour change is observed for allanalytes except melamine (Fig. 6A) with a bar diagram exhibit-ing absorbance at 414 nm (Fig. 6B). Calcium and iron have beeninterfering with current sensing strategy but gets eliminated dur-ing the milk pre-processing step and the same have been reportedpreviously [22].
3.1. Sensing mechanism based on interference biosynthesis ofsilver nanoparticles
Surface Plasmon Resonance of noble metal nanoparticles likesilver, gold and platinum nanoparticles play a key role in sensing ofdifferent analytes. Slight aggregation or change in size/shape leads
646 S.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650
F biosyc e has
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ig. 6. Selectivity test carried out using different analytes at 100 ppm by interferenceolour except melamine sample and corresponding B) bar diagram where melamin
o change in Surface Plasmon Resonance. As the concentration ofelamine increases there is absorbance decrease in the SPR of sil-
er nanoparticles at 414 nm while there is no increase at 600 nmFig. 2 and 4) which otherwise is present in other methods of syn-hesis followed by sensing [5] and hence indicates the absence ofggregation related plasmonic peak. Melamine may have reactedith parthenin and caffeic acid which are the major phytochemi-
al components of Parthenium leaf extract and thereby making theolecules unavailable as reducing agent leading to the differential
ormation of silver nanoparticles and thereby color change. Caffeic
cid has been previously used for synthesis of silver nanoparticles23]. Interference–synthesis based sensing of melamine has beenried using caffeic acid as a reducing agent and the UV–vis spec-roscopy sensing graph has been showed in Fig. 7. Thus caffeic acidnthesis based sensing using silver nanoparticles − A) photograph exhibiting similarthe least absorbance at 414 nm.
which is major phytochemical constituent of Parthenium is play-ing a key role in biosynthesis − interference sensing of melamineby Parthenium histerophorus. Synergistic action of caffeic acid withother chemical constituents present in the leaf extract for the syn-thesis of silver nanoparticles and simultaneous sensing cannotbe ruled out. Schematic representation of deduced mechanism ofbiosynthesis–interference sensing is presented in Fig. 8.
3.2. Interference sensing with raw milk
Raw milk spiked with different ppm concentration of melamineis subjected to interference biosynthesis based sensing. An initialpre-processing step as reported by Zhou et al., 2011 [20] withoutinvolving centrifugation has been carried out in two steps with
S.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650 647
Fig. 7. UV–vis spectroscopy graph exhibiting the spectral shift in SPR peak of silver nanoparticles synthesized using caffeic acid and with different ppm concentration ofmelamine, which confirms the role of Caffeic acid (a major phytochemical of Parthenium) in interference biosynthesis based sensing.
synth
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Fig. 8. Schematic representation of the interference bio
light modification. TCA and NaOH have been utilised in subse-uent steps to lyse proteins. It has been observed that there is colour
hange and subsequent spectral change/decrease in absorbance at24 nm due to interference biosynthesis based sensing which is notbserved in milk without melamine (Fig. 9A&B). This colour changeesis of silver nanoparticle based sensing of melamine.
can be directly observed by naked eye and by spectral change till alimit of detection of 0.5 ppm of melamine which is well below the
safety limits of 1 ppm in India, UK and USA. A calibration curve hasbeen obtained by plotting absorbance at 424 nm against differentconcentrations of melamine to enable the detection of unknown648 S.C.G.K. Daniel et al. / Sensors and Actuators B 238 (2017) 641–650
Fig. 9. Sensing of melamine in direct milk (after milk pre-processing) spiked with melamine A − Colour Photograph, B − UV Vis Spectroscopy graph, C − Calibration Curveof absorbance versus melamine concentration (0.5–500 ppm) and inset shows recovery studies done with raw milk spiked with 5 ppm melamine.
Table 1Comparison of detection of limit of melamine and detection time by different methods.
Methods Detection limit (mol L−1) Time (Synthesis & Sensing for Nanoparticles) Reference
HPLC 3.2 × 10−9 30 min [24]LC–MS 2.1 × 10−8 24 h [25]GC–MS 8.0 × 10−9 7 h [26]FTIR 2.0 × 10−8 5 h [27]SERS 1.9 × 10−8 12 h [28]MIP-based sensors 1.3 × 10−9 1 h [29]Citrate capped AgNPs 2.3 × 10−6 8 h [30]p-nitroaniline modified AgNPs 7.9 × 10−7 2 h [31]Dopamine modified AgNPs 7.9 × 10−8 1 h [32]Crown ether modified AuNPs 4.7 × 10−8 2 days [33]Cysteamine modified AuNPs 7.9 × 10−6 2 h and 30 min [34]
−8
cd9
slbstmlt
CTA capped AgNPs 3.6 × 10Sodium D-gluconate stabilised AgNPs 5 × 10−7
Interference-Biosynthesis AgNPs 7.9 × 10−7
oncentration of melamine in raw milk (Fig. 9C). Recovery studiesone with 5 ppm melamine added to milk exhibited a recovery of6% (Fig. 9 Inset).
In Table 1, current method of biosynthesis − interference basedensing is compared with other existing methods based on theimit of detection and time taken for sensing. Current strategy ofiosynthesis − interference based sensing is comparably havingame limit of detection while synthesis and sensing time is faster
han other methods. In most of the methods, sensing is achieved byeans of nanoparticle synthesis, functionalizing nanoparticles fol-owed by detection, which consumes a lot of time. Also most of theechnique which involves synthesis/functionalization of nanopar-
2 h and 6 min [35]2 h [36]Instantaneous (less than 30 s) Our method
ticles require high temperature, while the current method involvessensing at room temperature.
4. Conclusion
Interference biosynthesis of nanoparticles based sensing of ana-lyte (melamine) has been demonstrated by utilizing the leaf extractof the weed plant Parthenium histerophorus as well as using its
major phytochemical constituent caffeic acid. LOD for melamineusing the proposed method is 0.1 ppm in water and 0.5 ppm in rawmilk which is well below the EU, US and Indian norms for the safetylevels of melamine in food products. The morphology (size andnd Ac
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hape) of the nanoparticles has been modified significantly leadingo change in SPR. Corresponding change in UV–vis spectroscopy haseen observed for different ppm concentration and analysed. Directilk spiked with different concentration (5–500 ppm) of melamine
as also been detected after initial milk pre-processing step. A cali-ration curve has been plotted to enable the estimation of unknownoncentration of melamine in raw milk and also a recovery of 96% ischieved using raw milk spiked with 5 ppm of melamine. Melamineensing based on interference biosynthesis is quicker than synthe-is followed by sensing due to the fact that the former is done in aingle step while the latter has to be done in dual steps. Biosynthesislong with sensing reported here has made possible the realizationf a robust integrated sensor for field applications. Present tech-ique of interference biosynthesis based sensing can be used as alatform to sense various other analytes.
cknowledgements
Authors would like to acknowledge SPARSH: Social Innova-ion Programme for Products: Affordable and Relevant to Societalealth project funded by BIRAC-Biotechnology Industry Researchssistance Council for the financial assistance. Authors would also
ike to acknowledge Robert Bosch Center for Cyber Physical Sys-ems, IISc for their support.
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Biographies
S.C.G. Kiruba Daniel is a Post-Doctoral Fellow under the mentorship of Dr. Sai SivaGorthi in the Department of Instrumentation and Applied Physics at the IndianInstitute of Science (IISc), Bangalore. He completed his doctorate in Nanotechnol-ogy from Anna University, Chennai, India in 2015. He has been involved in greennanotechnology based development of products. Currently at IISc, he is involved inmicrofluidic nanotechnology for the development of sensing devices. His researchinterest include nanotechnology and microfluidics based point of care sensingdevices. His nanotechnology based device has been shortlisted for best ten innova-tions from India through Indo – Swiss AIT Program and got trained in EPFL, Lausanne.
Currently he is having 220 citations and h- index of 8.Ms. Lourdes Albina Nirupa Julius is a project associate working under the guidanceof Dr. Sai Siva Gorthi in the Department of Instrumentation and Applied Physics atthe Indian Institute of Science (IISc), Bangalore. She completed her Masters fromIndian Institute of Technology, Madras. She has been involved in the development
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point-of-care diagnostic devices with the combination of optics, microfluidics, andelectronics. His research interests include Imaging Flow Cytometry, Microfluidics
50 S.C.G.K. Daniel et al. / Sensors a
f point of care diagnostic devices for rapid malaria detection. Her interests includeicrofluidics, optics and point of care diagnostic devices. She is the recipient ofandhian Young Innovation Award for the year 2015.
ai Siva Gorthi is working as an Assistant Professor in the Department of Instrumen-ation and Applied Physics at the Indian Institute of Science (IISc), Bangalore. Prioro joining IISc, he was a post-doctoral fellow of the Rowland Institute at Harvardniversity, where he developed multiple imaging modalities for recording informa-
ion of fast flowing cells in microfluidic devices. He obtained his doctorate in Optical
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Metrology from EPFL (Swiss Federal Institute of Technology), Lausanne, Switzerland,in 2010. Currently at IISc, part of his group is focusing on the development of various
and Droplet-microfluidics Instrumentation, and Optical Metrology. He is recipientof BIRACs BIG (Biotechnology Ignition Grant) Innovator award in 2014, and DBTsInnovative Young Biotechnologist Award (IYBA 2013).