Form 1

2019 Report Form for Collaboration with
Research Center for Biomedical Engineering

Year/month/date
NumberDate /Month/Yeardate:

To Chairman, Board of Directors, Research Center for Biomedical Engineering

Applicant Affiliation: Discipline of Electrical Engineering, IIT Indore, India
Title: Associate Professor
Name: Dr. Vipul Singh
Address: Molecular & Nanoelectronics Research Group (MNRG), Discipline ofElectrical Engineering, Indian Institute of Technology Indore, Simrol, Khandwa
Road, Indore, Madhya Pradesh, 453-552, India.
Phone: +91-732-4306-594
Fax:
E-mail: vipul@iiti.ac.in
Report Form for Collaboration Research

Research Theme

Research and Development of SERS Based Sensor for Residual Fungicide Detection

Research Area

1. Biomaterials
2. Bioengineering
3. Functional molecules
4. Chemistry/Electrical Engineering/Mechanical Engineering/Materials Science

Research Period

From: Date/month/Year
0 1 / 0 6 / 2 0

1

9

~

To: Date/month/Year
3 1 / 0 3 / 2

0

2

0

Applicant Organization
Name
Dr. Vipul Singh

Department
Discipline of Electrical Engineering, IIT
Indore, Simrol, Khandwa Road, Indore,
Madhya Pradesh, India.

Title
Associate
Professor

Ms. Tejswini K Lahane

Discipline of Electrical Engineering, IIT
Indore, Simrol, Khandwa Road, Indore,
Madhya Pradesh, India.

Ph.D. student

SynthesisofAg
Nanoparticles and Ag
Nanowire network

Mr. Anil Yadav

Discipline of Electrical Engineering, IIT
Indore, Simrol, Khandwa Road, Indore,
Madhya Pradesh, India.

M.S. (Research)
student

Working on Researchand development of
SERS based sensor

Ms.
Nagarajan

GSST, Shizuoka University, Japan

Ph.D. Student

FDTD
Simulation,
Raman Spectroscopy

Anitharaj

Role
Overallmanagement/executionof the researchCollaboration Partners in the
Research Center

Prof. Hiroshi Inokawa, RIE, Shizuoka University
Dr. Hiroaki Satoh, RIE, Shizuoka University

Research Results (Including Purpose, Results, Figures, etc.)

Chlorpyrifos is one of the most widely-used organophosphate crystalline insecticide. It is used to protect
more than 50 crops from insect, many of which are dietary staples. However, chlorpyrifos residues on fruits
bring risks to human health e.g. affects nervous system, headache, diarrhoea, lung cancer etc. Therefore, it
has become quite obligatory to detect and remove chlorpyrifos from the foodstuff before consuming.
Numerous methodologies have been proposed to detect chlorpyrifos pesticide residues over the fruit
surfaces, such as high-performance liquid chromatography (HPLC) [1], gas chromatography (GC), mass
spectrometry coupled with gas chromatography (GC–MS) [2] or liquid chromatography, biosensor method[3] etc. All above mentioned techniques are costly and time consuming. Thus, it is required to develop
simple and cost-effective techniques for efficient detection of chlorpyrifos. Surface Enhanced Raman
Spectroscopy (SERS) has been emerged as a highly efficient and reliable technique, which is capable ofdetecting even a small trace amount of pesticide residues in food [4,5,6]. In this process, interaction between
pesticide molecules and novel metal nanoparticles leads to significant enhancement in the Raman scattering.
Raman scattering is the inelastic collision of photons with the molecules and the difference between theincident photon energy and emitted photon energy is equal to the difference between the vibrational energy
levels. Raman spectroscopy gives the blueprint of a molecule with the shift in frequency of emitted photon
known as Raman shift. But the intensity of Raman spectra is feeble as a very small number of photons i.e.
one in million collides inelastically with the molecule. Metal nanostructures assisted Raman spectroscopy ofmolecules shows a significant enhancement of 106 in Raman intensity known as Surface Enhanced Raman
Spectroscopy (SERS). The enhancement in Raman intensity can be product of two phenomenon occurring
simultaneously, a) electromagnetic enhancement and, b) chemical enhancement. When incident photon
interacts with the metal nanoparticle or rough metallic surface, it can enhance the local electric field throughsurface plasmon resonance phenomenon which leads to the enhancement in Raman intensity.

Fig. 1 (a) SPR Phenomenon and (b) The phenomena of Surface Enhanced Raman Scattering (SERS)
Localised Surface Plasmon Resonance (LSPR) enhances electromagnetic coupling at the interfaces ofnanostructures within an extremely confined area. This phenomenon can provide many plasmonic ‗‗hotspots‘‘ in a unique nanostructure for surface-enhanced Raman scattering (SERS). Hotspots are locations in
the vicinity of the plasmonic nanostructures where the local optical field is enhanced tremendously whencompared to its surrounding [14]. Consequently, any molecule present in a SERS-active hot-spot will exhibit
an immense enhancement in its Raman scattering signals. there by resulting in high sensitivity for thedetection of target molecules with ultra-low concentration. Also, a significant time has been devoted byresearchers in developing SERS substrates with high specific surface area and SERS hotspots.

To detect chemical analyte successfully, the following requirements must be met for SERS [15]:A good substrate is essential. It must have a roughened surface to give good enhancement as well as be
reproducible and robust with a good lifetime.

The analyte must absorb on the surface effectively. It should have a higher SERS cross-section than
any likely interferents.

Various substrates are used for SERS application such as glass [16], silicon, porous silicon [17], PDMS [19].
SERS active substrates such as anodisc can also be used [18] add up in the enhancement of Raman
intensity.
Here, we have used anodics with pore size of 100 nm, Fig 2 shows the FESEM (Field emission scanningelectron microscope) image of anodisc which shows its porosity.
Metals such as Ag, Au, Cu, Pt etc. have negative real and small positive imaginary dielectric constants andcan generate the LSPR phenomenon. Among them, Ag and Au nanoparticles have distinguishing features of
LSPR, which can lead to their high sensitivity and excellent uniformity as SERS substrates. Ag shows
stronger EM coupling than Au, therefore, Ag was the choice for fabricating metal based substrate forpesticide detection.
SERS coupled with silver colloidal nanoparticle was used to detect paraquat on fruit and vegetable skins [7].
It was used in the detection of phosmet residues in apple extracts with gold nanosubstrate [8]. Chlorpyrifos
was detected in tea leaf apples, orange peels, rice, spinach, etc [12]. Rapid detection of omethoate residues inpeach extract was performed with silver nanoparticle-based SERS [9]. Using SERS, the thiabendazole fromthe chemically treated citrus fruits skins were detected and monitored [10]. Carbaryl pesticide in apples
extract was detected with combined SERS and chemometric method [11], whereas chlorpyrifos on tomato
surfaces and in tomato extract was detected by surface-enhanced Raman spectroscopy (SERS) [13].

Fig.2 SEM images of Anodisc of front and back side.
Objective of our work is to optimize the synthesis of silver nanoparticles and silver nanowire and fabricate
SERS sensor by using anodisc as substrate over which different silver nanostructuture could be used todetect Pesticides.
Chemicals and Reagents Used:
Silver nitrate (AgNO3, 99.99%,Sigma Aldrich), Tri Sodium Citrate (Sigma Aldrich),DI ,Ethylene
Glycol(EG , 99.99%,Sigma Aldrich),Polyvinylpyrrolidone (PVP ,MW 130000) NaCl , KBr (sigma Aldrich),Reagent Ethanol and Acetone , Anodisk 100nm pore diameter.
Synthesis of Silver Nanoparticles:
There are many ways to synthesize silver nanoparticles (Ag NPs) such as physical, chemical and biologicalmethods. Chemical reduction is most frequently applied for the preparation of stable, colloidal dispersions
Ag NPs in water or organic solvents.
Synthesis of AgNPs using sodium citrate:
AgNps were synthesized by chemical reduction using Sodium citrate as reducing agent as well as stabilizer[5]. The average size, size distribution, morphology, composition and structure of particles were estimatedby Field emission scanning electron microscopy (FESEM), EDX and UV/Visible absorption spectroscopy.
In this process 70mg of Silver nitrate were dissolved in 400ml DI water, after that it was continuously boiled
and simultaneously stirred at 500 rpm. After boiling the silver nitrate solution, 1 wt. % aqueous sodiumcitrate solution was rapidly injected into the flask. In fig. 4 the color changes after adding sodium citrate isshown which corresponds to different size of the Ag NPs during the growth process. After further boiling for
30 min, it was cooled down to room temperature. To remove excess citrate ions from AgNP solution it wascentrifuged for 15 min with 3000 rpm. Overall growth mechanism of AgNPs is shown in Fig.5.

Fig. 3 Shows different steps involved in the synthesis of Silver Nanoparticles

Fig. 4 shows the changes in the color of the colloidal solution during synthesis

Fig.5 growth mechanism of Ag NPs caped by citrate ions [26].
FESEM-EDX analysis of silver NPs reduced by sodium citrate
FESEM was used to investigate the size and shape of silver nanoparticles. The SEM images of the
AgNPs are shown in Fig.-6. Upon morphological investigations it was found that the spherical particles withaverage size of 30- 50 nm were formed. Here sodium citrate was used as reducing and capping agents. In
growth solution. Moreover, from EDX spectra shown in Fig.-7 it is clear that silver nanoparticles werereduced by sodium citrate and has wt.% of 89.6%.

Fig. 6 Shows FESEM images of Ag nanoparticle synthesis using Sodium citrate

Fig. 7 EDAX data for Ag nanoparticle using Sodium citrate
UV-Vis of silver nanoparticles
The UV-Vis absorption spectra (Fig. 8.) shows a plasmonic peak at 414 nm which corresponds to the particle
size of 30-50 nm. Hence, this investigation is in agreement with the previously estimated size by FESEM
images. The full width half maximum (FWHM) of the peak is direct evidence of dispersity of thenanoparticles, where a large FWHM of can be attributed to polydispersity.

Fig. 8 UV-Vis Absorption spectra for silver nanoparticles dispersed on Anodisc
Synthesis of Silver Nanoparticles using polyol Method
Polyol method is used to synthesize monodispersed AgNPs. Size and shape control of silver
nanoparticles can easily possible due to polyol method by varying molar ratio of PVP and silver nitrate. Sizeand shape of silver nanoparticles can be controlled effectively by the polyol method by varying molar ratio

of PVP and silver nitrate. The SERS phenomena is highly dependent on the size and shape of thenanoparticle. Therefore, polyol method was chosen to synthesize the Ag nanoparticles with control on sizeand shape.
In this method ethylene glycol is used as a solvent as well as reducing agent, whereas, Polyvinyl pyrrolidone
is used as capping agent. To prepare the solution, 0.17g AgNO 3 and 1.2 g of PVP were dissolved in separate
10ml Ethylene Glycole (EG) solvent at room temperature. The PVP solution was continuously heating andstirring at 160 ℃ and 500 rpm for 1hr. Consequently, AgNO3 solution was added drop by drop (1ml/5min)
while continuous heating and stirring of the solution. Thereafter, the solution was kept for heating at 160 ℃
for 4hrs and allowed to cool at room temperature. The as-synthesized nanoparticles in the solution were
ultrasonicated and centrifuged three times at 5000 rpm for a duration of 20 min each in acetone and ethanol.
Finally, residue product was rinsed in ethanol solvent and kept in oven at 40℃ for 5 hrs.
SEM-EDX analysis of silver NPs synthesized by Polyol method
As shown in the FESEM images (Fig. 9) the nanoparticles synthesized using Polyol method havemonodispersed state with a particle size distribution in the range 90-100 nm. Whereas, the EDX spectra
analysis shows of silver content to 97.1% by weight.

Fig. 9 FESEM Images and EDAX data of Ag nanoparticles synthesized using Polyol synthesis
Synthesis of Silver Nanowire (Polyol method)

AgNWs structure has high specific surface area with flexibility which can provide an effective adsorption
surface for the target molecules. Therefore, AgNWs offer the possibility for use in SERS detection.
However, the random spatial clusters of AgNWs cannot generate uniform electromagnetic (EM) coupling in
the network structure. In contrast, AgNPs that are evenly distributed in the AgNW network can not only
generate more hot spots but also improve the uniformity of EM coupling [21]. The perfect structure cangenerate plasmonic hot spots via both the strong EM coupling among AgNWs and the strong EM coupling
among AgNPs and AgNWs [21].
In this work we have synthesized AgNWs using salt mediated polyol method for SERS based pesticide
detection. Firstly, 75mg of PVP was dissolved in 10ml of EG with simultaneous heating and stirring thesolution at 160 ℃ for 1hr. Thereafter, three solutions were prepared viz: NaCl with molarity of 3.5 mM, KBr
with molarity of 1.8 mM and AgNO3 of molarity 30.5 mM in 5 ml EG as solvent, respectively, for whichwere used as additives in the PVP solution. Subsequently, 40 µl of NaCl and 20 µl of KBr solutions wereadded in the PVP while continuous heating and stirring. After 20min-30min AgNO3 solution was added dropby drop (1ml/5min) while the process of heating and stirring of the mixture at 160 ℃ was performed for3hrs. Consequently, the solution was allowed to cool down at room temperature. To perform the purification,
the as-synthesized NWs were ultrasonicated and centrifuged for 3 times at 5000 rpm for 20 min in acetone
and ethanol, respectively. Finally, the purified colloidal solution of NWs was mixed in ethanol which wasagain dispersed on glass substrate for further characterization.
The Fig. 10 shows the FESEM images of the obtained NWs. From the analysis of the NWs images the aspectratio of the synthesized NWs was estimated to be 500. This much high aspect ratio NWs has very diverse
applications like in transparent electrodes.

Fig. 10 FESEM Images is Silver Nanowire synthesized using Polyol
UV- Vis of silver nanowire
The absorption spectra in Fig. 11 shows that the thin film has very feeble absorption corresponding to thevisible light. Whereas, the absorption peak is observed to be at 370 nm. This peak may be assigned to thetransverse plasmon mode of AgNWs. The exact position of this plasmon band is extremely sensitive to thenanoparticle size and shape.

Fig.11 UV-Vis Absorbtion spectra for silver nanowire dispersed on glass
Fabrication of SERS sensor
The pesticide solution was prepared by adding 1 mM chlorpyrifos in 80 µl of Methanol: DI (1:1) mixture.
In order to fabricate pesticide sensor, colloidal solution of silver nanoparticles was dispersed over theanodisc and allowed to dried it at room temperature for 1hr. Afterwards, chloropyrifos solution wasdispersed on anodisc and dried it for 2 hr at room temperature.
Raman Spectra of silver nanostructures and pesticide adsorbed silver nanostructures
For the purpose of SERS based pesticide detection the NPs synthesized by sodium citrate assisted growth
was preferred. The nanoparticles synthesised by this method has smaller feature size which is liable toprovide more hot spots. Raman spectroscopy was performed for the pristine NPs dispersed over the anodisc
and Fig. 12 shows the Raman spectra for the same. The Raman spectroscopy of chlorpyrifos, dispersed over
AgNPs, results in the phenomena of SERS was performed. The Fig. 12 shows the spectra corresponding
SERS phenomena.
In the Fig. 12 overall enhancement in Raman spectra of Ag nanoparticles with adsorption of chlorpyrifos
pesticide can be attributed to SERS phenomena. Along with AgNPs peaks there are several other peaks inchlorpyrifos adsorbed AgNPs Raman spectra. These peaks might have emerged because of bond stretchingin pesticide assisted Ag nanoparticles. The peak at 340 cm-1 corresponds to N-cyclopropyl bending vibrationand the peak at 411 cm-1 could be assigned to P–O–C stretch. Subsequently, a peak at 631 cm-1 withsignificant intensity is because of P=S stretching vibration [13]. Further, a feeble peak is also observed ataround 1240 cm-1 could be assigned to Cl-ring vibrations in chloropyrifos. Further as we can see all the
peaks for Ag nanoparticles which are highly dependent on synthesis process and capping agents are alsoavailable in pesticide assisted Ag nanoparticles also.

Fig. 12 Raman spectra of silver nanoparticles (synthesized by sodium citrate )and chloropyrifos adsorbed on
silver nanoparticles.
Surface Enhanced Fluorescence by Silver Nanoparticles
The PL from noble metal nanoparticles was reported for gold, copper and silver [22][23][24]. All thesemeasurements revealed the maximum of the PL band close to the interband absorption edge and thus the PL
was attributed to the interband radiative transitions [22-26]. The PL was observed only from nanoparticles
smaller than 40 nm in diameter and from nanorods with a high aspect ratio, which suggests a strong size andmorphology dependence of the PL spectra. When the size of the particles decreased, the intensity of the PL
increased indicating a strong influence of the surface the nanoparticles on the PL spectra.
The strong local electric field close to the surface of the particles enhances the Raman scattering frommolecules functionalized on its surface [25] and luminescence from molecules functionalized on the surfaceof the particles or close to a rough metal surface [26]. This suggests that the strong local electric field
induced by the SPR excitation in the particle can also enhance the quantum yield of the photoluminescencefrom the particles.
In Fig. 13 the excitation spectra of AgNPs and chlorpyrifos adsorbed AgNPs are shown. The excitation
wavelength was fixed at 325 nm and the emission spectra was recorded. Silver nanoparticles showedemissions peaks at 390 nm, 490 nm and 450 nm. Chlorpyrifos adsorbed on the surface of silver nanoparticles
show enhancement and red shift of 490 nm emission peak by about 20 nm with respect to the AgNPs
emission.

Fig.13. PL Spectra of silver nanaoparticles synthesized by sodium citrate.
Conclusion(s): We have successfully synthesized silver nanostructures.
 Silver nanoparticles synthesized by sodium citrate reduction gives nanoparticles size in the range
30-50nm with agglomeration.
 Silver nanoparticles synthesized by polyol method gives monodispersed nanoparticles with size of
nanoparticles 90-100 nm.
 Silver nanowire by salt mediated polyol method results in aspect ratios of 500.
 Anodisc as substrate gives porosity and roughness for silver nanoparticles which helps for Raman
enhancement.
 The enhancement in Raman spectra was observed when chloropyrifos adsorbed on silver
nanoparticles.
 Hence, SERS mediated pesticide detection was achieved by AgNPs.
 Also, we have observed PL enhancement for silver nanoparticles.
Future Work:
1) Analysis of Raman spectra for AgNWs as well as combination of AgNPs and AgNWs.
2) Raman spectroscopic analysis of chlorpyrifos adsorbed over AgNPs synthesized by different
methods.
3) Optimization of silver nanoparticle size and deposition on anodisc for improvement inenhancement of Raman scattering.
4) Detection of different pesticides and achieve repeatability.
References:[1] Beale DJ. et al., “Detection of s-triazine pesticides in natural waters by modified large-volume direct
injection HPLC”, Talanta, Vol. 82(2) (2010), 668–674.
[2] Sinha SN. et al., “Effect of dissociation energy on ion formation and sensitivity of an analyticalmethod for determination of chlorpyrifos in human blood, using gas chromatography–massspectrometer (GC–MS in MS/MS)”, Int. J. Mass Spectrom., Vol. 253 (2006), 48–57.
[3] Kuswandi B. et al., “An optical fiber biosensor for chlorpyrifos using a single sol–gel film containing
acetylcholinesterase and bromothymol blue”, Talanta, Vol. 74(4) (2008), 613–618.
[4] Fan Y. et al., “Analyses of phosmet residues in apples with surface-enhanced Raman spectroscopy”,
Food Control, Vol. 37(1) (2004), 153–157.
[5] Minwoo L. et al., “Sub nanomolar Sensitivity of Filter Paper-Based SERS Sensor for Pesticide
Detection by Hydrophobicity Change of Paper Surface”, ACS Sens., Vol. 3 (2018), 151−159.
[6] Sebastian S. et al., “surface Enhanced Raman Spectroscopy Concept and Chemical Application”,

Angew. Chem. Int. Ed., Vol. 53 (2014), 2 – 42.
[7] Fang H. et al., “Ultrasensitive and quantitative detection of paraquat on fruits skins via
surface-enhanced Raman spectroscopy”, Sens Actuators B Chem., Vol. 213 (2015), 452–456.
[8] Fan Y. et al., ―Analyses of phosmet residues in apples with surface-enhanced Raman spectroscopy‖,
Food Control, Vol. 37(1) (2014), 153–157.
[9] Yaseen T. et al., ―Detection of omethoate residues in peach with surface-enhanced Raman
spectroscopy”, Food Anal Methods, Vol. 11(9) (2018), 2518–2527.
[10] Müller C. et al., “Detection of thiabendazole applied on citrus fruits and bananas using surface
enhanced Raman scattering”, Food Chem., Vol.145(7) (2014), 814–820.
[11] Fan Y. et al., “Determination of carbaryl pesticide in Fuji apples using surface-enhanced Raman
spectroscopy coupled with multivariate analysis.”, LWT Food Sci Technol., Vol. 60(1) (2015), 352–357.
[12] Feng S. et al., “Development of molecularly imprinted polymers-surface-enhanced Raman
spectroscopy/ colorimetric dual sensor for determination of chlorpyrifos inapple juice”, Sens Actuators B Chem., Vol. 241 (2017), 750–757.
[13] Pei Ma. et al., “Rapid quantitative determination of chlorpyrifos pesticide residues in tomatoes by
surface‑enhanced Raman spectroscopy European”, Food Research and Technology, Vol. 246 (2020),
239–251.
[14] Wang A. et al., “Review of Recent Progress of Plasmonic Materials and Nano-Structures for
Surface-Enhanced Raman Scattering.”, Materials., Vol. 8 (2015), 3024–3052.
[15] McNay G. et al., “Surface-Enhanced Raman Scattering (SERS) and Surface-Enhanced ResonanceRaman Scattering (SERRS)”, A Review of Applications. Appl. Spectrosc., Vol.65 (2011), 825–837.
[16] Zhang L. et al., “Self-assembly Ag nanoparticle monolayer film as SERS Substrate for pesticide
detection.”, Appl. Surf. Sci., Vol. 270 (2013), 292−294.
[17] Chan S. et al., “Surface-Enhanced Raman Scattering of Small Molecules from Silver Coated Silicon
Nanopores”, Adv. Mater., Vol. 15 (19) (2003), 1595−1598.
[18] Huang Z. et al., “Large-area Ag nanorod array substrates for SERS: AAO template assisted
fabrication, functionalization, and application in detection”, PCBs. J. Raman Spectrosc., Vol. 44 (2),240−246.
(19) Cho et al., “Ultrahigh-Density Array of Silver Nanoclusters for SERS Substrate with HighSensitivity and Excellent Reproducibility”, ACS Nano., Vol. 6 (1) (2012), 249−255.
[20] M. Kamali et al., “Controllable Synthesis of Silver Nanoparticles Using Citrate as Complexing
Agent: Characterization of Nanopartciles and Effect of pH on Size and Crystallinity”, Iranian journal ofChemistry and Chemical Engineering, vol. 31(4) (2012), 21-28.
[21] Wei W.et al., “Improving SERS hot spots for on-site pesticide detection by combining silver
nanoparticles with nanowires”, J. Mater. Chem. C, Vol.6 (2018), 8793—8803.
[22] Mona B. et al., ―The lightning gold nanorods: fluorescence enhancement of over a million comparedto the gold metal”, Chem. Phys. Lett., vol. 317 (2000), 517.

[23] Oleg A. et al., “Influence of annealing conditions on si’ze and optical properties of copper
nanoparticles embedded in silica matrix‖, Mater. Sci. Eng., Vol. 137 (2007), 247.
[24] Dhrubajyoti B. et al. “Size selective photoluminescence in poly(methyl methacrylate) thin solid
films with dispersed silver nanoparticles synthesized by a novel method”, Chem. Phys. Lett., Vol. 420(2006), 115.
[25] Fan Y. et al., “Measurement of the Distribution of Site Enhancements in Surface-Enhanced Raman
Scattering‖, Science Vol. 321 (2008), 388.
[26] A. F. et al., “Enhanced fluorescence by surface plasmon coupling of Au nanoparticles in an organic
electroluminescence diode”, Appl. Phys. Lett., Vol. 90 (2007), 173116.
[27] V. T. et al., “Synthesis of Silver Nanoparticles via Chemical Reduction and its Anti-bacterial
Activities in Wastewater of Shrimp Pond‖, IJERT, Vol. 5 (2016).

List of Publications Related to the Collaboration Research

The results are still in infancy we are working to improve further the performance of the SERS based sensor.

List of Presentations ( Conference, Meeting, etc)

One Poster presented at the Annual meeting for the 2019 cooperative research at Research Center ofBiomedical Engineering at Tokyo Institute of Technology. Abstract for the same was submitted.
List of Awards

Registration of research-theme continuation for next year
Prior consent from the collaboration partner in the Research Center is necessary.

Yes

No

Research plan for the next year (from April 1, 2020 to March 31, 2021), if the collaboration research is continued.
Background:Pesticides and Fertilizers are widely used in modern day agriculture. In order to meet the demand for food for thebooming world population, extensive usage of pesticides and fertilizers has become more common for gettinghigh yield of crops by protecting them against attack of insects and diseases. An estimate suggests that nearly 3
billion kilograms of pesticides are applied every year around the world. Furthermore, due to lack of knowledge
and proper enacting of laws in under developed countries farmers tend to apply pesticides in quantities which are
more than the prescribed limits. These pesticide molecules can enter into the food chain through direct adsorption
on the plant‘s leaves, fruits or vegetables surfaces, or can also enter into the soil and thereby the undergroundwater, leading to contamination of soil as well as water. Furthermore, the problem is more acute for countries like
India, where the agriculture essentially relies on Monsoon or the underground water for irrigation purposes. The

long term effects of pesticides entering into the food chain can be highly detrimental [1-4]. In this work wepropose to develop SERS based sensor for detection of two pesticides molecules namely Diazinon (anorganophosphate insecticide) and Thiram (a Fungicide) which acts against fungal diseases in seeds and plants
and also acts as an animal repellent. While Diazinon is used in agriculture to control insects on fruits, vegetables,nuts and field crops, however, it is also extensively used for household insects. Human exposure to Diazinon is
known for its severe effects on the nervous system. On the other hand exposure to Thiram, especially uponinhalation can also lead to dizziness, nausea, headaches and other GI related complications. It is also reported toaffect the thyroid, kidney and liver adversely.
Surface Enhanced Raman Scattering (SERS) as a phenomenon was first observed in 1973 [5], for Pyridine
adsorbed on roughened Silver electrode. However, it was only in 1997 when single molecule SERS was observed
and the technique gained attention of scientific community [6]. It is a unique technique that combines the modern
laser spectroscopy with exciting optical properties of metallic nanostructures and thereby results in highly
amplified Raman signals when molecules are attached to a nanometer sized Gold or Silver structures [7-10].

Fig.1 (a) Shows the Antistokes and Stokes shifted Raman spectroscopic signals (b) The effect of Localized Surface
Plasmon arising due to coherent oscillations of electrons near a Gold nanoparticle.

The prime significance of the technique lies in the fact that it effectively combines the structural information
content of Raman Spectroscopy (which typically consists of Stokes and Antistokes shifted signals, as shown in
Fig. 1(a)), with localized surface plasmons giving rise to ultrasensitive detection limits. SERS is an analyticaltechnique, that provides information on surface and interface processes, thus opens up exciting opportunities in
the field of biomedical and biophysical spectroscopy. SERS as a technique also overcomes one of the major
limitation of Raman spectroscopy emanating due to its extremely small cross-section of Raman process, which isnearly 12-14 orders below fluorescence cross sections. Surface Plasmon Polaritons (SPPs) are coherent
oscillations of electrons that are excited by electromagnetic radiation at metal dielectric interfaces, which typicallyarises on the surface of coinage metals viz. Gold and Silver as shown in Fig. 1(b). The Localized Surface Plasmon
Resonance (LSPR) effect involves the confinement of SPPs in nanostructures of dimension comparable towavelength of light. LSPR exhibits significant enhancement of localized electromagnetic coupling at theinterfaces of nanostructures within an extremely confined area. The overall intensity of a SERS signal depends on
both the incoming field ωinc and outgoing field (ωinc- ωvib). Thus the overall intensity of SERS signal is given by

ISERS  E inc (inc ) E(inc  vib ) . Moreover since the incident radiation at ωinc and the Stokes Raman shifted22

radiation (ωinc- ωvib) are in resonance with the plasmon peak of the metal nanostructure thus, (ωinc- ωvib) ≈ ωinc.
4

Hence the overall SERS scattering signal can be approximated as ISERS  E (inc ) , and the above
approximation has been found to hold good for blue and green region of electromagnetic spectrum. Further, it

1
r

3

may be recalled that the electric field strength due to a dipolar radiation scales with E(r)    , which directly

12

1
implies that the SERS signal intensity is given by I SERS    , indicating the fact that SERS is a highlyr
surface selective effect. Infact the sensitivity of the technique is so high that even the orientation of molecules
relative to the surface normal can be detected with ease. It also must be noted here that for a reasonable
electromagnetic enhancement factor arising due to LSPR effect give by β ~ 100, the overall enhancement in the
SERS signal is observed to be of the order of β4 ~ 108, nearly a billion times enhancement of otherwise weak
Raman signal.
Goal:
In this proposal we intend to develop a SERS based sensor. Some of the key requirements for the development ofsuch a sensor are that it should be developed on an appropriate substrate viz. substrate that does not give
Fluorescence, the enhancement of the signal as discussed above can be improved by efficient coupling of LSPR
arising out of metal nanostructures and finally the chemical interaction of the molecule adsorbed on the metal
nanoparticle substrate. In this proposal we intend to utilize the Anodic Aluminum Oxide (AAO) as a templatefor SERS substrate.

Fig. 2 Shows the nanoporous Anodized Aluminum Oxide (AAO) membrane, its sectional view and Silver nanoparticles
dispersed over its surface and later adsorbed with molecules for development of proposed biosensor.

Objectives of the proposed work are the following:1) Electrochemical growth of porous AAO using Aluminum foil.
2) Optimization of growth parameters to obtain wide variety of aspect ratio of these nanoporous AAO
membranes, to be used as SERS substrate.
3) Development of Ag nanoparticles of different shapes and sizes by chemical reduction method.
4) Adsorption of Thiram/Diazinon molecule on Ag nanoparticle.
5) Finally development of SERS based biosensor, and its optimization w.r.t. aspect ratio of the porous AAO
substrates and size of metal nanoparticles.
Methodology:
The proposed methodology to achieve the above set objectives of the proposal includes electrochemical
anodization of Aluminum foil, synthesis and dispersion of Ag/Au nanoparticles on the surface of AAO, adsorptionof molecules on metal nanoparticles substrate and finally characterization of the sensor using SERS technique. It
has been well reported in the literature that electrochemical anodization of Aluminum can be carried out todevelop aluminum oxide giving rise to two different types of Al2O3, namely the barrier type and the porous type.
Well-ordered nanoporous AAO templates having honeycomb structure have been applied for the development ofordered Silver and Gold nanostructures as SERS-active substrate. These well-ordered 2D nanostructures of AAO
have been utilized as a template for development of metal nanostructures, later used as an effective SERS

substrate. The honeycomb nanostructures of AAO can be easily grown by a single step electrochemical
anodization of polished aluminum foil. The typical steps involved are electrochemical polishing of the Al
substrate in a mixture of Perchloric acid (HClO4) and ethanol, thereby resulting in smoothly finished Aluminum
foil as shown in Fig. 3 (a). Later these electro polished Al substrates are taken in a two electrode electrochemicalcell for the anodization of Al into Al2O3 [11-15]. Using sulphuric acid the electrochemical anodization of
Aluminum proceeds to generate porous honeycomb network as shown in Fig. 3 (b). It has already been reported
that due to the repulsive forces between the neighbouring pores resulting in mechanical stress drives theself-assembly process of formation of honeycomb nanostructures. By selectively controlling the electrochemical
process parameters the aspect ratio and diameter of the pores can be selectively controlled to range a hexagonalclose pack array of pores ranging in diameter from 20 nm – 500 nm. These would later be utilized for SERS
substrate.

Fig. 3 shows schematic (a) electro-polished surface of Aluminum foil, (b) growth of hexagonal close pack honeycomb
like structures of AAO.

Once a nanoporous AAO template is obtained, it would be dispersed with a colloidal solution of Ag nanoparticles.
These nanoparticles in turn would be separately synthesized using chemical route. Although a wide variety ofmethods have been proposed for synthesis of Ag nanoparticles [16-19]. However, using the chemical reduction
method called Turkevich method, in which AgNO3 solution is reduced by using trisodium citrate. Using this typeof bottom up approach Ag nanoparticle ranging in the size of 10 nm -100 nm can be readily synthesized. By
Additionally using surfactants we can control the growth kinetics and stability of the Ag nanoparticles in thesolution phase. Finally the device would be fabricated by dispersing the synthesized nanoparticles over thesurface of the nanoporous AAO, over which the Thiram/ Diazinon molecules (in the form of their solutions inwater/organic solvents) would be adsorbed. Upon excitation by CW laser the SERS signal can be obtained as isalso shown in Fig. 4(a). The interface of Ag nanoparticle and the molecule and the underlying interfacial
processes are shown in Fig. 4 (b & c) respectively. As mentioned earlier the SERS signal is strong enough owing
to the LSPR coupling effects, it is this LSPR coupling that we sought to increase by using nanoporous structuresoffered by AAO template.

Fig. 4 (a) shows the Ag Nanoparticle coated with molecules dispersed on to the surface of AAO substrate utilized as a

SERS based sensing device, (b) the adsorbed molecule on the surface of Ag nanoparticle and (c) shows the Energy
levels/transfer processes involved at the interface of Ag nanoparticle and the molecules adsorbed over it.

By controlled optimization of both the size of the nanopores of AAO and nanoparticles of Silver, we intend toobtain high sensitivity SERS substrate. Moreover the optimum aspect ratio of these nanoporous structures is
expected to provide high enhancement factor, leading to high strength of the SERS signal. More recently
researchers have shown the presence of hot spots to be critical for high enhancement of SERS signal. The hot
spots are highly spatially localized regions exhibiting extreme field enhancements [1, 6]. Although the probabilityof finding hot spots is lesser. However, by utilizing nanoporous structured template the likelihood of hot spots
consisting of Ag nanoparticles can be maximized. Hence high EM enhancement is expected. Furthermore
dispersion of a mix of Ag nanoparticles and Ag nanowires could even lead to further enhancement of the SERS
signal, thereby leading to improved limit of detection with enhanced sensitivity with improved uniformity.
Schedule/timeline of the project:

Activity

Time in quarters
June‘20 – September‘20

October‘20 – December‘20

January‘21 – March‘21

Electrochemical
polishing/ anodization of Al foil
Characterization of
nanoporous Al2O3
substrates
Synthesis &Optimization of Ag
Nanoparticles
Adsorption ofThiram/Diazinon
molecule on Ag
nanoparticle surface
Fabrication &Characterization of
SERS based Sensors
The expected results/outcomes from the proposal are as follows: Optimization of electrochemical growth conditions for Anodic Aluminum Oxide w.r.t. the aspect ratio of
the nanoporous substrates thus obtained.
 Optimization of synthesis conditions for Ag nanoparticles to obtain nanoparticles with variety ofdiameters and shapes.
 Understanding the energy transfer mechanism i.e. Forster or Dexter between the metal nanoparticles andthe adsorbed molecules on to its surface.
 Utilization of hot spots by using mix of Ag nanoparticles and Ag nanowires for large electromagnetic
enhancement leading to strong SERS signal.
Demonstration of high sensitivity SERS based sensor with improved detection limit well below sub nM range is
expected.
List of References:
1. W. Wei, et al., J. Mater. Chem. C, Vol. 6 (2018) pp. 8793.
2. M. Lee, et al., ACS Sensors, Vol. 3 (2018) pp. 151.
3. X. T. Wang, et al., Appl. Phys. Lett., Vol. 96 (2010) pp. 053104.
4. A. Kim, et al., Nanotechnology, Vol. 26 (2015) pp. 015502.
5. D. L. Jeanmaire, et al., J. Electroanal. Chem. Interf. Electrochem., Vol. 84 (1977) pp. 1.
6. S. Nie & S. R. Emory, Science, Vol. 275 (1997) pp. 1102.

7. A. Campion & P. Kambhampati, Chem Soc. Rev., Vol. 27 (1998) pp. 241.
8. S. Schluker, et al., Angew Chem. Int. Ed., Vol. 53 (2014) pp. 2.
9. M. Moskovits, J. Raman Spectrocsocpy, Vol. 36 (2005) pp. 485.
10. J. R. Lombardi & R. L. Birke, J. Chem. Phys., Vol. 136 (2012) pp. 144704.
11. J. Chen, et al., J. Food Sci., Vol. 80 (2015) pp. N834.
12. L. Assand, et al., Rev. Sci. Instrum., Vol. 86 (2015) pp. 073902.
13. J-H Lim, et al., J. Mater. Chem., Vol. 20 (2010) pp. 9246.
14. Q. Xu, et al., Prog. Mater. Sci., Vol. 95 (2018) pp. 243.
15. J. Zhang, et al., Mater Chem. Phys., Vol. 122 (2010) pp. 295.
16. M. Muzamil, et al., IOP Conf. Ser: Mater. Sci & Engg, Vol. 60 (2014) pp. 012034.
17. Y. Li, et al., J. Am. Chem. Soc., Vol. 127 (2005) pp. 3266.
18. P. Zhao, et al., Coord. Chem. Rev., Vol. 257 (2013) pp. 638.
19. M. Khan, et al., Dalton Trans., Vol. 47 (2018) pp. 11988.
List of Recent Publications of Dr. Vipul Singh (relevant to theme of LSPR and nanostructure synthesis that
relates to the project proposal):1. J. Agrawal, T. Dixit, I. A. Palani and V. Singh, Scripta Materialia, Vol. 163 (2019) pp. 1.
2. P. Rajagopalan, M. J. Raj, N. Prasanth, V. Singh, I. A. Palani and S-J Kim, ACS Appl. Mater. Interf., Vol. 11(2019) pp. 6078.
3. T. Dixit, A. Tripathi, K. L. Ganapathi, I. A. Palani, M. S. R. Rao and V. Singh, IEEE Electron Dev Lett.,Vol. 40 (2019) pp. 255.
4. N. Patra, V. Singh, I. A. Palani, Mater. Res. Exp., (2019, in press).
5. N. Patra, S. K. Karaturi, N. J. Vasa, D. Nakamura, M. Higashihata, V. Singh and I. A. Palani, J. Alloy &Comp., Vol. 770 (2019) pp.1119.
6. J. Agrawal, T. Dixit, I. A. Palani, M. S. R. Rao and V. Singh, Semicon. Sci. & Technol., Vol. 33 (2018) pp.
095021.
7. J. Agrawal, T. Dixit, I. A. Palani, M. S. R. Rao and V. Singh, Phys. Stat. Sol. RRL, (2018) pp. 1800241.
8. J. Agrawal, T. Dixit, I. A. Palani, M. S. R. Rao and V. Singh, J. Phys D: Appl. Phys., Vol. 51 (2018) pp.
185106.
9. T. Dixit, I. A. Palani and V. Singh, RSC Adv., Vol. 8 (2018) pp. 6820.
10. T. Dixit, I. A. Palani and V. Singh, J. Phys. Chem. C, Vol. 121 (2017) pp. 3540.