Detection_of_Nitroexplosives_by_Surface_Enhanced_Raman_Spectroscopy_on_Colloidal_Metal_Nanoparticles

Detection of Nitroexplosives by Surface Enhanced Raman Spectroscopy on Colloidal Metal Nanoparticles
by Marcia del Rocío Balaguera-Gelves
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Chemistry UNIVERSITY OF PUERTO RICO MAYAGüEZ CAMPUS 2006
Approved by: ________________________________ Samuel P. Hernández Rivera, Ph.D. President, Graduate Committee ________________________________ Luis A. Morell Cruz, Ph.D. Member, Graduate Committee ________________________________ Julio G.Briano Peralta, Ph.D. Member, Graduate Committee ________________________________ Sandra Coutin, Ph.D. Representative of Graduate Studies ________________________________ Francis Patrón, Ph.D. Chairperson of the Chemistry Department __________________ Date __________________ Date __________________ Date __________________ Date __________________ Date

ABSTRACT
Gold and silver colloids have been synthesized by chemical reduction methods, and they have been used for detecting molecules in solution with high sensitivity and molecular specificity. The present study focuses on metallic nanoparticles of silver and gold
colloids with ~60-80 nm particle size in surface enhanced Raman and surface enhanced resonance Raman scattering (SERS). The nanoparticles were characterized using techniques such as UV-Vis spectroscopy using a Varian Cary-100 UV-visible double beam and Scanning Electron Microscopy (JEOL JSM 6460LV). Microscopy images were taken with 1μL of colloids. The
colloids were developed for the identification of trace levels of nitroexplosives with detection limits down to femtomolar concentration. Detection of TNT deposited on gold and silver colloids was achieved at a wavelength of 785 nm with a laser operating at 6W power. TNT dye was detected at a wavelength of 532 nm with laser power set at 170 mW by Resonance Raman Scattering (SERS) at concentration levels as low as 1.1 X 1017
grams at pH 10. The excitation source was a diode-pumped 532 nm green laser with a
variable output power of up to 0.1 W (Millennia II laser from Spectra Physics). A Renishaw Raman Microspectrometer RM2000 with Leica objectives with 10xs magnifications was used. The spectra were obtained in the 100-3500 cm-1 range
acquiring 10 scans with 30 seconds of integration time. The azo structures, obtained by derivatizing the TNT, are highly colored derivatives which contain a functionality that

will enable a strong bond with the metal surface.
Recent experimental data have
demonstrated the effect of metal nanostructures coated with hydrophilic copolymers (silver on thin-polymer film coated glass slide). Results indicated a decrease in the intensity of the SERS in TNT and TNT dye which was still detectable by the enhanced presence of the NO2 out-of-plane bending modes at 820 and 850 cm-1 and the NO2 stretching mode at 1300-1370 cm-1. It was also observed that the polymer film does not interfere with the signal of SERS in the film and some stretching bands of TNT were detected. In conclusion we found that polymers with carboxylic side chains like 2hydroxyethylmethacrylate (Methacrylic Acid) cause the silver colloid surface layer to replace citrate ions by chemical species of like charge and similarly with aril carboxyl groups in polymer addition.

RESUMEN
Se han sintetizado coloides de oro y plata por métodos de reducción química y han sido utilizados para detectar moléculas en solución con alta sensitividad y especificidad molecular. Este estudio se enfoca en nanopartículas metálicas de coloides de plata y oro en tama?os de partícula de ~60-80 nm usando dispersión amplificada por superficie y resonancia amplificad por superficie Raman (SERS).
Las nanopartículas fueron caracterizadas usando técnicas tales como espectroscopia UVVisible usando un instrumento Varian Cary-100 de doble haz y un microscopio de rastreo de electrones (JEOL JSM 6460LV). Imágenes en el microscopio se tomaron a 1μL de coloides. Los coloides fueron desarrollados para la identificación de niveles traza de nitroexplosivos con límites de detección hasta concentraciones femto-molares. La
detección de TNT depositado en coloides de oro y plata se consiguió a una longitud de onda de 785 nm con un láser operando a una potencia de 6 W. El tinte de TNT fue detectado a una longitud de onda de 532 nm con una potencia de láser de 170 mW por dispersión de resonancia Raman (SERS) a niveles de concentración tan bajos como 1.1 X 10-17 gramos a pH = 9. La fuente de excitación fue un láser verde bombeado por diodo a 532 nm con una potencia de salida variable de hasta 0.1 W (laser Millennia II de Spectra Physics). Un microespectrómetro Raman Renishaw RM2000 con objetivos Leica con magnificación de 10xs fue usado. Los espectros se obtuvieron en el intervalo de 100-

3500 cm-1 adquiriendo 10 rastreos con 30 segundos de tiempo de integración. Las estructuras azo, obtenidas de la derivatización de TNT, son derivados altamente coloreados que contienen funcionalidad que permitirá la formación de un enlace fuerte con la superficie del metal. Datos experimentales recientes han demostrado el efecto de las nanoestructuras del metal cubiertas con copolímeros hidrofílicos (plata en laminilla de vidrio cubierta con capa polimérica fina). Los resultados indican una disminución en la intensidad de SER(R)S en TNT y tinte de TNT que aún era detectable por la presencia de los modos amplificados de torsión fuera del plano del NO2 a 820 y 850 cm-1 y el modo de estiramiento del NO2 a 1300-1370 cm-1. Se observó también que la capa polimérica no interfiere con la se?al SERS en la capa y algunas bandas de estiramiento del TNT fueron detectadas. En conclusión se encontró que polímeros con cadenas laterales
carboxílicas como 2-hidroxietilmetacrilato (ácido metacrílico) causan que la capa de superficie del coloide de plata reemplace iones citrato por especies químicas de igual carga y de igual forma con grupos aril carboxilo en adición polimérica.

To God, my mother Nohemy Gelves and my family.

ACKNOWLEDGEMENTS?
I want to thank God who gave me the hope, faith and love to breathe every day. A special thanks to Dr. Samuel P. Hernández-Rivera my advisor for his boundless patience and constant encouragement. He has been a great guiding force during the course of my studies and has also shaped me as an individual. I am very thankful to Dr. Luis Morell for helping me, for always extending your hand to me and giving me your example and friendship. I would like to thank Dr. Luis Rivera for allowing me to use the lab facilities and for the great interest that he has shown in my work. I thank Dr. Miguel Castro for providing me with SEM facilities. To my family, Nohemy Gelves, Jacqueline, my brothers, for their understanding and their support all these years. Also to my nieces: Julieth Tatiana and Monica. Thanks to Eliseo for his love and unconditional support. To my friends: Jacqueline Indira, Edwin de la Cruz, and Alvarito, for their friendship. Collaboration with the Center for Chemical Sensors Development of the University of Puerto Rico–Mayagüez sponsored by the Department of Defense, University Research Initiative–Multidisciplinary University Research Initiative (URI– MURI) Program, under grant no. DAAD19–02–1–0257 is also acknowledged. Finally, I would like to acknowledge the University of Puerto Rico, Mayagüez Campus and department of Chemistry for the opportunity to study in Puerto Rico.

Table of Contents
ABSTRACT RESUMEN ACKNOLWLEDGEMENTS TABLE OF CONTENTS TABLE LIST LIST OF IGURE 1 CHAPTER I INTRODUCTION 2 CHAPTER II THEORY 2.1 Preparation of Metal Nanoparticles 2.2 Stability of Colloidal Solution 2.2.1 Noionic Polymer 2.3 Properties of Nanoparticles 2.4 Surface Enhanced Resonance Raman Scattering 3 CHAPTER III THE PROBLEM 3.1 Investigated Compound 3.1.1 2, 4, 6-Trinitrotoluene (TNT) 3 .1.2 TNT Dye 3.1.3 Adenine 4 CHAPTER IV PREVIOUS WORK 5 CHAPTER V METHODOLOGY 5.1 Reagents and Chemicals 5.2 Cleansing Method 5.3 Preparation of Silver Colloids 5.4 Preparation of Gold Colloids 5.5 Preparation of Thin Silver Polymer by Addition (Method 1) 5.6 Preparation of Tin Silver Polymer Layer (Method 2) 5.7 Synthesis and Characterization of TNT Azo dyes 5.8 Preparation of 2, 4, 6 trinitrotoluene and TNT Dye Dissolution 5.9 Sample Preparation for Analysis by SER(R)S 5.10 Instrumentation 6 CHAPTER VI RESULTS AND DISCUSION 6.1 Characterization of Silver Colloids 6.1.1 Plasmon Absorption Band for Silver Colloids 6.2 Plasmon Absorption Band for Gold Silver Colloid 6.3 Characterization of Silver Colloids and Silver Polymeric Film 6.4 Characterization of TNT Dye for UV-vis 6.5 Comparison of silver Colloid and Thin Film for detection of adenine 6.6 Studies of Surface enhanced Raman spectroscopy (SERS) of 2, 4, 6 - TNT 6.7 Studies of SERRS Enhanced scattering of TNT Dyes and Laser Power Intensity 6.8 Changes in SERRS Signal Areas with Laser Power Intensities (mW) for Silver colloid With TNT Dye 6.9 Quantitative Multivariate Analysis 2 4 8 9 11 12 14 18 18 19 23 26 30 32 33 33 34 34 36 38 38 39 40 40 40 41 42 43 43 44 46 46 47 50 51 55 56 58 63 64 66
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6.9.1 Temporal Stability of TNT in silver Substrate 6.9.2 Quantitative Detection of TNT 7. CHAPTER VII CONCLUSIONS 8. REFERENCES
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List of Tables
Tables Page
TABLE 6.1 Vibrational Frequencies of Adenine ......................................................................................... 57
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List of Figures
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Figures and Schemes Page 15 18 19 20 28 29 34 34 34 34 39 41 42 43 44 45 46 48 49 50 52 53 54 55 56 58 59 60 61 62 63 64 Figure 1 SERS Model Scheme 1 top down Method for Nanofabrication Scheme 2 Bottom Up Method for Nanofabrication Figure 2 Proposed model of the double layer region under condition where anions are specifically adsorbed Figure 3 UV–visible absorption spectra for 9, 22, 48 and 99 nm diameter Au nanoparticles Figure 4 SERS phenomenon: Illustration of the excitation of the excitation of the LSPR Of a spherical nanoparticle by incident electromagnetiradiation Figure 5 Structure 2, 4, 6-Trinitrotoluene (TNT) Figure 6 Structure TNT Dye Figure 7 Structure of Adenine Figure 8 Synthesis of silver colloids a yellow-blue color resulted Figure 9 Gold Colloids λmax: 531nm Figure 10 Silver thin polymer film coated glass slide (a). Silver bulk polymer layer; (b). Silver polymer with uniform pores; (c). Silver polymer; (d) Thin silver polymer layer Figure 11 Reduction of TNT followed by azo dye formation. (i) Fe/AcOH, (ii) HCl, NaNO2, (iii) NaOH, 8HQ Figure 12 TNT solutions dissolved high purity water. Concentration at pH 9.0 of: (1): 1x10-2 M; (2):1x10-3; (3)1x10-4; (4)1x10-5; (5)1x10-6; (6)1x10-7; (7)1x10-8; (8)1x10-9; (9) 1x10-11; (10) 1x10-12 M. Figure 13 Scheme of SERS sample preparation Figure 14 Renishaw Raman Microspectrometer (RM2000). Figure 15 Particle size of silver colloid (less than 80 nm) Figure 16 UV-Vis spectra of formation of silver nanoparticles of different sizes. a) Silver colloids λmax = 418 nm; b) Silver colloids λmax = 440 nm; c) Silver nanowires λmax = 428 Figure 17 UV-Vis spectra of formation of silver nanoparticles at different pH Figure 18 UV-Vis spectra of formation of gold colloids Figure 19 Formation of silver-polymer films on glass substrates Figure 20 Different shapes of silver and gold colloids Figure 21Comparison UV-Vis spectra of formation Plasmon Absorption Of silver colloids and silver thin polymeric film Figure 22 TNT dye 9x10-5M has a sharp peak at 419.02 nm in the UV- Vis spectra Figure 23 Adenine to detect for SERS in silver film Figure 24 Adenine to detect for SERS in silver colloid Figure 25 SERS of TNT on silver colloids (420 nm), A: 1X10-3 M; B: TNT-Solid Figure 26 SERS of the TNT at: a) 1x10-12 g; b) 1x10-13 g; c) 1x10-14 g; d) 1x10-15 g; e) 1x10-16; g) f: 1x10-17M compared with TNT bulk at pH 10 Figure 27 SERS of the TNT at a: 1x10-12 g, b: 1x10-13 g, c: silver thin film to pH 10 Figure 28 Spectra of TNT at 1X10-12 g at pH 10 Figure 29 SERS of TNT at: a) 1x10-12 g; b) 1x10-13 g; c) 1x10-14 g compared with TNT at pH: 9 Figure 30 SERRS of TNT dye at a: 1x10-12 g, power supply different compared at pH 9
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Figure 31 SERRS of TNT dye at a: 1x10-12 g, power Laser intensities different compared at pH 9 Figure 32 Calibration curve of TNT using peak areas. TNT 1x10-3M has a sharp peak at 416.65 nm in the UV- vis spectra Figure 33 a) Curve of calibration of TNT high concentration range between 1x10-7 and 1x10-9 g; b) Curve of calibration of TNT low concentration range between 1x10-14 and 1x10-17 g
65 68 69
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CHAPTER I INTRODUCTION
Revolutionary technological change is coming and emerging technologies can make substantial contributions to the biotechnology, nanotechnology, robotics-artificial
intelligence and nanoscale fields. In the 20th century, nanoparticles play a very important role in many areas of chemistry, physics, and materials science. Nanoscience involves materials with some critical property that is attributable to an internal structure with at least one dimension limited to between 1-100 nm [5]. Nanoparticles can exhibit unique chemical properties due to their limited size and optical characteristics related to their shape, their enormous surface-to-volume ratio, electron plasmon resonance, and quantum confinement effects, very different from the bulk or the atomic state [6]. Nanosized particles are known by several names: clusters, colloids, sols, nanoparticles and nanocrystals. Michael Faraday carried out the first scientific study on colloidal Au, Ag and other metal aerosols and hydrosols in 1857. He established that the colloidal metal sols or pseudo solutions are thermodynamically unstable, and that the individual gold nanoparticles must be stabilized kinetically against aggregation [7].
Jeanmaire and Van Duyne demonstrated that the magnitude of the Raman scattering signal can be greatly enhanced when the scatterer is placed on or near a roughened noblemetal substrate. Strong electromagnetic fields are generated when the localized surface plasmon resonance (LSPR) of nanoscale roughness features on a silver, gold, or copper 14

substrate is excited by visible light [8], showed on Figure 1. When the Raman scatterer is subjected to these intensified electromagnetic fields, the magnitude of the induced dipole increases, and accordingly, the intensity of the inelastic scattering increases. This enhanced scattering process is known as surface-enhanced Raman (SER) scattering
Figure 1. SERS Model. When electromagnetic radiation with the same frequency is incident upon the nanostructure, the electric field of the radiation drives the conduction electrons into collective oscillation (Plasmon Resonance).
In addition, high sensitivity with SERS combines high information for establishing molecular identity and structural characteristics is observed whose intensity exceeds by a moderate factor (~106) [8]. Surface-enhanced resonance Raman scattering (SERRS) occurs when the laser excitation wavelength, overlaps with an electronic absorption band, thereby amplifying the Raman scattering intensities of the totally symmetric vibrational modes of the chromophore.
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Modern security systems require detection and identification of traces of explosives at very low detection limits: down to pico and femto-gram levels. In this respect, silver and gold nanoparticles are known to be excellent substrates for Raman spectroscopy of explosive materials. It has been specifically reported that Surface-Enhanced Raman Scattering (SERS) and (SERRS) are sensitive spectroscopic techniques achieving these limits of detection. One of the main disadvantages associated with using SER(R)S as an analytical technique is the difficulty associated with producing reproducible substrates: it has limited applicability when the molecule of interest is not adsorbed directly onto the substrate. Variation in the fabrication processes used to produce SERS-active substrates leads to inconsistent optical properties and, accordingly, discrepant enhancement factors. Because most of the current nanofabrication techniques implemented to create SERS-active substrates exhibit some degree of irreproducibility, enhancement factors can fluctuate by up to an order of magnitude for substrates fabricated with seemingly identical methodology [8]. Research interests focus on the phenomenon that occurs when dyes are absorbed on metallic colloids in aqueous suspensions for the enhanced detection of TNT and TNT dye in silver and gold colloids. The synthesis of colloidal solutions of silver and gold nanoparticles was achieved by the reduction of AgNO3 and KAuCl4 with sodium citrate as chemical method. Steric
stabilization of those colloids was achieved with the use of copolymers. Polymeric thin films of these nanoparticles could be used in applications such as reflective coatings, sensors, and
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as substrates for surface-enhanced Raman spectroscopy, and materials with high catalytic activity and specificity. [3]. These films were prepared by polymerization of 2-hydroxyethyl methacrylate with methacrylic acid (Method 1). With this unsaturated monomer it was necessary to use a particular initiator of free radicals which permits chain reactions and polymerization of the thin films covered by nanoparticles of gold and silver. At the same time, they have the property of being hydrophilic and by swelling they allow the adsorption of the analyte in an aqueous state. The other procedure employed (Method 2) incorporated the use of polyvinyl pyrrolidone and polyethylene glycol as copolymers. The Scanning Electron Microscopy (SEM) technique was used to provide microstructural information on the metal film synthesized and the ability to tune the nanocoating structure and spectral and electronic properties can be used for applications such as sensors used in the detection of explosives. TNT was characterized by surface-enhanced Raman scattering (SERS) and TNT dye by SERRS which integrates high chemical sensitivity with spectroscopic identification and has enormous potential for applications involving ultra-sensitive chemical detection. Spectra were obtained using Renishaw Raman Microspectrometer RM2000 system. We used this instrument for the vibrational spectroscopy measurements operating at a wavelength of 785 nm in gold colloids and in silver colloids, TNT and TNT dye were detected at a wavelength of 532 nm, the laser power used was170 mW by Resonance Raman Scattering (SERRS) at lower concentration levels of 1.1 X 10-17 grams at pH = 9. 17

CHAPTER II THEORY
In the advanced field of nanotechnology, one goal is to make metal colloidal or nanostructures and nanoarrays with special properties for application as SERS substrates. These are used for many purposes, such as, chemical and biological sensing, catalysis, photography, electronics, photonics and optoelectronics [9]. In recent years, methods have been developed for the preparation of these nanostructures. They can be generated by a number of preparation methods that typically are described as physical and chemical methods. 2.1 Preparation of Metal Nanoparticles The methods typically used to synthesize metals nanoparticles are generally divided into two categories: a. The top down approach: This method involves the continuous division of bulk matter into nanoparticles. A disadvantage of this method is the manipulation of small amounts of atoms at a time resulting in low fabrication throughput.
Scheme 1. Top down Method for Nanofabrication.
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b. Bottom-Up approach: This method involves the continuous building up of nanoparticles from molecular or atomic level.
Scheme 2. Bottom Up Method for Nanofabrication.
This technique includes evaporation under high vacuum [10]. Use of metal vapor techniques is limited because the operation of the apparatus is demanding and it is difficult to obtain a narrow particle size distribution. Among these are the solvated metal atom dispersion method (SMAD) [11], the laser ablation method in metal colloids [12] and electrochemical reductions methods (13). The “bottom up” methods of wet chemical nanoparticle preparation rely on the chemical reduction of metal salts, electrochemical pathways, or the controlled decomposition of metastable organometallic compounds.
2.2 Stability of Colloidal Solutions The stability of metal colloids in aqueous solution tended to diminish with time because of two basic modes of stabilization which have been distinguished; these are electrostatic and steric stabilization [14]. Electrostatic stabilization involves the
Coulombic repulsion between the particles caused by the electrical double layer formed
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by ions adsorbed at the particle surface (e.g., sodium citrate and the corresponding counterions). Due to their double layer structure, it is well known that small metal particles exhibit double layer charging (capacitive charging) in liquid electrolytes [15]. The inner layer also called the compact Helmholtz or Stern layer contains solvent molecules and sometimes other species as molecules or ions (Figure 2).
ψM
Metal
- - - Diffuse layer - - -- - Specifically adsorbed anion - - M q - x x1 2
Solvated
IHP OHP ψ1 ψ2
--
-
δi δd
Figure 2. Proposed model of the double layer region under conditions where anions are specifically adsorbed. Adsorbed ions are called the inner Helmholtz plane (IHP), which is at distance x1. Solvated ions can approach the metal only to a distance x2; the locus of center of these nearest solvated ions is called the outer Helmholtz plane (OHP). The interaction of the 20