Silver-graphene Oxide Composite for Optical Sensor

Published Date: 30 Nov 2017

• KHOSRO ZANGENEH KAMALI

ORIGINAL LITERARY WORK DECLARATION FORM

ABSTRACT

In this work, a [email protected] oxide ([email protected]) nanocomposite-based optical sensor was developed for the detection of biomolecules such as dopamine (DA), ascorbic acid (AA), and uric acid (UA). An aqueous solution of [email protected] was prepared using a simple chemical reduction method, and it showed a characteristic surface plasmon resonance (SPR) band at 402 nm. The SPR features of the [email protected] nanocomposite were used for the detection of DA, AA, and UA. The SPR intensity-based limits of detection (LoDs) of DA, AA, and UA were 49 nM, 634 nM, and 927 nM, respectively. The SPR band position-based LoDs of DA, AA, and UA were 30 nM, 1.64 M, and 2.15 M, respectively. The present optical sensor was more sensitive to DA than to UA and AA. The interactions of the biomolecules with [email protected] were studied based on the density functional theory (DFT), and it was found that DA had more interaction than AA and UA. This novel [email protected] nanocomposite is simple to prepare and showed excellent stability and sensitivity toward the detection of biomolecules.

The similar material is used for colorimetric detection of Mercury(II) ions (Hg(II)) that is able to show existence of 100 µM Hg(II) ions in solution by naked eyes. The development of this optical sensor for Hg(II) using silver nanoparticles (Ag NPs) is based on the decrement in the localized surface plasmon resonance (LSPR) absorption of the Ag NPs and the formation of silver-mercury (AgHg) amalgam. It is observed that increasing Hg(II) ions concentration in the solution results in the decrease of LSPR intensity and decolouration of the solution. The existence of GO prevents the agglomeration of Ag NPs and enhances the stability of the nanocomposite material, enabling this material to be used in industrial and real sample applications.

ABSTRAK

Di sini, oksida perak @ graphene (Ag @ GO) berdasarkan nanokomposit-sensor optik telah dibangunkan untuk mengesan biomolekul seperti dopamine (DA), asid askorbik (AA), dan asid urik (UA). Larutan akueus Ag @ GO telah disediakan dengan menggunakan kaedah pengurangan kimia yang mudah, dan ia menunjukkan satu ciri plasmon permukaan resonans (SPR) band di 402 nm. Ciri-ciri SPR daripada Ag @ GO nanokomposit telah digunakan untuk mengesan DA, AA, dan UA. Had keamatan-pengesanan (LoDs) bagi SPR berdasarkan daripada DA, AA, dan UA adalah masing-masing 49 nM, 634 nM, dan 927 nM,. The band SPR berdasarkan kedudukan-LoDS daripada DA, AA, dan UA adalah masing- masing 30 nM, 1.64 uM, dan 2.15 uM. Sensor optik masa kini adalah lebih sensitif kepada DA daripada UA dan AA. Interaksi daripada biomolekul dengan Ag @ GO dikaji berdasarkan ketumpatan teori fungsional (DFT), dan didapati bahawa DA mempunyai interaksi lebih daripada AA dan UA. Novel ini Ag @ GO nanokomposit adalah mudah untuk menyediakan dan menunjukkan kestabilan yang sangat baik dan kepekaan terhadap pengesanan biomolekul.Bahan yang sama telah digunakan untuk pengesanan ''colorimetric'' ion Mercury(II), (Hg(II)) yang mampu dilihat dengan kewujudan 100 μM ion Hg(II) dalam larutan dengan mata kasar. Pembangunan sensor optik bagi Hg(II) menggunakan nanozarah perak (Ag NPS) adalah berdasarkan pengurangan pada penyerapan Ag NPs resonan plasmon permukaan setempat (LSPR) dan pembentukan amalgam perak-merkuri (AgHg). Dapat diperhatikan bahawa peningkatan kepekatan ion Hg(II) memberikan hasil pengurangan pada intensiti LSPR dan perubahan warna. Peningkatan jumlah ion Hg(II) pada satu tahap membawa perubahan dalam morfologi Ag NPs dan pembentukan amalgam AgHg yang mempengaruhi LSPR Ag NPS dan menjadikan perubahan warna pada [email protected] Kehadiran GO menghalang penggumpalan Ag NPS dan meningkatkan kestabilan bahan nanokomposit yang membolehkan bahan ini untuk digunakan dalam industri dan aplikasi sampel sebenar.

ACKNOWLEDGEMENTS / DEDICATION

TABLE OF CONTENTS

Table of Contents

SILVER-GRAPHENE OXIDE COMPOSITE FOR OPTICAL SENSOR APPLICATIONS

ORIGINAL LITERARY WORK DECLARATION FORM

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS / DEDICATION

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

LIST OF SYMBOLS AND ABBREVIATIONS

LIST OF APPENDICES

CHAPTER 1: INTRODUCTION

CHAPTER 2: LITRETURE REVIEW

2.1. Plasmonic band of metal Nanoparticles

2.2. Graphene Oxide

2.3. Sensor

2.3.1. Electrochemical sensor

2.3.2. Surface enhanced Raman scattering

2.3.3. Optical sensor

2.4.2 Amalgamation and LSPR

CHAPTER 3: MATERIALS AND METHODS

3.1. Chemicals and Reagents

3.2. Preparation of [email protected] Nanocomposite

3.3. Characterization Techniques

3.4. Optical Detection of Biomolecules

3.5. Optical Detection of Hg(II) ions

CHAPTER 4: RESULTS AND DESCUSSIONS

4.2. Optical Sensing of Biomolecules using [email protected] Nanocomposite

4.2.1. Morphological Studies of [email protected] after Addition of Biomolecules

4.2.2. Raman Studies of [email protected] Nanocomposite

4.2.3. Computational Studies

4.3. Optical sensing of Hg(II) ions

4.3.1. Optical properties of [email protected] nanocomposites

4.3.2. Optical sensing of Hg(II) ions by [email protected] nanocomposite

4.3.3. Mechanism for the Amalgamation based detection of Hg(II) ions with [email protected] nanocomposite

4.3.4. Characterization of [email protected] nanocomposite before and after addition of Hg(II) ions

4.3.5. Selectivity of [email protected] nanocomposite based optical sensor

4.3.6. Practical application

CHAPTER 5: CONCLUSION AND DISCISSION

REFERENCES

Supplementary

Appendix

LIST OF FIGURES

Figure 1: UV-vis absorption spectra of (a) AgNO3 (b) GO, and (c) [email protected] nanocomposite. Inset: Photograph obtained for the aqueous solution of synthesized [email protected] nanocomposite.

Figure 2: (A) Absorption spectra obtained for [email protected] nanocomposite upon each addition of 100 nM DA. (B) Plot of absorption intensity vs. DA concentration. (C) Plot of Id vs. DA concentration. (D) Plot of λmax vs. DA concentration.

Figure 3: (A) Absorption spectra obtained for [email protected] nanocomposite upon each addition of 5 µM AA. (B) Plot of absorption intensity vs. AA concentration. (C) Plot of Id vs. AA concentration. (D) Plot of λmax vs. AA concentration.

Figure 4: (A) Absorption spectra obtained for [email protected] nanocomposite upon each addition of 5 µM UA. (B) Plot of absorption intensity vs. UA concentration. (C) Plot of Id vs. UA concentration. (D) Plot of λmax vs. UA concentration.

Figure 5: TEM images of (A) as-prepared [email protected] nanocomposite and after additions of (B) AA, (C) UA, and (D) DA.

Figure 6: Raman spectra of (a) [email protected] and (b) [email protected] with 1-M additions of (b) DA, (c) UA, and (d) AA.

Figure 7: Electron density map and energy gap of HOMO and LUMO energy levels for Ag and DA, UA, and AA adducts, respectively calculated by DFT methods.

Figure 8: Absorption spectra for the (a) AgNO3, (b) GO and [email protected] nanocomposite.

Figure 9: Absorption spectral changes observed for the [email protected] nanocomposite (A) before and (B) after the addition of 200 µM Hg(II) ions. Inset: The digital photographic images taken for the corresponding solution.

Figure 10: (A) Absorption spectral changes observed for [email protected] nanocomposite upon each addition of 100 nm μM of Hg(II) ions to the solution. (B) Plot of changes in the absorption intensity maximum at λLSPR of [email protected] nanocomposite against various Hg(II) ions concentr

Figure 11: (A) Schematic explain the function of GO in the detection Hg(II) ions. (a) Addition of Hg(II) ions into a solution containing [email protected] nanocomposite. (b) Adsorption of Hg(II) ions on the surface of GO. (c) Interaction of Hg(II) ions with Ag NPs and formation of AgHg amalgam. (B) Schematic representation for the formation of AgHg amalgam and its influence in absorption spectra of the Ag NPs present in the [email protected] nanoparticles.

Figure 12: Overview and high magnification TEM images obtained for the [email protected] nanocomposite before (A andB) and after addition of 200 µM Hg(II) ions (C and D).

Figure 13: X-ray diffraction patterns obtained for the [email protected] nanocomposite (a) before and (b) after addition of 200 µM Hg(II) ions.

Figure 14: XPS spectra obtained for the AgHg amalgam particles and their corresponding (A) Ag 3d and (B) Hg 4f regions of core-level spectra.

Figure 15: Cyclic voltammograms recorded in 0.1 M phosphate buffer solution with pH 7.0 at a scan rate of 50 mV s−1 for the GC electrode coated with the solution containing [email protected] nanocomposite (A) before and (B) after addition of 200 µM Hg(II) ions.

Figure 16: Difference in percentage of Ag NPs absorbance peak reduction observed for [email protected] nanocomposite in the presence of 200 µM Hg(II), Na(I), K(I), Mn(II), Ni(II), Zn(II), Co(II), Cu(II), Fe(II) and Fe(III) into the individual solutions. Inset: Photograph taken after the addition of 200 µM of Hg(II) ), Na(I), K(I), Mn(II), Ni(II), Zn(II), Co(II), Cu(II), Fe(II) and Fe(III) into the individual solution.

LIST OF TABLES

Table 1: Analytical performances of [email protected] nanocomposite for the detection of DA, UA and AA in human urine sample.

Table 2: Comparison of the sensing performance of some of the Ag NPs towards Hg(II) ions.

Table 3: Determination of Hg(II) ions in different water samples by using [email protected] nanocomposite.

LIST OF SYMBOLS AND ABBREVIATIONS

DAdopamine

UAuric acid

AAascorbic acid

LoD limit of Detection

LSPRlocalized surface plasmon resonance

SPRsurface plasmon resonance

SERSsurface enhanced resonance plasmon scattering

mmili

µmicro

nnano

Mmolar

HPLChigh-performance liquid chromatography

NPsnanoparticles

Hg(II) ionmercury (II) ion

GOgraphene oxide

rGOreduced graphene oxide

GCEglassy carbon electrode

eVelectron volt

DFTdensity functional theory

HRTEMhigh resolution transmission electron microscope

XRDX-ray diffraction

XPSX-ray photoelectron spectroscopy

FESEMfield emission scanning electron microscope

a.u.arbitrary unit

LIST OF APPENDICES

CHAPTER 1: INTRODUCTION

For several decades, silver (Ag) nanoparticles have been attracting attention because of their excellent optical and electronic properties, high catalytic activity, and biocompatibility. Hence, they are used in a wide range of applications such as catalysis ¹, solar cells ^2,3, and optical ⁴ and electrochemical sensors ⁵. Ag nanoparticles possess a sharp absorption in the visible region (400–500 nm), depending on the size of the nanoparticles. This absorption feature arises from the surface plasmon resonance (SPR), which is the absorption of light by the nanoparticles because of surface vibrations between atoms ^6–8. This SPR feature allows Ag nanoparticles to be used in optical sensors for the detection of toxic metals ⁹, biomolecules ¹⁰, and organic compounds ¹¹. The addition of any analyte to the Ag nanoparticles leads to assembled/aggregated nanoparticles. This influences the SPR absorption band and is extensively used to follow the molecular recognition processes.

Dopamine (DA) is an important catecholamine that belongs to the family of excitatory chemical neurotransmitters. It plays an essential role in the functioning of the drug addiction, cardiovascular, renal, central nervous, and hormonal systems, and in Parkinson’s disease ¹². Thus, it is very important to develop a simple sensor for the detection of a sub-micro-molar concentration of DA. In recent years, the detection of biomolecules such as uric acid (UA) and ascorbic acid (AA) in human fluids such as urine and serum has gained considerable attention ¹³. A deficiency or excess amount of UA in the body causes several diseases, including Lesch/Nyhan syndrome, hyperuricaemia, and gout ¹⁴. Cardiovascular disease and kidney damage result from an elevated UA concentration in serum ¹⁵. Analytical methods such as high-performance liquid chromatography (HPLC) ¹⁶, spectrofluorimetry ¹⁷, spectrophotometry ¹⁸, mass spectrometry ¹⁹, and electrochemical sensors ²⁰ have been reported for the detection of these neurotransmitter molecules. However, the existing detection methods have several limitations such as the need for expensive equipment, well-trained operators, and tedious sampling and time-consuming procedures. Alternatively, an optical sensor platform is more attractive for sensing a wide range of analytes. It is cost effective, portable, has a rapid response, and can provide real-time analyses. Recently, Ramaraj and his coworker reported a silicate-Ag nanoparticle-based optical sensor for the detection of DA, UA, and AA with LODs of 5, 5, and 1 nM, respectively ⁹ (Figure 1).

Most commonly, Ag nanoparticles are synthesized using various chemical and physical methods, which are not eco-friendly and suffer from problems that include the poor reproducibility and stability of the Ag nanoparticles due to colloidal aggregation ²¹. In order to overcome such limitations, considerable efforts have been made to prepare Ag nanoparticles on polymer ²², silicate sol-gel ¹¹, and graphene nanosheets ²³. Among these, Ag-graphene has a large surface area and strong van der Waals force between the graphene and Ag nanoparticles, which significantly reduces nanoparticle aggregation. In addition, the high interfacial interactions ensure the stability of the Ag nanoparticles ²⁴. In this study, graphene oxide-supported Ag nanoparticles were prepared using a simple chemical reduction method and used in an optical sensor for the detection of biomolecules such as DA, AA, and UA (Figure 2). The present synthetic method for the for preparation of [email protected] nanocomposite has advantages over other methods ^{11, 21-24} such as, long term stability, high homogeneity, rapid and ease of preparation and avoids any surfactant, stabilizers.

Silver nanoparticles (Ag NPs) attracted much attention due to its biocompatibility, high catalytic activity, anti-bacterial activity, electronic and optical properties ^25–28. The Ag NPs possess a principal absorption band in the region of 400 nm due to the localized surface plasmon resonance (LSPR)^29,30. This SPR feature of Ag NPs is aroused due to the collective oscillation of electrons on the surface of the Ag NPs that are excited by incident electromagnetic waves ³¹. The SPR band position and intensity mainly depend on the size, shape and refractive index ³². This SPR band of Ag NPs is more sensitive to the surrounding environment and it significantly influences the band position and intensity. Based on the changes in the LSPR band position and intensity, an optical sensor platform with Ag NPs was developed to detect the wide range of analytes, including biomolecules ³³, nitroaromatics ³⁴, phenolic compounds ³⁵, and heavy metal ions ³⁶.

Among the investigated analytes, heavy metal ions especially Hg(II) ions are more often monitored with Ag NPs through the optical sensing method owing to its high toxicity and solubility in water³⁷. Mercuric (Hg(II)) ions are mainly released into the atmosphere from solid waste incineration, power plants, and bumping fossil fuels³⁸ that pollute water, soil and air ^39,40. The existence of Hg(II) ions in water causes serious damage to the brain, nervous system, kidneys and endocrine system of living organisms⁴¹. Developing a system for detecting Hg(II) with high sensitivity and selectivity against other common metal ions dissolved in water is a challenge in recent years ^42–47. From an environmental point of view the development of an inexpensive, simple, selective and sensitive method of detection of Hg(II) becomes highly important.

There are many types of sensors invented to detect Hg(II) in the environment. Some studies reported the detection of Hg(II) ions using electrochemical methods ^48,49. Although they achieved a very high limit of detection (LoD), they need to use expensive apparatus and complicated setup. For the electrochemical testing of Hg(II) sample, the fabrication of an electrode is necessary, and that is a very tedious process. In addition, the detection of Hg(II) using fluorescence spectrometry has been widely investigated ^50,51. Although this method is simple and is able to detect trace amounts of Hg(II) ions in solution, it requires expensive equipment to work. In this respect, colorimetric sensors are cheaper and do not require tedious preparation methods, colorimetric sensors have the advantage that the existence of Hg(II) is easily discernible to the naked eye without being affected by other possible dissolved ions ^52–59. Recently, Hg(II) ion sensing was reported with noble metals such as Au and Ag by utilizing the size/interparticle distance-dependent optical properties and high extinction coefficients ^60–62. The interaction between surfactants and metal NPs results in changes to the refractive index of these NPs and the LSPR band ^32,33. They also may electrostatically repel the analyte, preventing it from interacting with the metal NPs and reduces the sensitivity of the sensor. In this study, the Ag NPs was prepared by using a simple chemical reduction agent and stabilized on graphene oxide (GO) sheets. Subsequently, the [email protected] nanocomposite was used to develop a colorimetric sensor for the detection of Hg(II) with the naked eye and an optical sensor also developed based on the LSPR changes upon the addition of various Hg(II) concentrations. This significant change in the LSPR of the Ag NPs is due to change in the morphology through the formation of AgHg amalgam. Selectivity in the detection of Hg(II) in the presence of various environmentally relevant metal ions was also studied.

CHAPTER 2: LITRETURE REVIEW

2.1. Plasmonic band of metal Nanoparticles

Most probably gold nanoparticles (NPs) synthesized in the 5th or 4th century BC in China and Egypt regions ⁶³. From that time, gold NPs have been used in both medicine and aesthetic aspects. As the result of the interesting optical properties of gold NPs, they were used for changing color of glass ⁶⁴, pottery and ceramics ⁶⁵. Faraday got interested about the optical properties of gold NPs and reported about the range of colors of gold nanoparticles colloidal solutions from ruby red to amethyst in 1857. Then he studied the factors influencing the color of gold NPs solutions and concluded that ‘‘the mere variation in the size of particles gave rise to a variety of resultant colors’’. Other than their optical properties, many applications of metal nanoparticles have been found in biochemistry, catalysis and sensors. For instance, one of the anti-odour commercialized devices in Japan is using the technology of immobilizing gold nanoparticles in oxide matrixes as active oxidation catalysts ⁶⁶. In defenitions, nanoparticles are particles composed of number of atoms, ranging from 3 to 10^{7 67}. Nanoparticles feature properties are different from atoms or bulk material due to their size. The metallic nanoparticles larger than 2 nm possess a strong and broad absorption band in the UV-visible spectrum that is called surface plasmon resonance (SPR) band. This absorbance has discovered by Gustav Mie and known as Mie resonance ⁶⁷. For smaller nanoparticles, quantum effects become more prominent and LSPR disappears. All metal nanoparticles possess the mensioned optical property, but the series of Au, Ag and Cu have very intense LSPRs. Other than that, their easy synthesis methods and their robustness to environmental conditions made silver and gold NPs to widely be used in this field. The LSPR features such as position, shape and intensity are strongly depends on various factors, to mention: the changes in the interparticle distance of the NPs and , and the changes in the refractive index of the local surrounding environment ⁶⁸.

There are indeed other types of plasmonic signals, such as the surface plasmon resonance band produced by planar metallic films, in reflection or transmission, some of them being called plasmon polaritons. Though the resulting physics is extremely exciting and the recent discoveries numerous, it is out of the scope of present thesis and they will not discuss further.

In recent years, many theories were adopted by both physicists and chemists in order to give a clear description of the SPR band and on the main factors impacting its position, broadness and intensity. Many works has done to overview the existing plasmon band theories ⁶⁷, and explain the SPR band by Mie and effective medium theory ⁶⁹. There are some researches to explain optical propertiese of NPs with arbitrary shape by Maxwell equation theory ⁶.

The phenomenon on absorbance of certain wavelength of light observed in transmission of light through metal nanoparticles in solid or solution phase, is called localized surface plasmon resonance band (LSPR).Nanoparticles intract with incident light in certain frequency that result global scattering of it. This observation can be explained by the collective resonance of the conduction electrons of the nanoparticle, due to interaction of electrons in nanoparticles with light. The evaluation of all parameters of material, specially its dielectric constant is necessary for understanding and study this phenomena. Usually, dielectric constant of nanoparticles count same as its bulk form and confinement effects and defects induced by edges or impurities will be neglected. For this aim a study on electrostatics in bulk metal by using Maxwell equations is necessary. In formulating the dielectric constant with known parameters, the Drude model, which describes the motion of free electrons in a metal can be applied. Then the question of the nanoparticles will be addressed: the conditions for conducting electrons resonance will be determined by several means. The determination of the frequency of the absorption maximum (denoted , the frequency of the Mie resonance), the height of this maximum and the width of the peak will be the ultimate goal of the calculations. The different geometrical confinement effect of free electrons on each material caused the electronic motion for nanoparticles vary material by material. Indeed, here the nanoparticles can be seen as a cationic network in which a cloud of conducting electrons (or free electrons) moves and oscillates. Nanoparticles dimensions are very small compared to the wavelength of the UV-visible light for which the phenomenon is observed and also comparable to the mean free path of electrons. The surface plasmon band is known to the resonance of the electronic cloud with the incident wave and the mechanics of this phenomenon can be evaluated.

In the case of nanoparticles, the conditions that electron cloud can resonate needs to be calculated. For this aim, The dielectric constant of metal nanoparticles assumed to be the same as the bulk material. Some postulates then become incorrect, but in calculation we have to keep them as an approximation. For instance, the electron density in small particles (r = 0) is not uniform and the charge will accumulate in particle edges and surface. Other than that, since the size of nanoparticles is very small comparing to the wavelength of incident light, we can consider that all electrons in the nanoparticle face with the same field at the given time and the electric field is independent of position ⁶⁷. This hypothesis is known as the quasi-static approximation.

When the electric field incidents to the particle, it result the displacement of the electron cloud that leads to the creation of surface charges. The positive charge would be where the cloud is lacking and the negative charge would be where it is concentrated (Fig. 1). ⁶⁷ The therm “surface” is justified by the electron cloud charge mentioned in previous statements. However, we have to kep in mind that all the electrons are moving together (collectively) under the influence of electromagnetic field. This collective oscillation leads to plasmon polaritons, ⁶⁴ that is different with the free plasmon in the bulk metal.⁶⁷

The term “plasmon” was given to the SPR phenomenon by Shopper, due to the bounded gaseous plasmon oscillations.⁶⁷ The dipolar charge repartition imposes a new force on the electron cloud. The electrons undergo a restoring force which conflicts with the external electric field.

Figure.‎0.1. Schematic description of electronic cloud displacements in nanoparticles under the effect of a electromagnetic wave.

2.2. Graphene Oxide

Recently, chemically modified graphene (CMG) has been studied in the context of many applications, energy-related materials, such as polymer composites, ‘paper’-like materials, field-effect transistors (FET), sensors, and biomedical applications, due to its excellent electrical, mechanical, and thermal properties. ^70–72 Chemical modification of graphene oxide, which is generated from graphite oxide, has been a promising route to achieve mass production of CMG platelets. Graphene oxide contains a range of reactive oxygen functional groups, which renders it a good candidate for use in the aforementioned applications (among others) through chemical functionalizations.

Although graphene known a relative novel material of broad interest and potential,1,3 GO has a history that extends back many decades to some of the earliest studies involving the chemistry of graphite.4–6 The first, the British chemist B. C. Brodie was exploring the structure of graphite by investigating the reactivity of flake graphite in 1859. One of the reactions he performed involved adding potassium chlorate (KClO₃) to a slurry of graphite in fuming nitric acid (HNO3).7 Brodie determined that the resulting material was composed of carbon, hydrogen, and oxygen, resulting in an increase in the overall mass of the flake graphite. He isolated crystals of the material, but the interfacial angles of the crystal lattice were unable to be measured via reflective goniometry. Successive oxidative treatments resulted in a further increase in the oxygen content, reaching a limit after four reactions. Brodie found the material to be dispersible in pure or basic water, but not in acidic media, which prompted him to term the material ‘‘graphic acid.’’ Nearly 150 years later, ‘‘graphene’’ would take the physics and chemistry communities by storm. Nearly 40 years after Brodie’s seminal discovery of the ability to oxidize graphite, L. Staudenmaier improved Brodie’s KClO₃-fuming HNO3 preparation by adding the chlorate in multiple aliquots over the course of the reaction (also, with the addition of concentrated sulfuric acid, to increase the acidity of the mixture), rather than in a single addition as Brodie had done. This slight change in the procedure resulted in an overall extent of oxidation similar to Brodie’s multiple oxidation approach (C :O B 2 : 1), but performed more practically in a single reaction vessel.8 Nearly 60 years after Staudenmaier, Hummers and Offeman developed an alternate oxidation method by reacting graphite with a mixture of potassium permanganate (KMnO4) and concentrated sulfuric acid (H2SO4), again, achieving similar levels of oxidation.9 Though others have developed slightly modified versions, these three methods comprise the primary routes for forming GO, and little about them has changed. Importantly, it has since been demonstrated that the products of these reactions show strong variance, depending not only on the particular oxidants used, but also on the graphite source and reaction conditions. This point will be borne out in the discussions that follow. Because of the lack of understanding of the direct mechanisms involved in these processes, it is instructive to consider examples of the reactivities of these chemicals in other, more easily studied, systems. The Brodie and Staudenmaier approaches both use KClO3 and nitric acid (most commonly fuming [>90% purity]) and will be treated together. Nitric acid is a common oxidizing agent (e.g. aqua regia) and is known to react strongly with aromatic carbon surfaces, including carbon nanotubes.10,11 The reaction results in the formation of various oxide-containg species including carboxyls, lactones, and ketones. Oxidations by HNO3 result in the liberation of gaseous NO2 and/or N2O4 (as demonstrated in Brodie’s observation of yellow vapors).12 Likewise, potassium chlorate is a strong oxidizing agent commonly used in blasting caps or other explosive materials. KClO3 typically is an in situ source of dioxygen, which acts as the reactive species.12 These were among the strongest oxidation conditions known at the time, and continue to be some of the strongest used on a preparative scale. The Hummers method uses a combination of potassium permanganate and sulfuric acid. Though permanganate is a commonly used oxidant (e.g. dihydroxylations), the active species is, in fact, diamanganese heptoxide (Scheme 1).

This dark red oil is formed from the reaction of potassium permanganate with sulfuric acid. The bimetallic heptoxide is far more reactive than its monometallic tetraoxide counterpart, and is known to detonate when heated to temperatures greater than 55 1C or when placed in contact with organic compounds.13,14 Tro¨ mel and Russ demonstrated the ability of Mn2O7 to selectively oxidize unsaturated aliphatic double bonds over aromatic double bonds, which may have important implications for the structure of graphite and reaction pathway(s) occuring during the oxidation (see below).15 The most common source of graphite used for chemical reactions, including its oxidation, is flake graphite, which is a naturally occuring mineral that is purified to remove heteroatomic contamination.16 As such, it contains numerous, localized defects in its p-structure that may serve as seed points for the oxidation process. If Tro¨ mel and Russ’s observations on styrene can be applied to graphite, then it is likely that the oxidation observed is not that of aromatic systems, but rather of isolated alkenes. The complexity of flake graphite, and the defects that are inherent as a result of its natural source, make the elucidation of precise oxidation mechanisms very challenging, unfortunately. Few other oxidants have been used for the formation of GO, though Jones’ reagent (H2CrO4/H2SO4) is commonly used for the formation of expanded graphite, whose partially oxidized, intercalated structure is somewhere between graphite and true graphite oxide.17 The recent review by Wissler is an excellent, succinct source of further information on commonly used graphites and carbons, as well as the terminology used to describe these materials.16

Structure

Aside from the operative oxidative mechanisms, the precise chemical structure of GO has been the subject of considerable debate over the years, and even to this day no unambiguous model exists. There are many reasons for this, but the primary contributors are the complexity of the material (including sample-to-sample variability) due to its amorphous, berthollide character (i.e. nonstoichiometric atomic composition) and the lack of precise analytical techniques for characterizing such materials (or mixtures of materials). Despite these obstacles, considerable effort has been directed toward understanding the structure of GO, much of it with great success. Many of the earliest structural models of GO proposed regular lattices composed of discrete repeat units. Hofmann and Holst’s stucture (Fig. 1) consisted of epoxy groups spread across the basal planes of graphite, with a net molecular formula of C2O.18 Ruess proposed a variation of this model in 1946 which incorporated hydroxyl groups into the basal plane, accounting for the hydrogen content of GO.19 Ruess’s model also altered the basal plane structure to an sp3 hybridized system, rather than the sp2 hybridized model of Hofmann and Holst. The Ruess model still assumed a repeat unit, however, where 1 4th of the cyclohexanes contained epoxides in the 1,3 positions and were hydroxylated in the 4 position, forming a regular lattice structure. This was supported by Mermoux based on observed structural similarities to poly(carbon monofluoride), (CF)n,20 a structure that entails the formation of C–F bonds through the complete rehybridization of the sp2 planes in graphite to sp3 cyclohexyl structures.21 In 1969, Scholz and Boehm suggested a model that completely removed the epoxide and ether groups, substituting regular quinoidal species in a corrugated backbone.22 Another remarkable model by Nakajima and Matsuo relied on the assumption of a lattice framework akin to poly(dicarbon monofluoride), (C2F)n, which forms a stage 2 graphite intercalation compound (GIC).23 These individuals also made a valuable contribution to understanding the chemical nature of GO by proposing a stepwise mechanism for its formation via 3 of the more common oxidation protocols.24

The most recent models of GO have rejected the lattice based model and have focused on a nonstoichiometric, amorphous alternative. Certainly the most well-known model is the one by Lerf and Klinowski (Fig. 2). Anton Lerf and Jacek Klinowski have published several papers on the structure and hydration behavior of GO, and these are the most widely cited in the contemporary literature. The initial studies done by Lerf and coworkers used solid state nuclear magnetic resonance (NMR) spectroscopy to characterize the material.25 This was a first for the field as earlier models relied primarily on elemental composition, reactivity and X-ray diffraction studies. By preparing a series of GO derivatives, Lerf was also able to isolate structural features based on the material’s reactivity.26

2.3. Sensor

2.3.1. Electrochemical sensor

Electrochemical sensors are applicable in wide range of applications such as glucose monitoring and ion sensors which are mainly applied in the solid-state chemical sensors field ⁷⁴. An electrochemical sensor is able to produce an electrical output signal into digital signal for further analysis through a series of principal stages (Fig. X). Basically, the response obtained in electrochemical sensors is due to the interaction between chemistry and electricity which are based on potentiometric, amperometric, and conductivity measurements ⁷⁵.

Figure ‎0.2. Principal stages in the operation of an electrochemical sensor.

Electrochemical sensors are sensors that detect the electroactive analyte based on oxidizing and/or reducing of the analyte by applying electronic signals. Each electroactive material has oxidation and reduction peak at certain voltages, slightly variable by modifying the working electrode surface. Study on peak positions and intensity to determine the concentration of and type of analyte by electrochemical methods is called electrochemical sensings. Generally an electrochemical sensor consists of five parts: reference electrode, working electrode, a counter electrode, potensiastat and electrolyte. The duty of each part explained briefly in following lines:

Reference electrode: reference electrode is an electrode with standard voltage that is used as the reference to the voltage of the working electrode.

Working electrode: working electrode is an electrode has the duty to oxidize and/or reduce the electroactive analyte.

Counter electrode: counter electrode is an electrode that collects the charge induced in the system.

Potentiostat: potentiostat is the device that can able to apply controlled electric signals to electrodes and measure their electric response.

Electrolyte: electrolyte is an ionic solution that enables transfer of charges between electrodes. Elctroactive analyte should is added to the electrolyte to be test.

The advantages of electrochemical sensors over other type of sensors are low limit of detection (LoD) and selective sensing of analytes in many cases.

Metal nanocomposite modified electrodes are one of the widely studied electrodes used in the electrochemical detection of molecules. High electrocatalytic activity, excellent conductivity, and selectivity which make metal nanostructures based electrodes an excellent choice to be used as an active material in electrochemical sensors. Lots of works have investigated the application of metal nanoparticles modified electrode for the detection of molecules. The feasibility of metal nanoparticles to be synthesized with in sub 10 nm diameters these materials the merit of higher surface area as compared to various metal oxide nanoparticles ⁷⁵.

Anthough Ag NP is a good material for fabrication of working electrodes in electrochemical sensors, it has oxidation and reduction peaks in 250 mV and -0.05 mV that disable this material to be used for many of molecules that have the oxidation and/or reduction peaks in this reagion such as ascorbic acid (AA), uric acid (UA) and dopamine (DA).

2.3.2. Surface enhanced Raman scattering

The Raman spectra of pyridine on roughened silver were observed in 1974 ⁷⁶. The SERS field has hugely developed from their first enhancement on roughened silver electrodes to the current fields of sensing and imaging applications, single molecule detection, and extensions to ultra-high vacuum and ultrafast science ⁷⁷. The basic of SERS can be described by increasing the weak Raman scattering signal by aid of metal surface. There are several methods implemented by researchers to enhance the Raman scattering efficiency, to mention stimulated Raman processes and electronic resonance enhancement. However, the most significant amplification of the Raman signal comes from SERS ⁷⁸. After long debate and investigation on the SERS, scientist are now generally agreed on dominant contribution of the electromagnetism enhansment mechanism as the SERS principle ⁷⁹. The SERS enhancement comes from the amplification of the light by the excitation of LSPRs. The SERS enhancement occurs preferentially in gaps or sharp edges of coinage metals.The robust and reproducible structures of coinage metals that strongly enhance the electromagnetic field are desirable for SERS. The electromagnetic enhancement for SERS theoretically calculated to reach factor of ~ 10¹⁰ – 10¹¹ depending on the structure of the supporting plasmonic material ⁸⁰.

In most cases, the enhancement factor can be well approximated by the magnitude of the localized electromagnetic field to the fourth power ⁷⁹. Chemical enhancement, is the other mechanism involved in signal enhancement where the excitation wavelength is resonant with the metal-molecule charge transfer electronic states ⁸¹. Theoritically, the enhancement factor of 10³ found for chemical enhancement.

The SERS sensors are able to identify the materials with great accuracy, but they are unable to do it quantitively.

2.3.3. Optical sensor

Optical sensors are basically sensors that detect the analyte by measuring its changes in absorbance or emission spectrum. The emission based methods can be monitored by fluorescence spectroscopy measurement.

Following the advantages of the surface characteristics and high stability of the particles, gold and silver nanoparticles have been studied extensively as fluorescence quenchers ^82–84, the reason being manifold. The spherically shaped metal nanoparticles have no defined dipole moment as the dyes, so energy transfer to the nanometal stake splace for any orientation of the donor relative to the surface and the large absorption cross-section near the plasmon resonance enhances their performance as energy acceptors. Fluorescence quenching-based “turn-on” assay is one of the most important applications among various energy transfer based techniques, in which the fluorescence of the donor can be effectively quenched by the acceptor in the absence of the targets. The quenched fluorescence is “turned-on” upon addition of the target, and the restored fluorescence intensity is proportional to the concentration of the target ⁸⁵.

The other family of optical sensors is LSPR based optical sensors. These sensors are based on the changes in LSPR band of metal nanoparticles that are explained in detail in next part.

2.3.3.1 Localized Surface Plasmon resonance

2.3.3.2. Refractive index and LSPR

The optical properties of noble metal NPs have attracted interest ever since the Romans began using gold NPs as colorants in glasses. The color of colloidal metal NPs depends on both the size and shape of the particles, as well as the refractive index of the surrounding medium. To obviate these problems, some works have prepared gold NPs with a narrow size distribution in water and transferred them into a variety of organic solvents. Since the same particles are used in all cases, shape factors, structural defects, and other matrix effects are eliminated. The optical changes associated with the transfer of the colloid particles into organic solvents with different refractive indices to water are shown to be in quantitative agreement with Mie theory ⁸⁶.

A variety of synthetic methods have been developed to fabricate metal nanoparticles assembly based chemical sensors ^87,88. The localized surface plasmon resonance (SPR) based sensor exhibits good sensitivity with improved ease of fabrication and thus enhanced utility outside the laboratory ⁸⁸. The label-free optical sensor responds to the adsorbate induced refractive index changes near or on plasmonic nanostructures and this property was used to monitor the binding events in real time ^10,89. The development of the SPR sensor has gained considerable momentum for the detection of chemical and biological species ⁸⁹. A number of reports have been published on the sensing of analytes related to medical diagnostics, environmental monitoring, and food safety and security ⁸⁹. The SPR sensor has been discussed in reviews ⁹⁰ and articles ⁹¹as one of the main optical sensor technologies. The main advantages of the SPR sensors include sensitivity, selectivity, linearity, resolution, accuracy, reproducibility, dynamic range, and limit of detection ^89,92. The sensors based on LSPR changes on Ag NPs is also attracted attention for scientists due to their ease of characterization. The changes in LSPR can be monitored and compared with original data with simple UV-vis device.

2.4.2 Amalgamation and LSPR

As discussed before, the LSPR absorbance band of noble metal nanoparticles affected by both the size and shape of the nanoparticles, as well as their refractive index. Another approach to develop a sensor based on LSPR band of metal nanoparticles is to change their size and morphology by addition of mercury ions (Hg(II)). The sufficient electrochemical potential of Hg(II) ions (0.85V) to overcome the Ag(I)/Ag couple (0.8V) and enable redox interaction to happen at room temperature ⁹³. As the result, the morphology of the Ag NPs changes and AgHg NPs start to form. The Ag NPs get smaller as the consequence of the transformation of Ag NPs to AgHg NPs, the blue shift in absorbance spectrum happen. The other hand, AgHg NPs do not possess any LSPR band and the intensity of the LSPR band of Ag NPs starts reducing by formation of AgHg. This reduction in color can be easily observed by naked eyes. Since the electrochemical potential of other common metal ions to reduce to their 0 state are not sufficient to overcome the electrochemical potential of Ag to become Ag(I) ions, this material is highly selective toward Hg(II) ions.

CHAPTER 3: MATERIALS AND METHODS

3.1. Chemicals and Reagents

Graphite flakes were purchased from Ashbury Inc. USA. Chemical reagents such as silver nitrate (AgNO₃), sulphuric acid (H₂SO₄, 98%), phosphoric acid (H₃PO₄, 85%), potassium permanganate (KMnO₄, 99.9%), hydrogen peroxide (H₂O₂, 30%), 3-hydroxytyraminium chloride (dopamine, DA), L(+)-ascorbic acid (AA), and uric acid (UA) were purchased from Merck. Silver nitrate (AgNO₃) and sodium borohydride (NaBH₄) were received from Systerm, ferrous chloride tetrahydrate (FeCl₂.4H₂O), ferric chloride hexahydrate (FeCl₃.6H₂O), nickel sulphate (NiSO₄), zinc sulphate (ZnSO₄), Sodium sulphate (Na₂SO₄), potassium chloride (KCl), manganese sulphate (MnSO₄), cobalt acetate (Co_(asas)) and cooper (I) chloride (CuCl) bought from Merk. All other chemicals used were of analytical grade without further purification. All solutions were prepared by deionized water.

3.2. Preparation of [email protected] Nanocomposite

The [email protected] nanocomposite for biomolecule optical sensor was prepared as follows. Initially, GO was prepared by following the well-known simplified Hummer’s method ⁹⁴. A 4 mg quantity of GO was dispersed in 30 mL of DI water. Then, 85 mg of silver nitrate (AgNO₃) was separately dissolved in 10 mL of DI water and mixed with the GO solution, after which the mixture solution was sonicated for 15 minutes at 50 W using a horn sonicator in order to homogeneously mix Ag⁺ ions on GO surface and break GO sheets into smaller pieces in a cool bath. At the end of the sonication, 400 µL of 5 mM of freshly prepared sodium borohydride (NaBH₄) solution was added to the GO-AgNO₃ solution. After this addition, the color of the solution instantly became dark yellow as a result of the formation of silver nanoparticles. The prepared [email protected] nanocomposite was mixed with 30 mL of DI water and stored in a vial without any washing or purification. The similar material prepared for Hg(II) sensor by differing sonication time and power to 20 minutes and 80 W.

3.3. Characterization Techniques

The crystalline phase of the samples was analyzed via X-ray diffractometer (Siemens-D5000) with Cu Kα radiation (λ=1.5418 Å) at a scan rate of 0.02 degrees sec⁻¹. The morphology of the nanocomposite was examined with JEOL JEM-2100 F high resolution transmission electron microscopy. Optical absorption properties in the spectral region of 190–600 nm were assessed using a Thermo Scientific Evolution 300 UV-vis absorption spectrophotometer. The electrochemical

measurements were carried out using PAR-VersaSTAT 3 electrochemical analyzer (Princeton Applied Research, USA) with a conventional three electrode system under a nitrogen atmosphere at room temperature. The Raman spectra were collected using a Renishaw 2000 inVia system with an argon ion laser emitting at 514 nm.

3.4. Optical Detection of Biomolecules

The optical sensing of biomolecules, including DA, AA, and UA, using the [email protected] nanocomposite was performed using the Thermo Scientific Evolution 300 UV-vis absorption spectrophotometer. The absorption spectra of the [email protected] nanocomposite were recorded upon the addition of various biomolecule contents such as DA, AA, and UA. For the optical detection, 10 µM of optimized analyte concentration (DA/AA/UA) was added into 2 mL of the [email protected] solution, shaken well, and subjected to a constant resting time. The absorbance spectra of [email protected] solution with the addition of different concentration of analyte were recorded. Then, the difference in the absorbance was monitored by recording the absorbance spectrum.

3.5. Optical Detection of Hg(II) ions

The optical sensing of metal ions with the [email protected] nanocomposite was performed using Thermo Scientific Evolution 300 UV-vis spectrophotometer. The absorption spectra of the [email protected] nanocomposite were recorded upon the addition of various concentrations of metal ions. For optical detection, an optimized concentration of the metal ion solution was added in 2 ml of [email protected] solution, shaken well uniformly and subjected to a constant resting time before the absorbance spectrum of the solution was recorded.

CHAPTER 4: RESULTS AND DESCUSSIONS

The absorbance spectra of the AgNO₃, GO, and synthesized [email protected] nanocomposite are shown in Figure 1. The peak at 265 nm for AgNO₃ corresponds to the absorbance peak of Ag⁺ ions ^95,96. The visible peak at 230 and a shoulder at 300 nm in the GO absorbance spectrum are attributed to the π  π* of the aromatic C–C bond and the n  π* of the C=O bond transition, respectively ⁹⁷. The addition of NaBH₄ to the GO-AgNO₃ solution caused a reduction in the Ag⁺ ions into Ag⁰ and resulted in the formation of Ag nanoparticles on the GO. The existence of the nπ* transition of the C=O bond (absorbance at 300 nm) after the addition of NaBH₄ to the GO-AgNO₃ solution suggested that NaBH₄ produced a reduction in the Ag ions to Ag nanoparticles, but did not reduce the GO to rGO.

Figure 4.‎0.1: UV-vis absorption spectra of (a) AgNO3 (b) GO, and (c) [email protected] nanocomposite. Inset: Photograph obtained for the aqueous solution of synthesized [email protected] nanocomposite.

4.2. Optical Sensing of Biomolecules using [email protected] Nanocomposite

The biomolecule sensing abilities of [email protected] were investigated in absorbance-based titration experiments with different concentrations of the analytes (DA, AA and UA). The changes in the SPR band intensity and peak position of the [email protected] nanocomposite were used for the sensing of the biomolecules. The absorbance of the [email protected] sample recorded by the titration of DA with a concentration range of 100 nM up to 2 µM at intervals is shown in Figure 2, along with the obtained absorbance changes by this titration. Figure 2A shows the decrease in the absorption intensity and red shift in the absorbance peak from increasing the concentration of dopamine in 100-nM steps. The decrease in the absorption and red shift in the absorption maximum can be explained by the Mie theory⁹⁸, which suggests that any changes in the refractive index of the local environment around the surface of noble metal nanoparticles will introduce some changes in the absorption intensity and/or position of the SPR absorption band. An increase in the refractive index usually produces a red shift to a longer wavelength in the SPR absorption band ^10,98,99. The changes in the absorption intensity and/or position of the SPR of the [email protected] sample by the increasing concentration of dopamine are plotted in Figure 2(B and D). A linear relationship with a correlation coefficient of 0.9985 (n=20) for the regression equation (_shift (nm) = 403 + 0.0065 nM) was obtained for the [email protected] in the DAconcentration range of 100 nm to 2 M. The LOD was calculated to be 30 nM from the equation LOD = 3 × standard deviation of the regression line ()/slope (S).

Further, the decrease in the absorption intensity is plotted against the DA concentration (Figure 2B). It can be seen that the absorbance intensity of the [email prote