1.

INTRODUCTION

1.1. Nanomaterials

Analytical Chemistry now a days mostly uses nanomaterials, hence should be

produced economically and modestly in the current research field. The nanomaterial is an
emerging and the economically important field for various aspects [1,2]. As a way of
example, nanoscience can be defined as “an emerging area of the science that studies and
develops the materials in the nanoscale” whereas, nanotechnology “is the technology of
materials and structures, both in the nanometric level with a great variety of applications
thanks to the nanosize and the exceptional properties of the nanomatter” [3-5]. The term
“nanoparticles” (NPs) have been defined by several authors, but the most appropriate is the
IUPAC definition which considered the NPs as “particles of any shape with dimensions in the
range of 1x10−9 and 1x10−7 m” [6]. These NPs can be found naturally in the environment or
be synthesized by two methodologies, bottom-up and top-down as shown in Fig: 1.1. & Fig:
1.2. The bottom-up methodology is based on the formation of complex type of nanostructures
from molecules or atoms to reach the nanomatter size whereas in the top-down methodology
the synthesis is performed from bigger particles or even other nanomaterials as precursors
usually involving physical methods [7-9].

The widely accepted range for nanomaterial is between 1 and 100 nm [10-12]. This
range, also the quantum regime and that‟s why many nanomaterials exhibit quantum effects
that make them suitable for electronics, semiconductor technologies and quantum
technologies. So, even there are many different classes of nanomaterials, including thin films,
coatings and nanoparticles [13-15].

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 Fig: 1.1. Synthetic methodologies of nanomaterials.

Fig: 1.2. Difference between Top-down and Bottom-up Approach.

Taking into account of these definitions, we can consider nanoscience and

nanotechnology [NS&NT] as a multidisciplinary field which converges with other many

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areas and technologies including (nano)medicine, (nano)electronics, (nano)devices and
(nano)technological instrumentation [16,17]. For instance, the nexus between NS&NT and
analytical chemistry is the consideration of the nanomatter as tools or the object of study (the
analyte) in an analytical process to get a step further in innovation, simplification and
improvement of the analyses [18-20]. Many classifications related to the exploitation of
analytical nanoscience and nanotechnology (ANS&NT), some of them are going to be
commented in this thesis. The first one attends to the size of the material involved in the
analysis, being macro, micro or nano; thus the use of NPs confers a nanotechnological
character to the analytical process [21,22]. Another principle is the consideration of
nanomaterials as the analyte, that is, the determination of NPs in the consumer products and
environmental systems. Furthermore, a very common classification is based on the
exploitation of the unique properties of NPs, the nanosize and the combination of both, which
entails the definition of three main groups [23,24]. A nanometer is a unit of length in the
metric system, equal to one billionth of a metre (10-9), technology is the making, a usage, and
knowledge of tools, machines and techniques, in order to solve a problem or perform a
specific functions [25-28].

1.1.1. Nanometric analytical systems: which are related to the nanosize of the analytical
devices. They can be considered as the current trend towards miniaturization and many of
them are based on nanometric volume [29-32].

1.1.2. Nanotechnological analytical systems: which exploit the exceptional

physicochemical properties of NPs. The nanotechnology is involved in micro or macro
analytical systems [33-35].

1.1.3. Analytical nanosystems: which are a combination of both nanotechnological and

nanometric analytical systems and involve the exploitation of the NP properties and the
nanosize [36,37].

1.2. Classification of Nanoparticles

The classification is the most popular and takes into account the dimensionality which
is double classified depending on the dimensions (below 0 nm and above 100 nm) [39-40].
The three types of nanostructures with dimensions below 100 nm Fig: 1.3.

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 Fig: 1.3. Classification is based on the number of dimensions.

However, other authors classified the nanostructures based on the dimensions above 100
nm [41] show in Fig: 1.4.

 0D Zero dimension (Carbon dots)

 1D One dimension (Nanowires, Nanotubes)
 2D Two dimension (coatings)
 3D Three dimension (nanoporous material)

Fig: 1.4. Schematic representation of various dimensions (0D, 1D, 2D, and 3D) of nanomaterials.

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1.2.1. Zero-dimensional nanomaterials

Materials wherein all the dimensions are measured with in the nanoscale range (No
dimensions are larger than 100 nm). The most common representation of zero-dimensional
nanomaterials are nanoparticles.

Be amorphous or crystalline, be single crystalline or polycrystalline, be composed of

single or multi-chemical elements, exhibit various shapes and forms, exist individually or
incorporated in a matrix, be metallic, ceramic, or polymeric [42,43].

1.2.2. One-dimensional nanomaterials

One of the dimension is in the nanoscale range. This leads to needle like-shaped
nanomaterials. 1-D materials include nanotubes, nanorods, and nanowires. 1-D nanomaterials
can be: Amorphous or crystalline; Single crystalline or polycrystalline; Chemically pure or
impure; Standalone materials or embedded in within another medium; Metallic, ceramic, or
polymeric [44-47].

1.2.3. Two-dimensional nanomaterials

Two of the dimensions are not confined to the nanoscale, 2-D nanomaterials exhibit
plate-like shapes. Two-dimensional nanomaterials include nanofilms, nanolayers and
nanocoatings. 2-D nanomaterials can be: Amorphous or crystalline; Made up of various
chemical compositions; used as a single layer or as multilayer structures; Deposited on a
substrate; Integrated in a surrounding matrix material; Metallic, ceramic, or polymeric [48-
50].

1.2.4. Three-dimensional nanomaterials

Bulk nanomaterials are materials that are not confined to the nanoscale in any dimension.
These materials are thus characterized by having three arbitrarily dimensions above 100 nm.
Materials possess a nanocrystalline structure or involve the presence of features at the
nanoscale. In terms of nanocrystalline structure, bulk nanomaterials can be composed of a
multiple arrangement of nanosize crystals, most typically in different orientations. With
respect to the presence of features at the nanoscale, 3-D nanomaterials can contain
dispersions of nanoparticles, bundles of nanowires, and nanotubes as well as multinanolayers
[51-54].

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1.3. Carbon Dots (CDs)

Over the past decades, green chemistry has captured the imagination of many
chemists due to its clean and sustainable feature. Preparation of nanomaterials using nontoxic
chemicals, environmental friendly solvents, and renewable materials is the key issue that
should be taken into consideration in a green synthetic strategy [55]. To this end, a green
method using precursors directly from the nature is a very promising solution and would be
of great benefit to large scale synthesis and widespread applications. In the present work,
hydrothermal method is used, we have utilized a totally green approach toward the synthesis
of fluorescent carbon dots [56,57]. Recently, the carbon dots (CDs), which are a new class of
fluorescent nanomaterials with mainly sp3/sp2 hybridized carbons, have received much
attention owing to their good water solubility, excellent photo stability, low toxicity, and
favorable biocompatibility [58]. Carbon dots (CDs) are a recently developed material
belonging to the carbonaceous family and have attracted considerable attention in various
research areas. In this work, a facile and green method for the preparation of fluorescent CDs
by hydrothermal treatment and the application has been proposed [59,60]. On the basis of
fluorescence property, the prepared CDs can serve as an effective sensor for sensitive and
selective determinations. The excellent chemical and photochemical stability of CDs together
with their biocompatibility [61] give a clear advantage in the context of biological
applications, and thus making them a legitimate competitor to the conventional CDs with
comparable or even better performance [62-66].

Moreover, the surface of CDs can be readily modified with different functional
groups, which enables more prospects for tuning their physicochemical properties. Lately,
more effort has been put into the preparation of fluorescent carbon dots because of their
extraordinary photophysical and photochemical properties, good biocompatibility [67], and
the related broad applications in the areas of bioimaging [68], electrocatalysis [69], solar cells
[70], and sensors [71,72]. Several methods have been established for the preparation of CDs,
including etching with larger carbon materials, such as laser ablation [73], electrochemical
oxidation [74], thermal oxidation [75], microwave irradiation [76], hot injection [77], and
pyrolysis [78]. The water soluble fluorescent carbon quantum dots are emerging as new area
of research due to their distinct properties. They find extensive applications in different field
such as organic and inorganic fluorescent dye, optical limiting [79], LED-technolog [80,81],

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photocatalyst, bio-sensor [82], bio-imaging [83], bio-medical [84] and drug delivery [85]
show in Fig: 1.5.

Fig: 1.5. Different applications of CDs.

On considering the limitations of existing methods and conservancy of greener

environment, we have adopted a hydrothermal carbonization method to synthesize CDs. The
prepared CDs are less expensive and highly soluble in water. These CDs have diversified
benefits because of their sensing ability in low ppm with high sensitivity. The sensing study
was carried out using cyclic voltammetry techniques. The formation of CDs was confirmed
by structure and functional group characterization by using Fourier-transform infrared
Spectroscopy (FT IR) and Raman spectroscopy, the electronic transition state is confirmed by
using UV-Visible, photoluminescence properties confirmed by using PL spectroscopy. The
surface morphology confirmed by using TEM, the phase purity and crystallite size was
confirmed by using XRD analysis and particle size was confirmed by using DLS studies.

Carbon dots (CDs) materials have been synthesized from carbohydrates, waste peel
materials and natural gum materials. This points out to an important concept in the synthesis
of CDs where the hydrothermal treatment of substances which contain carbon, and oxygen
will in most cases give the fluorescent nature of CDs [86,87].

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 A large variety of synthetic techniques and starting precursors has been used to
synthesize carbon dots (CDs). Generally speaking, the synthesis methods can be divided into
top-down and bottom-up methods, all having certain advantages and disadvantages [88,89].
Top-down methods involve physical or chemical disruption of bulk carbon precursor usually
graphite or amorphous carbon. This can be achieved by laser ablation, arc discharge,
electrochemical synthesis and chemical oxidation in strong acids [90]. Bottom-up syntheses
generally do not require any specific starting precursor because CDs can be prepared with
bottom-up synthesis from any organic precursor that undergoes carbonization [91]. Bottom-
up syntheses offer simple and fast preparation of CDs with large number of surface functional
groups, and allow easy introduction of dopants which significantly affect the
photoluminescent properties of synthesized CDs. Bottom-up approach include solvothermal
synthesis, microwave synthesis and template supported synthesis [92]. In top-down approach
include thermal method, microwave assisted method, hydrothermal & aqueous method,
template method [93] show in Fig: 1.6.

Fig: 1.6. Bottom-up and Top-down methods

Use of different methods synthesis of CDs.

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1.3.1. Top down method

1. Arc discharge method

2. Laser ablation method
3. Electrochemical oxidation method
4. Chemical oxidation by strong acids
5. Ultrasonic synthesis

1.3.2. Bottom up method

6. Thermal decomposition method

7. Microwave assisted method
8. Hydrothermal & Aqueous method
9. Template method
10. Plasma treatment

Carbon (C) is one of the most widespread elements in nature and is responsible for
our existence. Carbon exists in nature in different allotrope forms such as diamond, graphene,
graphite and amorphous carbon. A large number of new carbon materials with well-defined
nanostructures have been synthesized, such as fullerenes, carbon dots, carbon nanotubes,
graphene, nano diamonds, etc. With regard to applications, carbon will continue to play an
important role [94,95].

Carbon (C) is one of the most important and unique elements with a huge chemical
diversity. Since carbon is the main building block of organic compounds, it is essential for all
living organisms. Carbon (C) and its chemical compounds are also important products in
chemical and energy industry. Pure carbon is an inorganic material which exists in multiple
allotropes with variety of properties [96]. Two best known crystalline allotropes of carbon are
graphite (sp2 hybridized carbon atoms) and diamond (sp3 hybridized carbon atoms), and also
amorphous carbon. In bulk, graphite and diamond can be considered to consist of infinite
carbon networks and they do not show photoluminescent properties. If the size of these
carbon networks is reduced to nanometer scale, the resulting structural changes usually
change electronic properties of the material, and thus its optical properties [97-99].

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1.4. Carbohydrates (CH)

The carbohydrates glucose, sucrose, cellulose were used as a model precursor for the
synthesis of fluorescent carbon dots. Since glucose, sucrose, cellulose only contains of carbon
(C), oxygen (O) and hydrogen (H) atoms, the synthesized carbon dots were considered
“undoped” CDs. Carbohydrates are broadly defined as polyhydroxy aldehydes or ketones and
their derivatives or as substances that yields one of these compounds. Functional groups
present include hydroxyl groups. Carbohydrates may be classified as monosaccharides,
disaccharides and polysaccharides depending on the number of monomer (sugar) unit they
contain. Classifications based on the number of sugar units in total chain. Monosaccharides
single sugar unit, disaccharides two sugar units and polysaccharides more than 10 units.
Saccharide comes from the Greek language and means “sugar”. Monosaccharides or simple
sugar, have from three to seven carbon atoms and one aldehyde or one ketone group if the
sugar has an aldehyde group, it is an aldose. If it has a ketone group, the sugar is classified as
a ketose [100-102].

1.4.1. Monosaccharides (Glucose)

Glucose is a sugar with molecular formula C6H12O6. It is a monosaccharide and a

hexose sugar (6 carbon atoms). The D-isomer (D-glucose) is also known as dextrose and
occurs widely in nature. Glucose may exist in the form of five different isomers where one of
the isomers represents an open-chain form and the other four are cyclic forms. They may
exist in a linear molecule or in ring forms [103]. They are classified according to the number
of carbon atom in their molecule. show in Fig: 1.7.

Fig: 1.7. Pictorial image and molecular structure of glucose.

Many forms exist as isomers, isomers are molecules which have the same empirical
formula (recipe) but have different structures (shapes) due to the arrangement of the atoms in
the molecule. This also gives them different properties. Glucose and fructose both have the
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empirical formula C6H12O6, but they have different structural formulas or shapes. We used
glucose as the carbon source to fabricate CDs. As a typical example, we present the results
based on glucose-derived CDs.

1.4.2. Disaccharides (Sucrose)

Sucrose is a sugar with molecular formula C6H22O11. When two monosaccharide are
combined by glycosidic linkage, disaccharides are formed. There fore they yield two
molecules of same or different monosaccharide on hydrolysis. The disaccharides just like
monosaccharide‟s are white crystalline sweet solids. However, even though they are soluble
in water they are too large to pass through cell membranes [104,105] show in Fig: 1.8.

Fig: 1.8. Pictorial image and molecular structure of sucrose.

1.4.3. Polysaccharides (Cellulose)

Polysaccharides are large molecules containing 10 or more monosaccharide units.

Carbohydrate units are connected in one continuous chain or the chain can be branched.
Polysaccharides are polymeric carbohydrate structures formed of repeating units (either mono
or di saccharides) joined together by glycosidic bonds [106]. These structures are often linear,
but may contain various degrees of branching. Polysaccharides are often quite heterogeneous,
containing slight modifications of the repeating unit. Low cost and sustainability are
important criteria for selecting carbohydrates as a carbon source to produce CDs [107,108].
show in Fig: 1.9.

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 Fig: 1.9. Pictorial image and molecular structure of cellulose.

1.5. Waste Peels (WP)

Waste peels are produced in large quantities in markets and constitute a source of
nuisance in municipal landfills because of their high biodegradability. In India fruit waste
peels constitute about 5.6 million tonnes annually and currently these waste peels are
disposed of by dumping on the out skirts of cities. Among the several processes that are being
used nowadays, the ones described are the following: thermal processes, evaporation,
membrane processes, anaerobic digestion, anaerobic co-digestion, biodiesel production and
composting. India is the world‟s second largest producer of fruits waste peels, it is well
known that the huge quantities of lignocellulosic biomass are produced every year during
cultivation, harvesting, processing and consumption of agricultural products [109-111].

1.5.1. Banana peel (BP)

Banana is a tropical fruit from plants of the musaceae family and is one of the most
consumed fruits in the world, representing an important fruit crop in tropical and sub-tropical
regions. India is the largest producer of bananas, with nearly 10% of the total world
production. The main banana residue is the fruit peel, which accounts for 30–40% of the total
fruit weight [112]. There are few industrial uses for banana peels, and they are a major
agricultural waste in different regions of the planet show in Fig: 1.10.

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 Fig: 1.10. Pictorial image of banana peel.

1.5.2. Orange peel (OP)

Oranges are one of the most popular fruits in the word. Orange peel is mostly
composed of cellulose, pectin, hemicellulose, lignin, chlorophyll pigments and low molecular
weight hydrocarbons show in Fig: 1.11.

Orange waste peels are one of the most underutilized natural resources and most
geographically diverse bio-waste residues on earth, and the huge remnants have never been
challenged to utilize extensively in an effective manner.

Fig: 1.11. Pictorial image of orange peel.

Orange waste peels are composite of carbohydrates including fructose, glucose,

sucrose and cellulose. In this respect, bio-waste materials can be considered as an effective
and potential alternative feed stock to fossil resources for a variety of chemicals [113].

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1.5.3. Pomegranate peels (PGP)

The Pomegranate peels makes up 50% of the fruit and contains a number of bioactive
compound. The peel majorly contains of anthocyanins, polyphenols, ellagic acid and
glycosides minarals such as potassium, magnesium and sodium. Pomegranate peels spherical
carbon dots are prepared via simple one step hydrothermal method. pomegranate is important
to the cosmeceutical development with its clinically proven anti-inflammatory, anti-oxidant
and anti-aging properties [114-116] shoe in Fig: 1.12.

Fig: 1.12. Pictorial image of pomegranate peel.

1.6. Natural gum (NG)

The polysaccharide gums are most abundant and naturally occurring gum material,
which are extensively used in industries either to form a gel or make a viscous solution or to
stabilize the emulsion systems. Water-soluble gums are known as „hydrocolloid‟. The
considerably growing interest in plant gum exudates from their diverse structural properties
and metabolic functions in food, pharmaceutical, cosmetic, textile and biomedical products.
Plant polysaccharide gums can be used as dietary fiber, texture modifiers, gelling agents,
thickeners, and emulsifiers, stabilizers, coating agents and packaging films and carbon
materials. These polysaccharide gums are also reported to exhibit biodegradable and bio safe
characteristics [117-119].

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1.6.1. Azadirachtaindica gum (AIG)

Azadirachtaindica gum was used because of availability, widely cultivated and fast
growing trees all over the India. It has versatile applications in pharmaceuticals and cosmetics
as it grows in all climatic conditions including tropical region. Neem gum, a typical plant
gum exudate from the tree (Melia azadirachta, Meliaceae) is the salt of a complex
polysaccharide acid [120,121]. It has been in pharmaceutical use in India for many centuries
[122] show in Fig: 1.13.

Fig: 1.13. Pictorial image of azadirachtaindica tree and gum.

1.6.2. Moringa oleifera gum (MOG)

Moringa oleifera (family of: moringaceae) is a middle size tree about lam in height
cultured throughout India. It is a multipurpose tree used as vegitable, spice a source of
cooking and consmetic oil and as a medicinal plant. This plant contains alkaloids,
anthocyanins, proanthocyanidins, flavonoids, and cinnamater. The moringa oleifera gum
contain D-glucuronic acid, L-rhamnose, D-mannose, D-xylose, L-arabinose, D-galactose and
leucoanthocyanin [123-125]. Moringa oleifera gum has been investigated as a potential
carrier for colon-specific drug delivery and sensing Fig: 1.14.

Fig: 1.14. Pictorial image of moringa oleifera tree and gum.

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1.6.3. Acacia Arabica gum (AAG)

Acacia Arabica gum also known as AAG, is a natural gum consisting of the hardened
sap of various species of the acacia tree. AAG is a complex mixture of glycoproteins and
polysaccharides and have been used in traditional medicine and day to day applications [126].
AAG is a naturally occurring nontoxic polysaccharide derivative. Acacia Senegal and acacia
seyal trees are the main source of AAG show in Fig: 1.15.

Fig: 1.15. Pictorial image of acacia arabica tree and gum.

1.7. Electrochemical sensor

Electrochemistry is the branch of chemistry which deals with the study of chemical
changes caused by the passage of an electric current and the production of electrical energy
by chemical reactions. It has an enormous application including environmental monitoring,
industrial quality control, battery industries, chemical sensors, and biomedical analysis.
Electrocatalysis using voltammetric techniques is characterized by current enhancement and
or a potential shift to lower values in the case of cyclic voltammetry [127].

Electrochemical sensors are devices that give information about the composition of a
system in real time by coupling a chemically selective layer (the recognition element) to an
electrochemical transducer. In this way, the chemical energy of the selective interaction
between the chemical species and the sensor is transduced into an analytically useful signal.
They attract great interest nowadays because they are easy to miniaturize and integrate into
automatic systems, without compromising analytical characteristics [128]. Fig: 1.16. shows
the basic mechanisms through which electrocatalytic reaction at electrodes modified with a
catalyst

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 Fig. 1.16. Basic mechanism of electrocatalytic reaction.

1.7.1. Riboflavin (RF)

The electrochemical sensing of Riboflavin and Quercetin.

Riboflavin (RF), the IUPAC names is 6,7-dimethyl,9-(d-1-ribityl) isoalloxazine (also

known as Vitamin B2) is a part of the vitamin B group a well-known water-soluble vitamin
and is essential to the human health. Vitamin B2 is a biochemical molecule widely existing in
food and pharmaceutical products. It is the major component of the cofactors flavin adenine
dinucleotide (FAD) and flavin mononucleotide (FMN), and it is required for a variety of
flavorprotein enzyme reactions including activation of other vitamins. Like all the B
vitamins, vitamin B2 plays a key role in energy production Fig: 1.17. RF is an essential
precursor of these coenzymes [129]. Vitamin B2 is a water-soluble vitamin that is flushed out
of the body daily, so it must be restored each day. RF is the composition of coenzyme and
involved in sugar, protein, fat metabolism, promoting growth and cell regeneration. RF can
be found in certain foods such as milk, meat, eggs, nuts, enriched flour, and green vegetables.
It is involved in promotion of skin, nails, hair's normal growth, and eliminate the mouth, lips,
tongue inflammation, also in promotion of vision, reduce eye fatigue. At the same time, RF is
used in a kind of phototropism, phototaxis, and photodynamic therapy and photosensitive
agent [130].

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 RF is a vitamin that is needed for growth and overall good health. It helps the body
break down carbohydrates, proteins and fats to produce energy, and it allows oxygen to be
used by the body.

Fig: 1.17. Pictorial image of riboflavin.

In this work, a CDs/GCE modified electrode has been fabricated for the
electrochemical sensing method. The electrochemical behavior of riboflavin at the modified
GC electrode was investigated by using cyclic voltammetry (CV), different scan rate, effect
of pH solutions and differential pulse voltammetry (DPV) methods. The sensor exhibited a
high sensitivity and fast response.

Quercetin (QC)

Flavonoids are natural products widely distributed in the plant kingdom and generally
present in the common human diet. Quercetin (QC) the IUPAC name is (3,3/,4/,5,7-penta
hydroxyl flavones), is a flavonoid of widespread occurrence in plants origin (caper, lovage,
broccoli, lettuce, spinach) and food of plant origin (onions, apples, various berries, tea). One
of the abundant flavonoid molecules widely exists in vegetables and fruits, especially in
traditional Chinese herbs. Furthermore, it can inhibit cancer cell proliferation and reduce
tumor development. However, an overdose of QC may lead to kidney cancer and the decrease
of glutathione s-transferase activity [131]. The modified glassy carbon electrode (GCE)
showed better electrochemical sensing performance when compared with bare electrodes.
The CDs is used to prepare electrochemical sensor for QC show in Fig: 1.18.

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 Fig: 1.18. Pictorial image of quercetin.

1.8. MCF-7 human breast cancer cell lines

The MCF-7 is a human breast cancer cell line that was first isolated in 1970 from the
breast tissue of a 69-years old Caucasian woman. The two mastectomies she received, the
first revealed that the removed tissue was benign. Five years later, a second operation
revealed a malignant adenocarcinoma in a pleural effusion from which tissue was taken that
would eventually result in the MCF-7 cell line show in Fig: 1.19. The donor was treated for
breast cancer with radiotherapy and hormonotherapy [132].

Fig: 1.19. Pictorial image of MCF-7 cell line.

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 MCF-7 (human breast cancer) cell lines were purchased from NCCS center, Pune,
India. MCF-7 cell lines were grown and maintained in suitable (DMEM -media and were
grown and sub cultured in medium supplemented with 10% fetal bovine serum,1% L-
glutamine.1% penicillin streptomycin antibiotic solution).

The CDs have great potential for use in a variety of biological and biomedical
applications with favorable properties, such as strong photoluminescence and robust stability,
but biocompatibility of CDs still remains a key concern for further biological applications,
particularly in live cells, tissues, and animals. It is still important to perform systematic and
reliable bio safety assessment of CDs before employing them in practical applications [133].

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