NANOPARTICLE COMPOSITION AND PROCESS OF PREPARATION THEREOF

Abstract

ABSTRACT NANOPARTICLE COMPOSITION AND PROCESS OF PREPARATION THEREOF A nanoparticle composition comprising a gold nanoparticle is provided. The gold nanoparticle is prepared using gold chloride trihydrate. The gold chloride trihydrate (HAuCI. · 3Hz0) is reduced 5 by adding 0.1 M oxalic acid (CzHzO•) and stabilized by 0.5M L-ascorbic acid (C6Hs06) with continuous stirring in constant temperature. Oxalic acid reduces into oxalate ions when the redox reaction occurs on the gold chloride trihydrate which converts into gold nanoparticles and Lascorbic acid which stabilizes the gold nanoparticles. Eventually, carboxyl group of oxalic acid and L-ascorbic acid have the ability of potential bond interaction towards the gold surface. Since, I 0 the synthesized AuNP-COOH nanoparticle~ having ekvated number of carboxyl group on its surface which affords more avenue for the attachment of any amine-based compounds includes proteins, peptides, drug molecules etc. Hence, the synthesized gold nanoparticle functionalized with carboxylic groups can be used in different applications.

Specification

NANOPARTICLE COMPOSITION AND PROCESS OF PREPARATION THEREOF
BACKGROUND
Technical Field
[000 I) The present invention pertains to the field of nanoparticle composition, specifically
5 it relates to a process for synthesizing functionalized gold nanoparticles (AuNPs). Specifically, the
present invention focuses on the use of oxalic acid and L-ascorbic acid to produce gold
nanoparticles with an increased number of furictionalized carboxyl groups on their surface.
Description of the Related Art
[0002) Gold nanoparticles (AuNPs) are widely recognized for their unique optical,
10 chemical, and physical properties, making them a cornerstone in various fields of nanotechnology,
including medicine, materials science, and diagnostics. Traditional methods of synthesizing
AuNPs typically involve the use of citrate or tannic acid as reducing agents, which help c~ntrol
the size and shape of the nanoparticles. While these methods are effective, there has been a growing
interest in "green" synthesis approaches that utilize safer, more biocompatible reducing agents·,
15 especially tor applications in biological systems.
[0003) The Turkevich method, one of the most commonly used approaches for gold
·nanoparticle synthesis, involves the reduction of gold ions (Au'•) using organic acids. However,
to enhance the surface functionality of gold narioparticles, particularly for biomedical applications,
further research is necessary to develop techniques that introduce specific functional groups to the
20 nanoparticle surface.
[0004) Functionalizing gold nanoparticles with carboxyl groups provides a versatile
platform for various applications, especially in targeted drug delivery, biosensing, and protein
binding. The carboxyl group (-COO H) is highly reactive and can form stable covalent bonds with
amine-containing molecules. Thus, carboxylated AuNPs are of particular interest for applications
25 that require the conjugation of proteins, peptides, or drugs.
[0005) Thus, there is a need for an efficient process of synthesizing gold nanoparticles that
are functionalized with carboxyl groups, using oxalic acid and L-ascorbic acid.
OBJECTIVES OF THE INVENTION
--::1' 30 [0006) The primary objective of this invention is to develop a process for synthesizing
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functionalized gold nanoparticles (AuNPs). Specifically, the invention focuses on the use of oxalic
acid and L-ascorbic acid to produce gold nanoparticles with an increased number of functionalized
carboxyl groups on their surface. These carboxyl-functionalized AuNPs offer enhanced surface
properties that facilitate the attachment of various amine-based compounds, such as proteins,
5 peptides, and drug molecules. The method can be used in a wide array of applications, including
biosensors, drug delivery systems, diagnostics, and bioimaging.
SUMMARY
(0007] In v1ew of the foregoing, an embodiment herein provides a nanoparticle
composition, comprising a gold nanoparticle that is synthesized by the reduction of gold chloride
I 0 trihydrate (1-!AuCI. · 31-!,0) using oxalic acid (C,H,O.) as a reducing agent and L-ascorbic acid
(C.HsO•) as a stabilizing agent, wherein a functionalized surface of the gold nanoparticle with
carboxyl groups (-COOI-I) is derived from oxalic acid and L-ascorbic acid, wherein the surface of
the gold nanoparticles has an elevated number of functionalized carboxyl groups, enabling
enhanced attachment of amine-based compounds, wherein the gold nanoparticles has a quasi-
15
20
25
30
spherical morphology with an average size of about I 0 nanometers (nm).
(0008] In an embodiment, the concentration of oxalic acid employed for the reduction is
about 0.1 M. In another embodiment, the concentration of L-ascorbic acid employed for
stabilization is about 0.5 M.
[0009] In yet another embodiment, the average size of the synthesized gold nanoparticles
is approximately I 0 nm. In yet another embodiment, the amine-based compound includes proteins,
peptides, or drug molecules, wherein the nanoparticle composition exhibits a quasi-spherical
morphology. In yet another embodiment, the gold nanoparticles are characterized by their negative
zeta potential, indicating electrostatic stability.
[00 I OJ In one aspect, an embodiment herein provides a process for prepanng gold
nanoparticles functionalized with carboxyl groups. The process includes the steps of: (i) dissolving
gold chloride trihydrate (1-!AuCI. · 31-!,0) in distilled water to form a gold chloride solution; (ii)
heating the gold chloride solution to about 70°C while continuously stirring; (iii) adding 0.1 molar
(M) of oxalic acid to the heated gold chloride solution, wherein oxalic acid acts as a reducing
agent, reducing Au'' ions into elemental gold (Au0
) and forming gold nanoparticles; (iv) adding
0.5 M L-ascorbic acid to the solution as a stabilizing agent, which prevents the aggregation of the
gold nanoparticles and enhances their stability by functionalizing the surface with carboxyl groups
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(-COOH); (v) continuing the stirring process to allow complete reduction and stabilization of the
gold nanoparticles; (vi) isolating the gold nanoparticles by centrifugation and rinsing with distilled
water to remove impurities; and (vii) drying and storing the gold nanoparticles, wherein the
resulting gold nanoparticles exhibit a quasi-spherical morphology and are functionalized with
5 carboxyl groups, enabling improved binding capabilities for amine-based molecules, such as
proteins, peptides, or drugs.
(00 I I] In an embodiment, the temperature of the gold chloride solution is maintained at
approximately 70°C during the reduction and stabilization process. The gold nanoparticles may be
separated by centrifugation at 12,000 rpm and stored at 4°C after drying. In another embodiment,
I 0 he synthesized gold nanoparticles arc rinsed with high-performance liquid chromatography
(HPLC) grade water to remove residual impurities. The carboxyl-functionalized gold
nanoparticles may be capable of binding with amine-based compounds, facilitating their
application in drug delivery, biosensing, or bioimaging. The process further comprises the step of
stirring the solution at 540 rpm during the addition of oxalic acid and L-ascorbic acid.
15 [00 12] The use of oxalic acid and L-ascorbic ~cid allows for a high density of carboxyl
N 20
groups (-COOH) on the surface of the gold nanoparticles. This enhanced surface functionalization
increases the ability of the nanoparticles to bind to amine-based compounds, such as proteins,
peptides, or drugs, making them highly effective for targeted biological applications. The gold
nanoparticles exhibit a quasi-spherical morphology with a controlled average size of
approximately I 0 nanometers. This uniform size and shape are advantageous for consistent
behavior in biological environments, such as in drug delivery or bioimaging. The stabilization of
the nanoparticles by L-ascorbic acid prevents aggregation, ensuring that the nanoparticles remain
stable in solution, which is crucial for applications in biomedical and pharmaceutical fields. The
gold nanoparticles possess a negative zeta potential, which indicates strong electrostatic stability.
This reduces the likelihood of nanoparticle aggregation in various environments, making them
suitable for long-term storage and use in biological applications.
E....
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30
(00 13) The described process of synthesis is straightforward, involving simple and
commonly available reagents like gold chloride trihydrate, oxalic acid, and L-ascorbic acid. The
process can be e_asily scaled up and is reproducible, making it suitable for industrial or large-scale
production of functionalized nanoparticles. The process also includes easy isolation techniques
like centrifugation, ensuring that the nanoparticles can be efficiently separated and purified. The
4
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functionalized surface of the gold nanoparticles makes them suitable for a wide range of
applications, including drug delivery, biosensing, and bioimaging. The ability to bind amine-based
molecules allows for the creation of nanoparticle-based delivery systems for drugs or other
therapeutic agents. In biosensing, the functionalized nanoparticles can be used for the detection of
5 biomolecules, while in bioimaging, they can serve as contrast agents due to their unique optical
properties.
(0014) Gold nanoparticles are known for their biocompatibility, meaning they ~re less
likely to cause adverse reactions when used in biological systems. The functionalization with
carboxyl groups enhances their compatibility for use in medical applications such as targeted
I 0 therapies or diagnostics. The use of mild reagents like oxalic acid and L-ascorbic acid, both of
which are naturally occurring and non-toxic, minimizes the risk of introducing harmful substances
into the synthesis process. This leads to a safer and more environmentally friendly production
method. The functionalized surface can be further modified to attach various molecules, allowing
customization for specific applications. This flexibility is crucial for developing tailored solutions
15 in nanomedicine, including targeted drug delivery systems, biosensors, or imaging agents for
specific diseases.
(00 15) These and other aspects of the embodiments herein will be better appreciated and
understood when considered in conjunction with the following description and the accompanying
drawings. It should be understood, however, that the following descriptions, while indicating
20 preferred embodiments and numerous specific details thereof, are given by way of illustration and
not of limitation. Many changes and modifications may be made within the scope of the
embodiments herein without departing from the spirit thereof, and the embodiments herein include
all such modifications.
BRIEF DESCRIPTION OF THE ORA WINGS
25 [0016] The embodiments herein will be better understood from the following detailed
description with reference to the drawings, in which:
(00 17) FIG. I illustrates a process of preparing gold nanoparticles functionalized with
carboxyl groups according to an embodiment herein;
(00 I R) FIG. 2 illustrates a UV-Visible absorption spectrum of the carboxyl-functionalized
30 gold nanoparticles (AuNPs-COOH) according to an embodiment herein;
5
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[00 19) FIG. 3 illustrates an XRD pattern of the carboxyl-functionalizcd gold nanoparticles
(AuNPs-COOH) according to an embodiment herein;
[0020) FIG. 4 illustrates a FTIR spectrum of the carboxyl-functionalized gold
nanoparticles (AuNPs-COOH) according to an embodiment herein;
[0021) FIG. 5 illustrates a Zeta potential distribution of the carboxyl-functionalized gold
nanoparticles (AuNPs-COOH) according to an embodiment herein;
[0022) FIG. 6 illustrates an EDX analysis of the carboxyl-functionalized gold
nanoparticles (AuNPs-COOH) according to an embodiment herein; and
[0023) FIG. 7 illustrates HRTEM images (A-C) and a SAED pattern (D) of the carboxy II
0 functionalized gold nanoparticles (AuNPs-COOH) according to an embodiment herein.
15
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0024) The embodiments herein and the various features and advantageous details thereof
are explained more fully with reference to the non-limiting embodiments that are illustrated in the
accompanying drawings and detailed in the following description. Descriptions of well-known
components and processing techniques are omitted so as to not unnecessarily obscure the
embodiments herein. The examples used herein are intended merely to facilitate an understanding
of ways in which the embodiments herein may be practiced and to further enable those of skill in
the art to practice the embodiments herein. Accordingly, the examples should not be construed as
20 limiting the scope of the embodiments herein.
[0025) As mentioned, there remains a process for synthesizing functionalized gold
nanoparticles (AuNPs). Specifically, the invention focuses on the use of oxalic acid and L-ascorbic
acid to produce gold nanoparticles with an increased number offunctionalized carboxyl groups on
their surface. These carboxyl-functionalized AuNPs offer enhanced surface properties that
25 facilitate the attachment of various amine-based compounds, such as proteins, peptides, and drug
molecules. The method can be used in a wide array of applications, including biosensors, drug
delivery systems, diagnostics, and bioimaging.
[0026] Embodiments herein provides a nanoparticle composition. The nanoparticle
composition includes a gold nanoparticle that is synthesized by the reduction of gold chloride
30 trihydrate (HAuCI. · 3H,O) using oxalic acid (C,H,O.) as a reducing agent and L-ascorbic acid
6
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(C.HsO•) as a stabilizing agent. A functionalized surface of the gold nanoparticle with carboxyl
groups (-COOH) is derived from oxalic acid and L-ascorbic acid. The surface of the gold
nanoparticles has an elevated number of functionalized carboxyl groups, enabling enhanced ·
attachment of amine-based compounds. The gold nanoparticles have a quasi-spherical morphology
5 with an average size of about I 0 nanometers (nm).
[0027] In an embodiment, the concentration of oxalic acid employed for the reduction is
about 0.1 M. In another embodiment, the concentration of L-ascorbic acid employed for
stabilization is about 0.5 M. In yet another embodiment, the average size of the synthesized gold
nanoparticles is approximately I 0 nm. In yet another embodiment, the amine-based compound
I 0 includes proteins, peptides, or drug molecules, wherein the nanoparticle composition exhibits a
quasi-spherical morphology. In yet another embodiment, the gold nanoparticles are characterized
by their negative zeta potential, indicating electrostatic stability.
15
20
25
30
[0028] The use of oxalic acid and L-ascorbic acid allows for a high ·density of carboxyl
groups (-COOH) on the surface of the gold nanoparticles. This enhanced surface functionalization
increases the ability of the nanopartjcles to bind to amine-based compounds, such as proteins,
peptides, or drugs, m'aking them highly effective for targeted biological applications. The gold
nanoparticles exhibit a quasi-spherical morphology with a controlled average size of
approximately I 0 nanometers. This uniform size and shape are advantageous for consistent
behavior in biological environments, such as in drug delivery or bioimaging. The stabilization of
the nanoparticles by L-ascorbic acid prevents aggregation, ensuring that the nanoparticles remain·
stable in solution, which is crucial for applications in biomedical and pharmaceutical fields. The
gold nanoparticles possess a negative zeta potential, which indicates strong electrostatic stability.
This reduces the likelihood of nanoparticle aggregation in various environments, making them
suitable for long-term storage and use in biological applications.
[0029] Gold nanoparticles are known for their biocompatibility, meaning they are less
likely to cause adverse reactions when used in biological systems. The functionalization with
carboxyl groups enhances their compatibility for use in medical applications such as targeted
therapies or diagnostics. The use of mild reagents like oxalic acid and L-ascorbic acid, both of
which are naturally occurring and non-toxic, minimizes the risk of introducing harmful substances
into the synthesis process. This leads to a safer and more environmentally friendly production
method. The functionalized surface can be further modified to attach various molecules, allowing
7
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customization for specific applications. This flexibility is crucial for developing tailored solutions
in nanomedicine, including targeted drug delivery systems, biosensors, or imaging agents for
specific diseases.
(0030] FIG. illustrates a process of preparing gold nanoparticles functionalized with
5 carboxyl groups according to an embodiment herein. At a step I 02, gold chloride trihydrate
(HAuCJ. · 3Hz0) is dissolved in distilled water to create a gold chloride solution. At a step I 04,
this solution is then heated to around 70°C while continuously stirred. At a step I 06, Oxalic acid,
at a concentration ofO.l M, is added to the heated solution. Acting as a reducing agent, oxalic acid
converts Au'• ions into elemental gold (Au0
), resulting in the forrnation of gold nanoparticles. At
I 0 a step I 08, 0.5 M L-ascorbic acid is introduced as a stabilizing agent. This prevents the aggregation
of the nanoparticles and enhances their stability by functionalizing the surface with carboxyl
groups (-COOH). Stirring is continued to ensure complete reduction and stabilization of the
nanoparticles. The gold nanoparticles are centrifuged at a step II 0 and rinsed with distilled water
to remove impurities at a step 112 to isolated the gold nanoparticles at a step 114. Finally, the gold
15 nanoparticles (which is in pinkish white brown) are dried and stored. These nanoparticles exhibit
a quasi-spherical morphology and are functionalized with carboxyl groups, which improve their
binding capabilities with amine-based molecules such as proteins, peptides, or drugs.
(0031] The gold nanoparticles are subsequently separated through centrifugation at 12000
rpm for 4°C. The precipitate is carefully rinsed with HPLC water to eliminate any other impurities
20 and stored at 4°C after drying.
25
30
Materials and Methods
[0032] Gold (Ill) chloride trihydrate (520918) (~99.9%), L-ascorbic acid (A4403), and
oxalic acid (241172) ~99% are purchased from Sigma- Aldrich and Bradford Reagent (cat. No
ML 178-1 PK) from Himedia. All studies and dilutions are conducted using sterile deionized (DI)
water with a resistivity of around -18.2 M, unless specified otherwise.
Synthesis of Carboxylated Gold Nanoparticles
[0033] AuNPs are synthesized from Gold(lll) chloride trihydrate (HAuCI~ · 3H20) by
chemical reduction method ofTurkevich (Turkevich et al., I '151) with minor moditication. Brietly,
500!11 of I% HAuCidl·hO is added to 50ml of dis. H20 with the constant temperature 70°C and
allowed for continuous stirring (5mins). Add 2m I ofO.I M C2H20• to the gold solution and allowed
8
ZF·ATFNT ~- ..... . ! l
0
-Q)
C)
Ill
D..
Q)
-1- N
E....
0
-LL. .....
-::1' en
M
CIO
0 .....
-::1'
-::1'
N
0
~
-::1'
M
-::1'
N
-.M... . -::1'
N
0
~
for 'vigorous stirring for 20mins (540rpm). Additionally, lml of 0.5M C6Hs06 is added to the
aqueous solution.
AuNPs Characterization
[0034) Epoch BioTek instrument is used to measure the UV- visible absorbance spectra
5 of AuNPs-COOH. The gold nanoparticles are subjected to FTIR spectrum measurement using an.
FTIR interferometer (FTIR-JASC04600), XRD pattern (RIGAKU ULTIMA IV), EDX (Quantum
FEG) and Zeta-potential (Anton Paar) analysis. TEM studies are conducted using a Hitachi (Model
H-7500) transmission electron microscope operating at 80 kV for AuNPs-COOH. For the
preparation of samples, a small amount of AuNPs-COOH is carefully placed onto carbon-coated
I 0 TEM grids and left to air-dry for 5 minutes at room temperature before evaluation. A particle size
(lmageJ) is used to examine the distribution of AuNPs-COOH particle sizes.
15
[0035) The use of oxalic acid and L-ascorbic acid allows for a high density of carboxyl
groups (-COOH) on the surface of the gold nanoparticles. This enhanced surface functionalization
increases the ability of the nanoparticles to bind to amine-based compounds, such as proteins,
peptides, or drugs, making them highly effective for targeted biological applications. The gold
nanoparticles exhibit a quasi-spherical morphology with a controlled average size of
approximately I 0 nanometers. This uniform size and shape are advantageous for consistent
behavior in biological environments, such as in drug delivery or bioimaging. The stabilization of
the nanoparticles by L-ascorbic acid prevents aggregation, ensuring that the nanoparticles remain
20 stable in solution, which is crucial for applications in biomedical and pharmaceutical tields. The
gold nanoparticles possess a negative zeta potential, which indicates strong electrostatic stability.
This reduces the likelihood of nanoparticle aggregation in various environments, making them
suitable for long-term storage and use in biological applications.
[0036] The described process of synthesis is straightforward, involving simple and
25 commonly available reagents like gold chloride trihydrate, oxalic acid, and L-ascorbic acid. The
process can be easily scaled up and is reproducible, making it suitable for industrial or large-scale
production of functionalized nanoparticles. The process also includes easy isolation techniques
like centrifugation, ensuring that the nanoparticles can be efficiently separated and purified. The
functionalized surface of the gold nanoparticles makes them suitable for a -.yide range of
30 applications, including drug delivery, biosensing, and bioimaging. The ability to bind amine-based
.9
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0
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C)
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5
molecules allows for the creation of nanoparticle-based delivery systems for drugs or other
therapeutic agents. In biosensing, the functionalized nanoparticles can be used for the detection of
biomolecules, while in bioimaging, they can serve as contrast. agents due to their unique optical
properties.
(0037] Gold nanoparticles are known for their biocompatibility, meaning they are less
likely to cause adverse reactions when used in biological systems. The functionalization with
carboxyl groups enhances their compatibility for use in medical applications such as targeted
therapies or diagnostics. The use of mild reagents like oxalic acid and L-ascorbic acid, both of
which are naturally occurring and non-toxic, minimizes the risk of introducing harmful substances
I 0 into the synthesis process. This leads to a safer and more environmentally friendly production
method. The functionalized surface can be further modified to attach various molecules, allowing
cust6mization for specific applications. This flexibility is crucial for developing tailored solutions
in nanomedicine, including targeted drug delivery systems, biosensors, or imaging agents for
speci fie diseases.
15 [0038] FIG. 2 illustrates a UV-Visible absorption spectrum of the carboxyl-functionalized
gold nanoparticles (AuNPs-COOH) according to an embodiment herein. The reduction of
HAuCk3H20 to gold nanoparticles is confirmed by UV-visible spectroscopy. The nanoparticles
formation can be easily identified by the distinct peaks observed around 540 nm, which indicate
the formation of gold nanoparticles. Additionally, the colour change further supports this
20 observation.
25
30
[0039] FIG. 3 illustrates an XRD pattern of the carboxyl-functionalized gold nanoparticles
(AuNPs-COOf-1) according to an embodiment herein. The crystalline nature of as prepared AuNPscOOH
is validated using XRD. The nanoparticle suspension is coated over a glass slide and further
used for characterization. The XRD pattern exhibited peaks that agree with Bragg's reflection of
AuNPs-COOH as reported in preceding study. Further, the XRD spectrum of AuNPs-COOH
nanoparticles shows distinct peaks of cubic phases (ICSD No. 98-005-0861 ). The XRD patterns
are formed at the diffraction peaks at 29 = 38.01, 44.37, 64.54, 77.54, 81.75 which could be
corresponded to the (I I I), (200), (220), (311 ), (222) lattice planes, respectively. These sharp peaks
at 38.0 I, 44.37 and 64.54 confirms that crystalline nature of gold. The average crystallite size is
30nm that is calculated using Debye's Scherer equations,
10
0_ . -- .. - z,t" -·f!;·tt:: N-~-- ·t·-ii-t-=,t-..:}~-- -----,-----
-::1'
0
. . r· ~
-Q)
C)
Ill
D..
Q)
5
D = KA/(f3hkiCOS0) (I),
where D is crystallite size, K known as shape factor, commonly a constant value of 0.9, A. is the
wavelength of Cu Ku, the source radiation (0.15406 nm) and f3hkl is the full width half maximum
of the Bragg peaks with respect to the hkl planes.
(0040] FIG. 4 illustrates a FTIR spectrum of the carboxyl-functionalized gold
nanoparticles (AuNPs-COOH) according to an embodiment herein. In order to determine the likely
molecule responsible for the subsequent expert stabilization of the gold nanoparticles, FTIR
measurements are repeated. In figure, the significant drop in peak intensity is seen between 3800
and 3100 cm·1
, where the absorptions in the 0-H vibrational stretching region are seen at 3399 em·
10 1
• The COOH groups are visible between 1830 and 1360 cm·1
• At 1651 cm·1
, a small amount of
the stretching in the carboxylic group is absorbed. The broad and strong band between I 000 and
500 cm·1 is attributed to the vibration of Au-01-1. This finding clearly indicates that the use of
oxalic acid and L-ascorbic acid in the Au nanoparticle preparation procedure has a major effect
and promotes the development of AuNPs-COOH.
15 [0041] FIG. 5 illustrates a Zeta potential distribution of the carboxyl-functionalized gold
nanoparticles (AuNPs-COOH) according to an embodiment herein. The figure shows the zeta
potential measurement of gold nanoparticles prepared through a chemical reduction method
(Turkevich). Zeta potential is a measure of the electrostatic charge on the surface of particles, and
in this case, it is used to assess the properties of gold nanoparticles. According to the results, the
20 zeta potential of the gold nanoparticles is found to be -14.2 mY in a neutral medium. The negative
zeta potential indicates the presence of negative charges on the surface of the gold nanoparticles.
These negative charges are attributed to the oxalic acid used and L-ascorbic acid in the synthesis
process, which serves a dual role as both a reducing and stabilizing agent for the gold nanoparticles.
The oxalic acid plays a crucial.role in preventing the aggregation and precipitation of the gold
25 nanoparticles over time.
· [0042] FIG. 6 i_llustrates an EDX analysis of the carboxyl-functionalized gold
nanoparticles (AuNPs-COOH) according t<) an embodiment herein. The elemental composition of
the sample is ascertained using energy dispersive X-ray analysis (EDX). A peak approximately at 2.5
keY has been identified as gold according to the graph, and a lesser peak near 2 keY further
30 supports the presence of gold. Between two gold peaks, there exists a minor peak indicating
II
-Q)
C)
Ill
D..
.Q)
carbon, presumably derived from oxalic acid. The examination confirmed the creation of
nanoparticles by revealing the presence ofC and Au in the samples. This suggests that the prepared
sample is very pristine.
[0043] FIG. 7 illustrates HRTEM images (A-C) and a SAED pattern (D) of the carboxyl-
5 functionalized gold nanoparticles (AuNPs-COOH) according to an embodiment herein. The figure
depicts the quasi-spherical morphology and uniformity of the as prepared AuNPs-COOH with
average size of -10 nm. And, the SAED pattern confirms the crystalline nature of the particle.
Formation of functionalized gold nanoparticles:
(0044] Carboxyl based organic compounds have been versatile application m vanous
I 0 fields like nanocomposite, polymer, medical chemistry, diagnostics, chemical sensing etc. In the
nanoparticle formation, oxalic acid act as a surface modifier which enhance the stability and
functionalization of gold particles. Therefore, oxalic acid reduction occurs on HAuCI4.3H20 which
is proposed as:
(0045] When the gold chloride is dispersed in dis.l-hO, gold ions {Au3•) and chloride ions
( 4Cn are released. Further, the reduction reaction of oxalate ions leads to the formation of gold
nanoparticles (Au0). Oxalic acid functions as a reducing as well as capping reagent which will
promote monodispersed formation of gold nanoparticles. Similarly, ascorbic acid is a major
5 compound to reduce the various metals. As a reduction, L-ascorbic acid could bind with gold ions
{Au3•) to form oxidized ascorbic acid and gold nanoparticles (Au0). Additionally, the oxidized
state of ascorbic acid has three carboxyl group which attracts and bind on the surface of gold
nanoparticles. It is observed that the number of carboxyl group (Coo-) may present on the surface
of gold nanoparticles due to the higher reactivity of oxalic acid. As a result, the gold nanoparticle
I 0 surface becomes functionalized due to carboxyl group of oxalic acid and L-ascorbic acid.

[0046] Subsequently, the characterization of functionalized AuNPs is performed. UVspectra
reveals strong absorption at 540nm and this spectrum corresponds to formation of gold
nanoparticles. The purity and crystalline structure of AuNPs can be verified via X-ray diffraction
(XRD), which provides _a general assessment of the particles size, ascertained by the Debye-
20 Scherer equation.
25
30
[0047] Generally, the researchers have been focused on functionalized gold nanoparticles
synthesize with -NH2, -COOH,-SH etc. for targeted drug delivery. On the other hand, the carboxyl
group can make strong interaction on the surface of gold nanoparticles which attract the polar
compound easily. Additionally, AuNPs can bind with proteins based on electrostatic, hydrophobic,
Van der Waals, and coordination force by including effective adsorption and more complex
interactions with protein-based compounds. Anionic groups (-COO-) on the nanoparticles and
cationic charged amino groups (-NH+) on the amino acid residue facilitate the electro-statical
integration towards the drug. Therefore, the surface functional properties of the AuNPs are further
confirmed by FTIR analysis. In our prediction, 0-H stretching vibration mode is observed as broad
peak at 3399.89 cm·1
• One strong peak absorbed at 1651 cm·1 belongs to carboxyl stretching
13
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vibration. This stretching vibration clearly confirmed the existence of carboxyl group on the
surface of AuNPs. Hence, it is anticipated that the particle surface may be negatively charged due
to the presence of carboxylate anions on particle surface.
[0048] In order to validate this, zeta-potential measurements are performed. The average
5 zeta-potential of AuNPs is measured to be -14.2 mY, demonstrating the particles with negatively
charged surface which causes the electrostatic repulsion to prevent the aggregation among the
particles. The elemental peak of C, Au (2.5 KeY) is observed together in EDX analysis. Further,
the TEM image of AuNPs showed monodispersed formation of sphere shape (-IOnm). Many
studies have been focused on colloid!ll metal particles to understand the immune diagnostics which
I 0 is based on the size and chemical properties of the nanoparticles. Most of the researchers reported
that the best immunological effects are achieved by AuNPs in nanospheres shape. Hence,
carboxylated gold nanoparticles could have wide applications in different fields.
15
ADVANTAGES OF THE INVENTION:
[0049] The use of oxalic acid and L-ascorbic acid allows for a high density of carboxyl
~
groups (-COOH) on the surface of the gold nanoparticles. This enhanced surface functionalization
increases the ability of the nanoparticles to bind to amine-based compounds, such as proteins,
peptides, or drugs, making them highly effective for targeted biological applications. The gold
nanoparticles exhibit a quasi-spherical morphology with a controlled average size of
approximately I 0 nanometers. This uniform size and shape are advantageous for consistent
20 behavior in biological environments, such as in drug delivery or bioimaging. The stabilization of
the nanoparticles by L-ascorbic acid prevents aggregation, ensuring that the nanoparticles remain
stable in solution, which is crucial for applications in biomedical and pharmaceutical fields. The
gold nanoparticles possess a negative zeta potential, which indicates strong electrostatic stability.
This reduces the likelihood of nanoparticle aggregation in various environments, making them
25 suitable for long-term storage and use in biological applications.
[0050] The described process of synthesis is straightforward, involving simple and
commonly available reagents like gold chloride trihydrate, oxalic acid, and L-ascorbic acid. The
process can be easily scaled up and is reproducible, making it suitable for industrial or large-scale
production of functionalized nanoparticles. The process also inclu(Jes easy isolation techniques
30 like centrifugation, ensuring that the nanoparticles can be efficiently separated and purified. The
14
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functionalized surface of the gold nanoparticles makes them suitable for a wide range of
applications, including drug delivery, biosensing, and bioimaging. The ability to bind amine-based
molecules allows for the creation of nanoparticle-based delivery systems for drugs or other
therapeutic agents. In biosensing, the functionalized nanoparticles can be used for the detection of
5 biomolecules, while in bioimaging, they can serve as contrast agents due to their unique optical
properties.
[0051] Gold nanoparticles are known for their biocompatibility, meaning they are less
likely to cause adverse reactions when used in biological systems. The functionalization with
carboxyl groups enhances their compatibilitY. for use in medical applications such as targeted
I 0 therapies or diagnostics. The use of mild reagents like oxalic acid and L-ascorbic acid', both of
which are naturally occurring and non-toxic, minimizes the risk of introducing harmful substances
into the synthesis process. This leads to a safer and more environmentally friendly production
method. The functionalized surface can be further modified to attach various molecules, allowing
customization for specific applications. This flexibility is crucial for developing tailored solutions
15 in nanomedicine, including targeted drug delivery systems, biosensors, or imaging agents for
speci fie diseases.
[0052] The foregoing description of the specific embodiments will so fully reveal the
general nature of the embodiments herein that others can, by applying current knowledge, readily
modify and/or adapt for various applications such specific embodiments without departing from
20 the generic concept, and, therefore, such adaptations and modifications should and are intended to
be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is
to be understood that the phraseology or terminology employed herein is for the purpose of
description and not of limitation. Therefore, while the embodiments herein have been' described in
terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein
25 can be practiced with modification within the scope of the appended claims.



1/We claim:
I. A nanoparticle composition, comprising:
· characterized in that, a gold nanoparticle that is synthesized by the reduction of gold
chloride trihydrate (HAuCI. · 3Hz0) using oxalic acid (C,H:,O.) as a reducing agent and L-ascorbic
acid (CoHsOo) as a stabilizing agent, wherein a functionalized surface of the gold nanoparticle with
5 carboxyl groups (-COOH) is derived from oxalic acid and L-ascorbic acid, wherein the surface of
the gold nanoparticles has an elevated number of functionalized carboxyl groups, enabling
enhanced attachment of amine-based compounds, wherein the gold nanoparticles has a quasispherical
morphology with an average size of about I 0 nanometers (nm).
I 0 2. The nanoparticle composition as claimed in claim I, wherein the concentration of oxalic acid
empl.oyed for the reduction is about 0.1 M.
15
20
3. The nanoparticle composition as claimed in claim I, wherein the concentration of L-ascorbic
acid employed for stabilization is about 0.5 M.
4. The nanoparticle composition as claimed in claim I, wherein the average size of the synthesized
gold nanoparticles is approximately I 0 nm.
5. The nanoparticle composition as claimed in claim I, wherein the amine-based compound
includes proteins, peptides, or drug molecules, wherein the nanoparticle composition exhibits a
quasi-spherical morphology.
6. The nanoparticle composition as claimed in claim I, wherein the gold nanoparticles are
characterized by their negative zeta potential, indicating electrostatic stability.
-C))
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7. A process for preparing gold nanoparticles functionalized with carboxyl groups, comprising the
steps of:
dissolving gold chloride trihydrate (HAuCI. · 3H,O) in distilled water to form a gold chloride
solution;
5 heating the gold chloride solution to about 70°C while continuously stirring;
adding 0.1 molar (M) of oxalic acid to the heated gold chloride solution, wherein oxalic acid
acts as a reducing agent, reducing Au'• ions into elemental gold (Au0
) and forming gold
nanoparticles;
adding 0.5 M L-ascorbic acid to the solution as a stabilizing agent, which prevents the
I 0 aggregation of the gold nanoparticles and enhances their stability by functionalizing the surface
with carboxyl groups ( -COOH);
15
continuing the stirring process to allow complete reduction and stabilization of the gold
nanoparticles;
isolating the gold nanoparticles by centrifugation and rinsing with distilled water to remove
impurities; and
drying and storing the gold nanoparticles, wherein the resulting gold nanoparticles exhibit a
quasi-spherical morphology and are functionalized with carboxyl groups, enabling improved
binding capabilities for amine-based molecules, such as proteins, peptides, or drugs.
20 8. The process as claimed in claim 7, wherein the temperature of the gold chloride solution is
maintained at approximately 70°C during the reduction and stabilization process, wherein the gold
nanoparticles are separated by centrifugation at 12,000 rpm in 4°C and stored at 4°C after drying.
9. The process as claimed in claim 7, wherein the synthesized gold.nanoparticles are rinsed with
25 high-performance liquid chromatography (HPLC) grade water to remove residual impurities,
wherein the carboxyl-functionalized gold nanoparticles are capable of binding with amine-based
compounds, facilitating their application in drug delivery, biosensing, or bioimaging .

I 0. The process as claimed in claim I, wherein the process further comprises the step of stirring
the solution at 540 rpm during the addition of oxalic acid and L-ascorbic acid.

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