Journal Pre-proof

Reduced graphene oxide-gadolinium oxide-functionalized paper based

immunosensor for electrochemical detection of gentamicin

Jayendra Kumar Himanshu, G.B.V.S. Lakshmi, Akhilesh Kumar Singh, Pratima R.

Solanki

PII: S2590-1370(24)00006-2
DOI: https://doi.org/10.1016/j.biosx.2024.100442
Reference: BIOSX 100442

To appear in: Biosensors and Bioelectronics: X

Received Date: 9 October 2023

Revised Date: 10 January 2024
Accepted Date: 16 January 2024

Please cite this article as: Himanshu, J.K., Lakshmi, G.B.V.S., Singh, A.K., Solanki, P.R., Reduced
graphene oxide-gadolinium oxide-functionalized paper based immunosensor for electrochemical
detection of gentamicin, Biosensors and Bioelectronics: X (2024), doi: https://doi.org/10.1016/
j.biosx.2024.100442.

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© 2024 Published by Elsevier B.V.

 Reduced graphene oxide-gadolinium oxide-functionalized paper based
immunosensor for electrochemical detection of gentamicin
Jayendra Kumar Himanshu1,2, G.B.V.S. Lakshmi1, Akhilesh Kumar Singh2, Pratima R.
Solanki1*
1
Special Centre for Nanoscience, Jawaharlal Nehru University (JNU), New Delhi-110067,
India.
2
Department of Biotechnology, School of Life Sciences, Mahatma Gandhi Central University,
Motihari, Bihar-845401, India.

*Corresponding author email: partima@mail.jnu.ac.in; pratimarsolanki@gmail.com

Abstract

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Excessive consumption of antibiotics like gentamicin (GEN) can lead to hostile effects as
antibiotic resistance. Therefore, the detection is important for which, reduced graphene oxide-

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Gadolinium oxide nanocomposite (rGO@Gd2O3 NC) was composed through co-precipitation
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method by using of rGO for the detection of GEN. The structural, morphological and functional
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group characterization were done using XRD, FT-IR, SEM and TEM techniques. The cyclic
voltammetry (CV) shows excellent electrocatalytic activity and superior performance
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towards GEN detection. Through the use of GEN monoclonal antibodies (anti-GEN) on
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a screen-printed electrode (SPE), a very sensitive electrochemical immunosensor was

fabricated. Covalent interactions were employed to construct the electrochemical
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immunosensor, while bovine serum albumin (BSA) was employed as a blocking agent on the
anti-GEN/rGO@Gd2O3/SPE electrode surface. The analysis of the CV response of the
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BSA/anti-GEN/rGO@Gd2O3/SPE bioelectrode demonstrated linear detection range from 1 pM

– 100 µM, along with limit of detection (LOD) of 0.424 pM and sensitivity of 44.87 µA pM-1
cm− 2. Additionally, rGO@Gd2O3 immunosensor, exhibited a good level of linearity with R2
value of 0.981. These findings indicate the excellent potential of the rGO@Gd2O3
electrochemical immunosensor for accurately detecting GEN in milk samples containing
spiked concentrations.

Keywords: - Gadolinium oxide; reduced graphene oxide; gentamicin; Screen-printed electrode.

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1. Introduction
Gentamicin (GEN), a type of aminoglycoside antibiotic, is frequently employed in the
treatment of bacterial infections, particularly those brought by gram-negative organisms. It
works by preventing the production of bacterial proteins to produce an antibacterial action [1].
The permissible limits of GEN in food products are primarily regulated by governmental
agencies in the United States, Food and Drug Administration (FDA) and in European Union,
European Food Safety Authority (EFSA) responsible for food safety. These regulatory bodies
establish Maximum Residue Limits (MRLs) for various veterinary drugs, including GEN, in
order to safeguard public health and ensure that the consumption of food products does not
pose undue risks due to the presence of harmful substances. In the European Union, provisional

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maximum residue levels for aminoglycoside antibiotics have been established at 200 µg/kg [2].
The European Authority for the Safety of Medical Products has stipulated a MRL of 100 ng/mL

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for GEN in milk [3]. Antibiotics are frequently given to animals, in animal husbandry and
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agricultural production to treat diseases in cattle, swine, and poultry [4]. These antibiotics will
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be consumed by humans in a certain amount from farm products, including milk, eggs, and
meat. However, excessive consumption of GEN can lead to ill effects like allergies, hearing
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loss and renal toxicity [5,6]. Therefore, it is essential to develop an efficient, affordable, and
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simple method for detecting GEN content in food. Researchers have investigated a lot of
technologies to detect antibiotics, including GEN, to determine the amount of antibiotics
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present in food and to ensure that the diet of humans is safe. Capillary electrophoresis [7],
liquid chromatography-mass spectrometry (LC-MS) [6], enzyme-linked immunosorbent assay
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[8], instance spectrofluorimetry [9] and high-performance liquid chromatography (HPLC)

[10,11] are among the conventional techniques. The aforementioned techniques detecting
antibiotics has advantages of its own, such as a high level of sensitivity for HPLC as well as
outstanding efficacy for LC-MS, but their practical use is constrained by complicated
processes, pricey equipment, and time-consuming steps. Therefore, alternate techniques to
create quicker, less expensive, and more precise technologies are needed. Electrochemical
nano-biosensors are one of the alternatives. Among various nanomaterials to be used in nano-
biosensors, the lanthanides family has displayed beneficial uses in different disciplines as
catalysis [12], adsorption [13], and energy storage [14] owing to its unique properties, such as
thermal stability, semiconducting, fluorescent and paramagnetic in nature. Gd (III), a member
of the lanthanide series, has seven unpaired electrons and exhibits significant paramagnetism;
as a result, it is used in optical applications, magnetic probes, and magnetic resonance

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imagining [15,16]. Among them rare earth oxide such as gadolinium and its oxide (Gd2O3)
used because of good thermal and chemical stability with large bandgap around 5.4 eV [17].
In addition, it also shows unique features like specific heat capacity, semi conductivity, low
toxicity, and biocompatibility but exhibits certain issue including electrode instability,
moderate sensitivity, and selectivity. Therefore, to overcome this problem integration of carbon
and its compounds with Gd2O3 enhances selectivity, sensitivity and stability. Carbon
compound as reduced graphene oxide (rGO) serves as a brilliant catalyst material demonstrates
its exceptional performance across multiple domains including supercapacitor [18], lithium-
ion batteries [19], photo degradation [20], and electrochemical studies [21–23]. Integration of
rGO with Gd2O3 show highly conductive, enhance surface area, chemical stability and thermal

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stability, biocompatibility, cost-effective, and simple synthesis process. Using an L-cysteine

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functionalized MoS2@MWCNT nanocomposite, Amit Kumar Yadav et al. reported that an

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immunosensing platform for the detection of GEN with the range of 1x10-6 - 40 mg/mL with
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LOD 0.039 mg/mL [24]. Elmorsy Khaled and colleagues conducted a potentiometric method
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for detecting GEN, demonstrating a linear range of 10-7 to 10-2 mol L-1 and LOD of 7.5 × 10-8
mol L-1 through the utilization of screen-printed sensors based on Calixarene/Carbon nanotubes
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[25]. In other report, Miranda-Andrades and colleagues reported a calorimetric sensor for the
detection of GEN with detection of limit of 0.4 ng mL−1 using spherical gold nanoparticles and
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gold nanorods [26]. A chemosensor designed for the detection of GEN exhibited a linear range
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from 0 – 100 × 103 nM and LOD 1280 nM, when employed silver nanoparticles coated with
epicatechin [27], was reported. Joshua C. Gukowsky and collaborators documented a
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colorimetric sensor capable of detecting GEN within the range 10 – 200 nM, LOD 12.45 nM,
by employing cysteamine-modified Au nanoparticles [28]. A colorimetric sensor for detection
of GEN with linear range from 120 to 280 nM using Au nanoparticles [29] was reported by
Shang Yan et al.

The purpose of this work is to fabricate a simple immunosensor based on disposable paper
SPEs modified with rGO@Gd2O3 NC for highly sensitive GEN detection over wide range 1
pM to 100 M. Our studies show good sensitivity and selectivity towards GEN antibiotics
detection. Our work approach is straightforward and affordable, involving the direct
immobilization of an antibody onto the active surface area of disposable SPEs. The
establishment of a stable amide bond results from covalent binding between the amino terminal
of the antibody and the carboxylated functional group on rGO, facilitated by EDC/NHS
coupling. This innovative immunosensor, based on rGO@Gd2O3, demonstrates exceptional

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sensitivity, an enhanced detection limit, prolonged storage stability, highlighting the antibody's
strong affinity for the antigen. The applicability in the real sample testing was confirmed by
testing spiked milk samples.

2. Experimental
2.1. Reagents
Gadolinium (III) nitrate was purchased from central drug house (P) Ltd. 1-(3-(dimethylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (EDC), Graphite powder, cholesterol, glucose, Vit-
C (ascorbic acid) and Bovine serum albumin (BSA) was purchased from Sigma Aldrich.
Ethanol and hydrogen peroxide (H2O2) were bought from Fisher Scientific. N-
hydroxysulfosuccinimide (NHS) and sodium phosphate monobasic anhydrous (NaHPO4),

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ciprofloxacin, sodium phosphate dibasic dihydrate (NaH2PO4), ofloxacin, potassium
ferrocyanide, sodium hydroxide pellets (NaOH), potassium ferricyanide, norfloxacin,

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gentamicin (GEN) were obtained from Hi-Media and SRL Limited respectively. Every
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chemical was used as an analytical grade without any treatment. To create a fresh 0.2 M
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phosphate buffer solution (PBS) with a pH of 7.4, a mixture of NaH2PO4 and NaH2PO4 was
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prepared using DI water, and the storage of solution was done at 4°C for further
experimentation. The gentamicin antibodies (anti-GEN) were purchased from MyBio-Source,
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USA. The preparation of dilution was carried out at pH 7.4 in PBS. All experiment was
performed in Distilled water obtained from PURELAB pulse water purification equipment.
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2.2. Synthesis of rGO@Gd2O3 NC

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Gadolinium (III) nitrate (0.116 M) used as a precursor for the formation of Gd2O3 NC with use
of sodium hydroxide (NaOH) (0.58 M) as a reducing agent for the synthesis of Gd2O3 by Co-
precipitation method. NaOH was added dropwise to gadolinium (III) nitrate solution under
constant stirring of 500 rpm till the pH reached to ~12. The precipitate was collected using
Whatman filter paper and then repeatedly washed with D.I. water to achieve a pH of
approximately 7. After filtration, the obtained precipitate was subjected to drying overnight at
80°C in an oven, followed by grinding in a mortar and pestle to achieve a fine powder of
gadolinium hydroxide. The resulting powder was then calcined for 2 hr at 350°C to obtain
Gd2O3.

Co-precipitation
Gd(NO3) 3 + 3NaOH Gd(OH)3 + 3NaNO3…………………………...(1)

2Gd(OH)3 Gd2O3 + 3H2O……………………………………...………(2)

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Graphene oxide (GO) was synthesized by modified Hummer’s method using graphite powder.
A mixture of H2SO4 /H3PO4 is added to graphite powder. After that, KMnO4 was added to the
above solution very slowly. The reaction is quenched by adding H2O2. A yellowish slurry
mixture was obtained which was then washed with DI water until pH 7 was reached and dried
at 70℃ to obtain solid GO. The reduction process of GO powder involved dispersing of GO
powder (400 mg) in 400 ml of DI water, followed by sonication for 3 hr. Next, 4g ascorbic acid
was added to the mixture while stirring continuously at 500 rpm for 30 min at 60℃. Afterward,
the reduced product was obtained and separated by centrifugation at 4000 rpm for 40 min.
Subsequently, 30 wt% H2O2 was introduced to the resulting black paste for complete oxidation
of ascorbic acid under stirring at 60℃ for 30 min. Once the stirring was completed, the resultant

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black paste was collected and washed three times with ethanol and DI water, respectively and

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finally dried in an oven at 120℃ for 24 hr to obtain rGO. The above-mentioned Gd2O3 and

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rGO were mixed in 10 ml of DI water at 2:1 weight ratio (200 mg, 100 mg respectively) and
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sonicated for 9 hr to disperse the nanomaterial. After that stirred mixture for 4 hrs then the
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resulting paste was dried in an oven at 80°C overnight and grind using a mortar-pestle to get a
fine powder of rGO@Gd2O3, which was then calcined at 650°C for 4 hr. Precursor
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concentration, reaction temperature, its concentration, calcination temperature and reducing

agent type in the post-processing [30] can all parameter affect the size of the rGO@Gd2O3 NC.
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This approach provides numerous benefits, including easy to use and requiring minimal
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equipment.
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2.3. Immunoelectrode on Screen printing electrodes (SPEs) fabrication

The paper-based SPEs were printed using graphite-based ink using the Graflica Flextronica
nano-print 1015 instrument from India, and was allowed to dry. After that sliver paste was
applied on the reference electrode. A hydrophobic insulating material paste is applied to
confine the buffer solution over the sensing surface. Freshly prepared 2:1:1 ratio solution at 50
µg/ml Anti-GEN in PBS buffer, 0.4 M EDC, and 0.1 M NHS were kept for 1 hr at 4°C.
rGO@Gd2O3/SPE electrode was coated uniformly with a 5 µL solution of activated Anti-GEN
solution throughout the immobilisation process, it was placed in a humid chamber at 25°C for
a duration of 4 hr. The carbonyl group of Anti-GEN and rGO@Gd2O3 interact electrostatically
to form a metal ester bond. Using PBS buffer (pH 7.4), the unbound Anti-GEN was washed
from the Anti-GEN/rGO@Gd2O3/SPE bioelectrode surface. 2% BSA (3 µL) was employed to
block non-specific sites on the surface of the bioelectrode (Anti-GEN/rGO@Gd2O3/SPE)
surface, and it was incubated in a humid chamber for a duration of 4 hr at 25°C. Finally, PBS

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buffer (pH 7.4) was used to wash the BSA/Anti-GEN/rGO@Gd2O3/SPE immunoelectrode and
it was kept at 4°C for future research.

2.4. Preparation of GEN dilutions

Various concentration of GEN was prepared in PBS with a pH of 7.4 ranging from 1 pM to
100 µM using serial dilution. The complete electrochemical study was carried out on
immunosensor using the CV technique in 7.4 pH PBS containing redox species. The sensing
was performed by adding the 5 L analyte solution at different concentrations one by one to
the immunoelectrode in presence of PBS containing redox species and measured the
corresponding CV curves. The measurements were conducted at room temperature (25oC). The

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potential range used for CV was -0.8 to +0.8 V. The developed immunosensor was used to the

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test real samples by spiking milk samples.

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2.5. Preparation of real sample
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Milk purchased from the market was used for spiked sample study. The milk sample (100 mL)
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was pre-treated, with 6 mL of 5 M methanol and 1 mL of 20 mM trichloroacetic acid by mixing
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and centrifuged at 10,000 rpm for 15 min at room temperature (RT). The resulting supernatant
was used as the test sample. The detection of GEN was examined in these real samples. The
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volume of analyte used to spike the milk was 5 µl at different concentrations of GEN (0 pM-
100 M). The investigation of the BSA/anti-GEN/rGO@Gd2O3/SPE immunoelectrode sensing
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was carried out utilizing these real spiked samples.

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2.6. Characterization techniques

The developed SPEs system was electrochemically characterised using an Autolab,
Potentiostat/Galvanostat electrochemical analyzer (EcoChemie, The Netherlands) equipment.
In PBS with a pH of 7.4 and 5 mM [Fe(CN)6] 3-/4- as species undergoing redox transformations,
CV was recorded. CV measurements were conducted within a potential range of -0.8 to +0.8
V at a scan rate of 50 mV/s. XRD was employed to gather data on the phase structure and
crystalline characteristics [Rigaku Miniflex 600 (Japan) diffractometer], an X-ray beam
monochromator (λ 1.5406 Å) with radiation of CuKα, and working parameters of 40 kV
voltage and 15 µA current that were conducted in 2θ = 10° to 60° at a scan rate of 5° per
minutes with a step size of 0.02° at room temperature (RT). Using Fourier transform infrared
spectroscopy (FT-IR) in the ATR mode (Perkin Elmer, Spectrum 1 US) with wavenumbers
ranging from 4,000 to 400 cm-1, the functional groups of rGO@Gd2O3 NC were determined.
Scanning electron microscopy (SEM), JSM-IT200 JEOL, Japan, was used to characterise the

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surface morphology of the rGO@Gd2O3 NC. To determine the crystallographic structure of the
rGO@Gd2O3 NC, high-resolution transmission electron microscopy (HR-TEM) (JEOLJEM-
2200 FS, Japan) was used. The overall charge on the surface of rGO@Gd2O3 NC was
determined by using Zeta analyzer (ZEECOM, Microtec Co., LTD., Japan). Dynamic light
scattering (DLS) (LS spectrometer, serial No. LS-0713-0035, Switzerland) was used to
determine the hydrodynamic size of rGO@Gd2O3 NC. X-Ray Photoelectron Spectroscopic
(XPS) (Kratos Analytical LTD. Model AXIS Supra) was used to determine the electronic state
and binding energy of rGO@Gd2O3 NC.

3. Results and Discussion

3.1. Chemical characterization of rGO@Gd2O3/SPE immunoelectrode

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3.1.1. XRD analysis
The X-ray diffraction (XRD) pattern of rGO@Gd2O3 NC was shown below in Fig. 1 (a) after

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calcination at 650°C for 4 h showing its crystalline structure and phase purity. At room
temperature, the XRD peaks were obtained at 2θ values of 19.6°, 28.0°, 32.5°, 34.6°, 42.1°,
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47.0°, 51.6°, 55.8°, and 57.3° corresponding to monoclinic structure of Gd2O3 of planes (002),
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(111), (112), (311), (601), (313), (020), (801), and (313), respectively. There are no other peaks
of rGO were observed in the composite, rGO was decorated with Gd2O3 nanoparticles. The
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observed values for the monoclinic phase of Gd2O3 was matching with JCPDS card no. 43-
1015. The significant crystalline phase of Gd2O3 is demonstrated by the strong, intense, and
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quite well-defined peaks. As the calcination temperature was set at 650°C, the enhancement in
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heights of XRD peaks and the decrement in FWHM was seen. Hence, the XRD analysis
of rGO@Gd2O3 NC affirms the existence of the monoclinic phase of Gd2O3, and with no
impurity peaks. The average crystallite size (D) of Gd2O3 was determined using the Scherrer
formula, resulting in a measurement of 20.84 nm.
D = Kλ/β cos θ…………………………………………….……….…... (3)
In this context, the symbol θ represents the angle of Bragg's diffraction, λ signifies the
wavelength of the target material Cu-Kα (1.540 Å), K denotes the dimensionless shape factor
(0.9), and β represents the full width at half maximum (FWHM) of the diffraction peak.

3.1.2. Fourier transform-infrared spectroscopy study

The FTIR spectra shown in Figure 1 (b) with a wave number range of 4000-400 cm-1 and
correspond to (A) rGO, (B) Gd2O3, and (C) rGO@Gd2O3 NC. Curve (A) demonstrates the
FTIR spectra of rGO, displaying only two peaks at 1211 cm-1 (C-O-H) and 1619 cm-1 (C=C),

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indicating successful reduction of GO. Curve (B) represents the FTIR spectra of Gd2O3,
showing stretching vibrations of -C=O (1487 and 1399 cm-1) and Gd=O (534 cm-1). Curve (C)
represents the FTIR spectra of rGO@Gd2O3 NC, exhibiting vibrations at 1505 cm-1, 1380 cm-
1
, and 493 cm-1, strongly indicating the formation of the rGO@Gd2O3 NC [31].
3.1.3. Zeta potential and hydrodynamic size measurements
Zeta potential measurement was employed to assess the collective surface charge of the
rGO@Gd2O3 NC. Zeta potential (Z) was approximated as the surface potential (ϕ) on a
uniformly charged sphere [32].
Z ≈ φ = 4π(σ/εκ) …………………………...………………………………… (4)
The zeta potential measurement of rGO@Gd2O3 NC showing its anionic characteristics, with

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a recorded value of 12.05 mV, as depicted in Fig. 1 (c) [33,34]. The hydrodynamic diameter of

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rGO@Gd2O3 NC was determined using Dynamic Light Scattering (DLS) analysis, as shown in

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Fig. 1 (d). The analysis revealed that the hydrodynamic size of rGO@Gd2O3 NC measured 172
nm.
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Fig. 1 (a) XRD spectrum of rGO@Gd2O3 NC, (b) FTIR spectra of (A) rGO (B) Gd2O3 and
(C) rGO@Gd2O3, (c) Zeta potential of rGO@Gd2O3 NC and (d) DLS study of rGO@Gd2O3
NC.

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3.1.4. XPS analysis

XPS used for the investigation of surface electronic states, composition, and binding energy of
rGO@Gd2O3 was conducted, as depicted in Fig. 2. The XPS spectra reveal distinctive signals
corresponding to the elements C, O, and Gd consistent with the findings reported in energy-
dispersive X-ray spectroscopy (EDX). In the high-resolution C 1s XPS spectrum, peaks at
281.89 eV, 285.79 eV, and 286.99 eV are discerned, indicating the presence of rGO. Within
the O 1s XPS spectrum, the signal at 526.02 eV and 528.75 eV is attributed to O2-.
Additionally, the Gd 4d XPS spectrum exhibits signals at 138.36 eV, 140.70 eV, and 144.36
eV corresponding to Gd 4d and below 100 eV corresponding to Gd 4f. Consequently, the XPS
analysis provides confirmation of the formation of rGO@Gd2O3.

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Fig. 2. The XPS spectra of (A) rGO@Gd2O3 NC (B) C 1s, (C) O 1s, and (D) Gd 4d.

3.1.5. Study of morphology and elemental composition of rGO@Gd2O3 NC

To find out morphology and elemental composition, SEM-EDX studies were performed. SEM
image shown in Fig. 3 (a) that clearly represents sheets structure of rGO decorated with Gd2O3

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nanoparticles. Since the size of Gd2O3 nanoparticles is too small, they were not visible in the
rGO@Gd2O3 NC image. Therefore, the elemental analysis using EDX was conducted, as
depicted in Fig. 3 (b). EDX confirms the presence of gadolinium, oxygen and carbon atom in
rGO@Gd2O3 NC, without any traces of residue. The atomic percentages and weight were
found to be 55.62 % and 26.02 % for carbon, 35.97 % and 22.42 % for oxygen and 8.42 % and
51.56 % for gadolinium, respectively. The composite material exhibited a homogeneous
distribution of carbon (C) represented in red, oxygen (O) represented in orange, and gadolinium
(Gd) represented in blue, as revealed by the EDX elemental mapping shown in Fig. 3 (c-e).
This confirms the formation of rGO@Gd2O3 NC. TEM was performed to study the
morphological information and crystallinity of rGO@Gd2O3 NC. Figure 3. (f) rGO@Gd2O3

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NC revealed that Gd2O3 nanoparticles decorated on rGO sheets. TEM analysis was indicating

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that successful formation of rGO@Gd2O3 NC with good agreement of SEM and XRD results.

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The selected area electron diffraction (SAED) patterns show clearly ring formation of
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rGO@Gd2O3, which reveals the polycrystalline nature (inset to Fig. 3g). The HRTEM image
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in Fig. 3 (g) shows two different d-spacing planes, in which 0.32 nm corresponds to rGO and
0.24 nm corresponds to Gd2O3 which further confirmed the formation of the NC.
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Fig.3. displays various aspects of the rGO@Gd2O3 NC. These include: (a) SEM image, (b)
EDX spectrum with corresponding quantitative analysis shown in the inset, (c-e) Elemental
mapping illustrating the distribution of different elements within the rGO@Gd2O3 NC, (f)
Low-resolution TEM image of the rGO@Gd2O3 NC, (g) High-resolution TEM image of the
rGO@Gd2O3 NC, and the corresponding selected area electron diffraction (SAED) pattern of
rGO@Gd2O3 NC (inset to Figure g).
3.2. Electrochemical studies of rGO@Gd2O3/SPE electrode

The CV was employed to investigate the electrochemical characteristics of rGO@Gd2O3/SPE,

Anti-GEN/rGO@Gd2O3/SPE and BSA/Anti-GEN/rGO@Gd2O3/SPE electrodes in PBS. The
electrochemical properties of electrodes were studied using CV in PBS. For this, a 50 µL of
ferri/ferrocyanide [Fe(CN)6]3−/4− in PBS pH 7.4 was dropped on SPE as a part of three electrode
system. The CV curves depicted in Fig. 4 (A) demonstrate a notable change in peak current
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along with a shift in peak voltage after the immobilization of Anti-GEN. This observation
signifies the effective binding of Anti-GEN molecules to the electrode surface. The FTIR
studies were done for further confirmation of the immobilization of Anti-GEN and BSA onto
the rGO@Gd2O3/SPE immunoelectrode as shown in Fig. 4 (B). FTIR spectrum of
rGO@Gd2O3/SPE immunoelectrode showing small peaks at 494 cm-1, corresponds to the
Gd=O bond in Fig. 4B (a). A peak at 1066 cm-1 in the FTIR spectrum of the Anti-
GEN/rGO@Gd2O3/SPE electrode indicates the presence of the stretching vibration of the C-N
(C-NH2) bond in primary amine bond. Additionally, there is a distinct peak at 1635 cm-1, which
is attributed to the deformation of the amino acid (NH3+) group, which was shown in Fig. 4B
(b). The results indicate that Anti-GEN has been effectively immobilized onto the

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rGO@Gd2O3/SPE electrode. The FTIR spectrum exhibited no shift in peak wavenumber;

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however, a decrease in intensity indicates nonspecific sites were blocked by BSA as shown in

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Fig. 4B (c). These results confirm the successful immobilization of BSA onto the Anti-
GEN/rGO@Gd2O3/SPE immunoelectrode.
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Fig. 4 (A) Electrode study of (a) rGO@Gd2O/SPE (b) Anti-GEN/rGO@Gd2O/SPE (c)

BSA/Anti-GEN/rGO@Gd2O/SPE (B) FTIR spectra of (a) rGO@Gd2O3/SPE (b) Anti-
GEN/rGO@Gd2O3/SPE (c) BSA/Anti-GEN/rGO@Gd2O3/SPE immunoelectrode.

3.3. Electro-kinetic study

The interfacial kinetics of rGO@Gd2O3/SPE electrodes were examined through electro-kinetic

studies using the CV technique at various scan rates (10 to 100 mVs− 1). The anodic (Ia) and
cathodic (Ic) currents (redox peak currents) exhibited a linear increase with the scan rate, and
their relationship was found to be directly proportional to the square root of the scan rate (ν1/2),

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as depicted in the insets of Fig. 5. Thus, the diffusion-controlled process of the electrochemical
reaction can be justified.

The diffusion coefficient (D) was estimated using Randles–Sevcik equation [35]:

Ip = (2.69 × 105) Cn3/2 D1/2 ν1/2 A

In the given equation, the peak current of the electrode is denoted by I.P., and it is influenced
by various factors including the number of electrons transferred (n), the surface area of the
working electrode (A) measured in cm2, the diffusion coefficient (D) expressed in cm2 s − 1, the
electrolyte concentration (C) set at 5 mM, and ν as a scan rate measured in mVs− 1. The
diffusion coefficient (D) was determined, yielding a calculated value of D = 2.02 x 10-11 cm2 s

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–1

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Fig 5. The electro-kinetic study involved in examining the relationship between the square root
of the scan rate v/s peak current, as well as the square root of the scan rate v/s peak voltage.
The corresponding linearity plot was obtained to visualize these relationships in inset.

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3.4. rGO@Gd2O3/SPE electrodes for electrochemical response study

The electrochemical response study of rGO@Gd2O3/SPE electrodes against GEN were

depicted in Fig. 6 (a). The response study of rGO@Gd2O3/SPE electrodes was performed using
CV in the potential range of − 0.8 to 0.8 V. The concentration range was changed from 1 pM
to 100 µM. As the concentrations of GEN increased, a decrease in the CV peak currents was
observed. The peak currents remained nearly constant at higher concentrations, indicating
electrode saturation in terms of available specific antibody sites. The linear calibration plots of
template concentrations and peak current (ΔI) for rGO@Gd2O3/SPE can be observed in the
insets of Fig. 6 (b).

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Fig. 6. (a) Electrochemical response studies measured using CV method on rGO@Gd2O3/SPE

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electrodes (b) Linear plot of response studies of rGO@Gd2O3/SPE electrodes.

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The sensitivity of the rGO@Gd2O3/SPE electrodes was determined by dividing the slope by
the surface area, resulting in a value of 0.1256 cm2. The sensitivity was 44.87 µA pM-1 cm− 2,
displaying linearity with R2 value of 0.978. Utilizing the calibration plot, the limit of detection
(LOD) was determined, which was found 0.424 pM.

3.5. Interferent, Shelf life, Reproducibility and Spiked studies of milk against BSA/Anti-
GEN/rGO@Gd2O3/SPE immunosensing platform

Fig. 7. (a) illustrates the specificity study of the immunosensor concerning various interferants,
including folic acid, glucose and BSA. The investigation was conducted on the immunosensor
electrodes using CV. The selectivity test involves, 5 µL of each interferants (including
antibiotics such as norfloxacin, ampicillin, amoxicillin, ciprofloxacin, moxifloxacin,
levofloxacin, tetracycline, and ofloxacin, as well as ions like Na+, Mg2+, Ca2+) with 5 µL of
GEN in an electrolyte solution. The response was then observed using CV, and the

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immunoelectrode demonstrated excellent selectivity against the various interferants. Only in
the case of GEN the value of current change in a remarkable way, and in the case of the other
interfering agents, nothing changed.

Fig. 7. (b) demonstrating the long-term stability of the immunosensor. The immunosensor is
essential in order to identify potential measurement drifts caused by aging. The
immunoelectrode's stability was assessed over a period of sixteen weeks using the CV method,
with measurements taken at weekly intervals. Fig. 7. (b) shows current was held on account of
the immunoelectrode until the twelve weeks. Later, peak current decreased, indicating that the
immunosensor has a remarkable twelve-week shelf life. The immunosensor required for this
experiment was produced on the same day and kept at 4 °C in the refrigerator for future use.

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Reproducibility was a crucial factor for the practical use of immunosensor. Six different
immunoelectrode’s developed under identical experimental circumstances. It was used

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separately for every experiment to -ptest the reproducibility of the BSA/anti-
GEN/rGO@Gd2O3/SPE immunoelectrode. Fig. 7. (c) illustrates that the reproducibility
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exhibited low values of RSD, which were found to be within suitable range of 2.02%.
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The response of GEN was investigated in spiked milk with concentrations ranging from 1 pM
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to 100 µM on the immunoelectrode BSA/Anti-GEN/rGO@Gd2O3/SPE. The concentrations of

GEN were spiked in milk samples, all measurements were carried out under the same
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conditions [Fig. 7 (d)]. In comparison to analyte sensing, the detection pattern remained
consistent in the spiked samples. These results confirmed the applicability of these BSA/Anti-
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GEN/rGO@Gd2O3/SPE immunosensing electrochemical sensors in real sample applications.

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Fig. 7. a) Interferent of rGO@Gd2O3/SPE electrodes b) Shelf life of rGO@Gd2O3/SPE

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electrodes c) Reproducibility of rGO@Gd2O3/SPE electrodes d) Response study of BSA/Anti-

GEN/rGO@Gd2O3/SPE immunosensing electrodes for different conc. of GEN in the Milk.
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The control study was carried out under the same circumstances as for the BSA/Anti-
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GEN/rGO@Gd2O3/SPE immunoelectrode’s to evaluate the function of rGO@Gd2O3/SPE

electrodes (without Anti-GEN) with various GEN amount that range from 1 pM to 100 nM. A
control study was done to observe the current response of the rGO@Gd2O3/SPE electrode to
different concentrations of GEN, as illustrated in Fig. 8. The experimental studies indicate that
there was no alteration in the CV current of rGO@Gd2O3/SPE electrodes, with an RSD value
of 0.58%.

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Fig. 8. Control study of rGO@Gd2O3/SPE electrodes
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Scheme 1. illustrated the outlines process of fabricating rGO@Gd2O3 NC on SPE platform, as
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well as the immobilization of anti-GEN with varying concentrations of the GEN analyte.
rGO@Gd2O3 NC have been synthesized using through co-precipitation method, dispersed in
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DI and drop casted on SPE electrode. The working electrode of the SPE comprised a
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hydrophilic carbon paste with surface functional groups that facilitated the effective deposition
of the NC onto the electrode surface. Further, an electrostatic interaction takes place between
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the carbonyl group of Anti-GEN and rGO@Gd2O3 forming metal ester bond [24]. The present
immunosensor is developed on paper based SPE, which itself contained three electrode system
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and consume very less amount of analyte and buffer solutions. It can be further converted to a
disposable portable sensing strip for electrochemical detection of GEN with good sensitivity
and selectivity.

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Scheme 1: Diagrammatic representation of formation of rGO@Gd2O3 NC (A) and the

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detection of GEN using rGO@Gd2O3/SPE based electrochemical immunosensor (B).
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Table 1: Comparison of the previously published literature with our rGO@Gd2O3/SPE
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electrodes platform.
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Range of
Incubation
S. linear Sensiti
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Nanomaterials Techniques LOD Time Ref.

No detectio vity
(mins)
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Electrophoretically
deposited L-
142.6
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cysteine CV/DPV/EI 0.039 mg/ 13.55

1. 25 min pM to [24]
functionalized S mL µA (log
2.8 µM
MoS2@MWCNT µg/mL)
-1
nanocomposite
Gold 10-1000
2. Colorimetric 300 nM 2 min - [36]
Nanoparticles nM
Disposable screen-
printed electrodes
potentiometri
incorporated with 10-7 to
c titration 7.5 × 10-8
3. multi-walled 0.05 min 10-2 mol - [25]
and FIA mol L-1
carbon nanotubes- L-1
measurement
PVC and
calixarene

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 Quantitative Quantitati
(centrifugati ve method
on) 0.048
Multi-walled -
4. Qualitative ng/mL - - [3]
Carbon Nanotubes
(filtration) Qualitativ
Immunoassa e method
ys 0.1 ng/mL
40-800
Poly(lactide-co- HPLC-mass
ng/ml and
5. glycolide) spectrometri - - - [37]
0.1-
microparticles c method
100µg/ml
Complementary
Metal-Oxide-
piezoresistiv
Semiconductor

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6. Bio-Microelectro - - [38]
microcantile (µg/mL) (µg/mL)
mechanical
ver
Systems (CMOS-

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BioMEMS) -p
Colloidal gold-
based
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7. ELISA 6 ng/mL - - - [39]
immunochromatog
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raphic assay
44.87 This
rGO@Gd2O3/ 1 pM to
8. CV 0.424 pM 3 min µA pM- wor
SPE electrodes 100 µM
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1
cm− 2 k

4. Conclusion
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In this study, we developed a cost-effective SPE based electrochemical immunosensor for

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gentamicin GEN detection, by modifying the SPE with rGO@Gd2O3 nanocomposite. The
utilization of SPE offered notable advantages, including cost-effectiveness, simplicity, and
minimal reagent requirements. The rGO@Gd2O3 NC, synthesized via co-precipitation, was
characterized using FT-IR, XRD, and SEM. The nanocomposite was successfully applied to
SPEs, enabling anti-GEN immobilization through EDC-NHS coupling. The immunosensor
exhibited high sensitivity (35.63 µA pM-1 cm−2) with a low LOD of 0.389 pM, detecting GEN
in a broad linear range (1 pM to 100 M, R2= 0.981). Analyzing spiked milk samples
demonstrated specificity and repeatability (RSD= 2.02%). Control studies confirmed the
specific interaction of GEN with the immunoelectrode. The immunosensor presents a
promising approach for accurate, cost-effective antibiotic detection, suggesting advancements
in biosensor technology. Moreover, this immunosensor has the potential to introduce a new
approach to developing highly sensitive, biocompatible, and rapid biosensors and biochip
devices.

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Author contribution statement

The research work was visualized by PRS and AKS. The research problem was conceptualized
by GBVSL and PRS. Data curation, formal analysis and experiments were carried out by
GBVSL, JKH. Writing was done by GBVSL and JKH. Supervision and validation were done
by PRS. Funding acquisition and project administration was done by PRS.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

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The authors are thankful to the Advanced Instrument Research Facility (AIRF), Jawaharlal

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Nehru University (JNU) and Central Research Facilities, IIT Delhi Sonipat Campus Rajiv
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Gandhi Education City, Rai, Sonipat – 131029, Haryana for facilitating the instrumentation
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facility. GBVSL is thankful to DST for funding through DST Women Scientist Project. This
work was supported by a grant (PAC-SCNS-PS-DBT-12151218-934) from the Department of
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Biotechnology, Government of India, India. The authors are thankful to 3kNano, a start-up
incubated at JNU-AIC FI, New Delhi for providing SPE.
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Highlights

• Immunosensor for detection of Gentamicin was developed, that has impact on global public
health concern.
• A paper based disposable electrochemical immunosensor using rGO@Gd2O3 NC, was
developed.
• Present immunosensor exhibited a wide linear response from 1 pM to 100 M with limit
of detection 0.424 pM and sensitivity of 44.87 µA pM-1 cm− 2.
• Present immunosensor has advantages as it has ease of fabrication, longer stability, cost-
effective, high specificity, and robust.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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