Materials Today Chemistry 26 (2022) 101129

Contents lists available at ScienceDirect

Materials Today Chemistry

journal homepage: www.journals.elsevier.com/materials-today-chemistry/

Recent developments and fabrication of the different electrochemical

biosensors based on modified screen printed and glassy carbon
electrodes for the early diagnosis of diverse breast cancer biomarkers
P. Lakhera a, b, V. Chaudhary a, b, e, A. Jha c, R. Singh d, P. Kush d, **, P. Kumar e, *
a
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
b
CSIR-Central Scientific Instruments Organization, Sector 30-C, Chandigarh e 160030, India
c
Dolphin PG Institute of Biomedical and Natural Sciences, Dehradun, Uttarakhand-249161, India
d
School of Pharmacy, Adarsh Vijendra Institute of Pharmaceutical Sciences, Shobhit University Gangoh, Saharanpur, India
e
Exigo Recycling Pvt. Ltd., Noida 201309, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history: Breast cancer (BC) is the second most common cancer among women, comprising 34% of all cancers,
Received 6 May 2022 affecting ~2.23 million women every year globally. Therefore, early diagnosis of BC is required for suc-
Received in revised form cessful treatment and hence an increased survival rate. Timely detection of various biomarkers subsisting
15 July 2022
in the blood, cells, tissues, or other biological fluids using techniques like radioimmunoassay (RIA),
Accepted 3 August 2022
Available online 6 September 2022
enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) can be advantageous in
this direction. Although all these methods are effective but they have their pros and cons, hence an
alternative non-invasive, cost-effective screening technique with high sensitivity and specificity is
Keywords:
Biosensing
required for the point of care (POC) diagnosis. Electrochemical (EC) biosensors are promising tools for
Detection POC diagnosis due to their simplicity, reliability, portability, low cost, fast response, ease of detection
Point of care even with a little sample requirement, magnified sensitivity, low detection limit, and POC testing
Challenges compatibility. Screen-printed electrodes (SPEs), and glassy carbon electrodes (GCEs) are widely used as
Nanomaterials electrodes in EC biosensors due to their easy miniaturization, and magnificent mechanical and electrical
Surface modifications properties, respectively. This comprehensive review focuses on the recent developments (covering the
period of 2015e2022) and fabrication of the different EC bioscaffolds based on modified SPEs and GCEs
for the early diagnosis of diverse BC biomarkers at different molecular levels like genomic, tran-
scriptomic, and proteomic formats. Further, circulating tumor cells (CTCs) and multiplex determination
of BC biomarkers are also discussed by using both electrodes. Considering the magnified sensitivity, most
of the reported biosensors have been made by surface modification of SPEs/GCEs with various nano-
materials (NMs), enzymes, and metals due to their unique properties. Further, the challenges associated
with these biosensors, and future perspectives have been also discussed. Therefore, this review will
impart relevant background for the design and fabrication of innovative SPE- and GCE-based POC devices
for diverse BC biomarker monitoring which would open the way for augmenting cancer diagnosis and
therapeutics. Generally, the principle and techniques remain similar while changing the analyte-bio-
recognition element (BRE), hence this review will be a proven guide for the development of similar kinds
of sensors for other analytes as well.
© 2022 Elsevier Ltd. All rights reserved.

1. Introduction

Cancer is one of the most prominent life-threatening diseases;

possesses more than 200 types; and affects approximately 60 hu-
man organs [1]. Annually, the incidence of new cancer cases in men
and women is 439/10,000. As per International Agency for Research
* Corresponding author.
** Corresponding author.
on Cancer (IARC) report, globally ~17 million cancer cases were
E-mail addresses: preetikush85@gmail.com (P. Kush), parveenkaushik7@gmail. reported in 2018 [2]. It is estimated that cancer cases and the
com (P. Kumar). number of cancer-related deaths are predicted to be 1,762,450 and

https://doi.org/10.1016/j.mtchem.2022.101129
2468-5194/© 2022 Elsevier Ltd. All rights reserved.
P. Lakhera, V. Chaudhary, A. Jha et al.
606,880 in 2022, respectively [3]. According to World Health Or-
ganization (WHO), cancer will be the leading cause of approxi-
mately 12 million deaths by 2030 [4]. Breast cancer (BC) is the
second most common cancer among women, comprising 34% of all
cancers, affecting ~2.1e2.3 million women every year globally [5].
In 2020, 2.261 million new BC cases were recognized and 0.685
million people died globally. In India only, the incidence of BC cases
is predicted to cross 2,05,000 and reach over 2,32,000 by 2025 [6].
The highest prevalence rates were observed in developed coun-
tries, whereas the lowest rates were reported in Africa (23/100,000
women) and East Asia (21/100,000 women), but this statistical data
can be underestimated due to the lack of comprehensive methods
of exact data collection in the related wide region [5].
Diagnosis and treatment of BC are difficult due to the similarity
between normal cells and cancer cells [7]. If BC is diagnosed at the
primary/early stage, a 5-year survival rate may extend to 90% in
developed countries [8], whereas A 5-year survival rate decreases
to 27.4% in metastatic BC patients [5]. Therefore, early diagnosis is
required for the successful treatment of BC with an increased
survival rate. Various techniques like mammography, sonography,
thermography, magnetic resonance imaging (MRI), molecular
breast imaging, positron emission tomography (PET), biopsy,
computerized tomography (CT), etc. have been used for the
screening of BC [7,9]. These all methods are effective but possess
certain limitations like invasiveness, costly, time-consuming,
requiring trained personnel to perform and interpret the imag-
ing studies, etc. (Table 1) [7,9e11]. Cancers can be early diagnosed
by the detection of various biomarkers present in the blood, cells,
tissues, or other biological fluids [1,12]. Biomarkers are certain
biologically relevant molecules (mutated gene, surface receptor
proteins, micro-RNA (miRNA), cancer antigens (CAs), cytokines,
exosomes, circulating tumor cells (CTCs), etc.) that are overex-
pressed in/or cancer cells, indicate the tumor formation/pro-
gression or metastasis in contrast to normal cells [10]. Various
biomarkers-based techniques like radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA), fluorescent in-situ
hybridization (FISH), and immunohistochemistry (IHC) are used
for diagnostic purposes [9,13]. These methods are selective
and sensitive but possess certain limitations such as being
expensive, complex, and multistep processes, and require
high-quality samples, etc. (Table 1). Therefore, an alternative
non-invasive, portable, cost-effective screening technique with
high sensitivity and specificity is required for the point of care
(POC) diagnosis.
Biosensors are widely used for the detection of various BC bio-
markers, biomedical analysis [14], environmental monitoring [15],
and food manufacturing industries [16] owing to their automation,
low cost, sensitivity, specificity, reliability, and reproducibility.
Moreover, biosensors are fast, inexpensive, and directly detect
various biomarkers in biological fluids with minimal invasion
[10,17]. Additionally, they can also be used as POC devices at doc-
tors' clinics/hospitals and homes. For biosensing applications, a
biosensor mainly comprises target molecules (biomarkers), bio-
receptor/biorecognition element (BRE), and bio-transducer. The
classification of biosensors mainly depends upon the bioreceptor
(enzyme, antibody, nucleic acid, cell), and transducer (electro-
chemical (EC), optical, thermal, mass, acoustic wave, field-effect
transistor, and quartz crystal microbalance) [7,18]. Among various
types of biosensors, EC biosensors are promising for POC diagnosis
due to their simplicity, reliability, portability, low cost, fast
response, ease of detection even with a little sample, magnified
sensitivity, low detection limit, and POC testing compatibility.
Moreover, these can be easily converted into electronic wearable
biosensors and could be used to obtain results via a portable device
such as a smartphone [15,19e23].
2
Materials Today Chemistry 26 (2022) 101129
Screen-printed electrodes (SPEs), and glassy carbon electrodes
(GCEs) are widely used as electrodes in EC biosensors due to their
low cost, easy miniaturization [12], easy surface functionalization,
and magnificent mechanical and electrical properties [24,25].
Moreover, in situ analysis can be appropriately carried out by using
SPEs due to their magnified sensitivity, a linear output, minimum
power consumption, and ease to operate at room temperature [26].
Further, the sensitivity and reproducibility of these two types of
electrodes can be augmented by surface modification with various
nanomaterials (NMs) [27]. Furthermore, the specificity of the
electrodes can be increased by the functionalization of the working
electrodes (WEs) with various specific recognition molecules like
enzymes, antibodies [28], nucleic acids [1,29,30], and aptamers
[12,31,32], etc.
There are various reviews about the different EC biosensors for
the screening of BC biomarkers [1,5,28,33e35], but few of them are
focused on SPEs-based screening of biomarkers [36e38]. Addi-
tionally, the bibliography about GCEs-based biosensors are exten-
sive, but as per the best of our knowledge, there is no review
emphasizing GCEs-based EC biosensor for BC biomarkers detection.
This comprehensive review highlights the fabrication principle,
configuration, and development of recent (covering the period of
2015e2022) SPEs and GCEs-based EC biosensors for various BC
biomarkers detection. Further, the challenges and future perspec-
tives have been also discussed. Therefore, this review will impart
relevant background for the design and fabrication of innovative
POC devices for diverse BC biomarker monitoring which would
open the way for augmenting cancer diagnosis and therapeutics.
2. Breast cancer pathophysiology and biomarkers
BC is a diversified disease with various subtypes followed by a
series of genetic mutations, and DNA damage [39]. BC is classified
into four subtypes based on antigen Ki-67, estrogen receptor (ER),
human receptor tyrosine-protein kinase erbB-2 (HER2), and pro-
gesterone receptor (PR) [5]. There are various risk factors associated
with the prevalence of BC like age, family history, geographic
location, low rates of breastfeeding, socioeconomic status, lifestyle
risk factors (smoking, alcohol, diet, obesity, and physical activity),
ionizing radiation, and increased breast density, etc. (Fig. 1) [40]. BC
most often originates from the terminal lobes (lobular) and duct
(ductal) of the mammary glands. Normal breast cells convert into
cancer cells, which are phenotypically similar to the normal ones.
Breast biopsy demonstrates the molecular and histological sub-
types, which play an important role in the therapeutical manage-
ment of breast cancer (Fig. 2) [5]. Globally, lobular and ductal
carcinoma covers 40e75% of all diagnosed BC cases [41], but ductal
carcinoma covers ~80% of all diagnosed BC [42]. However, some-
times BC can originate from the stromal tissues that integrate the
fatty and fibrous connective tissues of the breast [43].
BC biomarkers are classified into stage-dependent biomarkers
and overexpressed biomolecule-based biomarkers (Table 2)
[1,5,9,10]. Further, stage-dependent can be subclassified as diag-
nostic (healthy vs. BC) [44], prognostic (early BC vs. advanced BC)
[45], predictive (provide information regarding the particular
treatment success probability) [46], and pharmacodynamic/thera-
peutic biomarkers (a target biomolecule for the therapeutic mo-
dalities). Biomolecule-based biomarkers are more significant in
contrast to the prognostic or therapeutic but there is a correlation
between the prognostic and diagnostic value of these biomarkers
[10,47]. Moreover, BC biomarkers can also be classified as genomic
(DNA), proteomic (a protein molecule), transcriptomic (messenger-
RNA molecules (mi-RNAs), and metabolomic (low molecular
weight metabolites) based on their origin, function, and structure
[48]. Fig. 3 summarizes the origination of biomarkers from
P. Lakhera, V. Chaudhary, A. Jha et al. Materials Today Chemistry 26 (2022) 101129

Table 1
Various techniques used for the screening of breast cancer and their limitations.

Techniques Limitations

Mammography False-positive and negative results, uncomfortable involves breast compression, low sensitivity, and specificity in young
females (<40 years), can only detect 70% of breast cancers
Sonography Difficult to detect a deep-lying solid tumor, higher cost and highly experienced operators are needed to perform the
experiment
Thermography High cost, false negative, use of radiation, and sophisticated instrument
Magnetic resonance imaging Very expensive, some types of breast cancer escape detection, time-consuming process
Microwave imaging Complex procedure
Positron emission tomography Harmful ionizing radiation
Biopsy Probability to miss tumor cells; unnecessary surgery; can cause tumor metastasis; performed for late-stage; expensive
Computerized tomography Expansive scanner, low sensitivity, and risk of harmful radiation
Fluorescent in-situ hybridization Separate patients into positive and negative groups, semiquantitative results
Radioimmunoassay Complex, and time-consuming process along with radioactivity risk
Enzyme-linked immunosorbent assay Time-consuming, expensive, risk of false-positive, requires professional operators
Immunohistochemistry Complex, and time-consuming process required trained personnel for the analysis

tumorigenesis, migration, and accumulation to the different organs
together with the relationship between these biomarkers.
3. Electrochemical biosensors
An EC biosensor is a device used to measure the EC behavior of
the electroactive surface to provide quantitative or semi-
quantitative analytical information using an EC transducer. Based
on the transducer detection principle EC biosensors are grouped
into voltammetric, amperometric, impedimetric, conductometric,
and potentiometric (Table 3). The voltammetric transducers are
highly sensitive and widely used for biosensing, and measure the
current-potential relationship. In this technique, the current is
measured at various forms of potential such as potential sweep,
potential waveform, step potential, potential ramp, etc. is applied to
the WEs. This technique is further grouped into cyclic voltammetry
(CV), differential pulse voltammetry (DPV), linear sweep voltam-
metry (LSV), and square wave voltammetry (SWV). The ampero-
metric technique is also based on the current-potential relationship
but in this technique, the current is measured resulting from the
oxidation or reduction of electroactive material at a constant po-
tential [48,50]. Chronoamperometry is another amperometric
technique where a square-wave potential is applied to the WEs and
the current is measured as a function of time [50]. Electrochemical
impedance spectroscopy (EIS) is extensively used for the surface
characterization of electrodes especially modified with bio-
materials. Moreover, this technique is used to assess the electrode/
electrolyte interface and the binding kinetics between the elec-
trode and analytes. The immobilized biomolecules on the electrode
surface change the interfacial electron transfer resistance and

Fig. 1. Various risk factors associated with breast cancer.

3
P. Lakhera, V. Chaudhary, A. Jha et al.
Fig. 2. Origination of breast cancer from the terminal lobular unit (breast functional unit).
capacitance of the application system [50]. Conductometry is based
on the relationship between conductance and a bio-recognition
event. Potentiometry measures the potential difference between
the working and reference electrode. However, its sensitivity is
lower in contrast to other techniques due to the small potential
difference during the analytical reaction [51].
4. The emergence of screen-printed electrodes (SPEs) and
glassy carbon electrodes (GCEs)
SPEs and GCEs have been widely used as electrode materials for
the fabrication of various EC biosensors due to reproducibility, and
low cost. EC biosensors based on SPE and GCE provide great sta-
bility and sensitive results which helps in the early detection of
many diseases and biomarkers [53]. Various commercial SPEs and
GCEs in different configurations that exist with excellent perfor-
mance can also purchase from different suppliers (Table 4) [38,54].
This section summarizes the basic fabrication principle and
configuration of SPEs and GCEs for EC sensing.
SPEs are inexpensive, reproducible, and disposable EC devices
that can be easily fabricated in bulk without any treatment steps
beforehand leading to real-time and on-site sensing [55]. These
diversified materials have diagnostic applications in various areas
like the detection of environmental pollutants, organic, and inor-
ganic contaminants in food, medicines, and agriculture due to easy
miniaturization, portability, less sample requirement, and easy
surface functionalization [26,54,55]. Moreover, SPEs circumvent
some common problems such as tedious cleaning and memory
effects associated with the use of classical solid electrode materials
4
Materials Today Chemistry 26 (2022) 101129
[56]. Additionally, SPE can reduce the size of EC instruments to
small pocket-sized ones, resulting in personal and professional use
[38].
A typical SPE comprises a three-electrode configuration: (1)
working electrodes (WEs); (2) counter electrodes (CEs); and (3)
reference electrodes (REs). SPEs fabrication is a multistep process
that involves: (1) selection of the mesh or screen (significantly af-
fects the SPEs size and geometry; (2) selection and preparation of
the appropriate viscosity inks (3e10 Pa s); (3) selection of the
suitable substrate material; and finally (iv) printing, drying and
curing [38,57]. Precisely, SPEs are fabricated by a layer-by-layer
printing of ink on a substrate via using a mesh/sieve followed by
solidification and drying of ink under thermal/ultraviolet (UV) light
exposure. Further, the conductive track is insulated via coating with
protective non-conductive ink/paint (Fig. 4, a). Transparent flexible
plastic, paper, glass, alumina, or ceramic surfaces are commonly
used solid substrates [54,56]. During the SPEs printing, WEs and
CEs are mostly printed with carbon paste/ink (carbon nanotubes
[CNTs], graphene [Gr], fullerene, or graphite), whereas RE is painted
with silver (Ag) ink [58]. Moreover, SPEs can be personalized as per
the requirement of ongoing study by using carbon, gold (Au), and
platinum (Pt) [54,56]. Additionally, the composition of printing ink
may be modified by the addition of various substances such as
complexing agents, electrochemical (EC) signal modifiers, metal,
metal oxides, etc.
A glassy carbon (GC), also known as polymeric carbon or vit-
reous carbon is a non-graphitic sp2 bonded carbon allotrope pro-
duced by the controlled pyrolysis of organic polymers at high
temperatures (>2000
C) [59]. The GC microstructure comprises
P. Lakhera, V. Chaudhary, A. Jha et al. Materials Today Chemistry 26 (2022) 101129

Table 2
Common breast cancer biomarkers for the diagnosis of breast cancer within their normal and abnormal range.

Type of biomarkers Examples Normal range Abnormal range Ref

Biomolecule based Genomic/DNA BRCA1, BRCA2 e e [1]

Transcriptomic/micro-RNA miR-21, 16, 27a, 150, 191 200 aM to 20 pM [1]
Proteomic/Glycoprotein HER2 2e15 ng/mL serum 15e75 ng/mL [3]
EGFR 2e15 mg/L >75.3 mg/L [1]
CEA 2.5 mg/L in non-smokers, 5 mg/L in a smoker [1,49]
MUC1 <31 U/mL 3100 U/mL [3]
CA15-3 <30 U/mL 30e50 U/mL [49]
CA27.29 38 U/mL [1]
ER 0.06 mg/g cytosol [1]
PR 0.06 mg/g cytosol [1]
VEGF 1e177 pg/mL 18e328 pg/mL [3,49]
Stage dependent Prognostic ER e e [1,5,9,10]
PR e e
HER2 e e
BRCA1 e e
Ki67 e e
Osteopontin e e
Mammoglobin e e
Sirtuins e e
Autoantibodies e e
Diagnostic HER2 e e
CEA e e
BRCA1 e e
miR-21, 155, 222 e e
Therapeutic ER e e
PR e e
HER2 e e
CA15-3 e e
CA27.29 e e
Ki67 e e
miR-21 e e
CTC e e

Abbreviations: BRCA1: breast cancer types 1; CA: cancer antigen; CEA: carcinoembryonic antigen; CTC: circulating tumor cells; EGFR: epidermal growth factor receptor;
EpCAM: epithelial cell adhesion molecule; ER: estrogen receptor; HER2: human epidermal growth factor receptor-2; miR: micro RNA; MUC1: mucin 1; PR: progesterone
receptor; VEGF: vascular endothelial growth factor.

discrete fragments of curved carbon planes and exhibits both glassy reactivity, durability, impermeability, chemical inertness, and high
and ceramic properties (Fig. 4, b). It is widely used as electrode electrical conductivity [60]. Moreover, GCEs can be used in various
material in electrochemistry owing to its low cost, broad anodic high-aspect-ratio instruments due to their compatibility with the
potential window, magnified temperature resistance, low 3-dimensional (3D) microfabrication and easy surface

Fig. 3. Origination of biomarkers from tumorigenesis, migration, and accumulation to the different organs together with the relationship between these biomarkers.

5
 Table 3

P. Lakhera, V. Chaudhary, A. Jha et al.

Comparative analysis of commonly electrochemical techniques.

Technique Principle Most usage Advantage Limitation Sensitivity Ref

Voltammetry The current is measured at various Diagnosis of various cancer biomarkers Highly sensitive, inexpensive, and Substances having fM- nM [48,50]
forms of potential such as potential such as Osteopontin, IL-10, HER2, PSA easily miniaturized electrochemical activity can be
sweep, potential waveform, step CA153, and tumor cells like HEp-2, measured, and interference in
potential, potential ramp, etc. is applied HT29, HCT sensitivity due to non-Faradaic
to the working electrodes. current
CV: Investigation of the electrochemical
behavior of electroactive material, and
measures the current with ramp-off
potential.
DPV: Measurement of current between
two points of each pulse concerning
potential which is applied in the pulse
form. The height of the DPV curve is
directly proportional to the analyte
concentration.
LSV: The current is measured by the
varied potential at a constant rate
through scanning.
SWV: The current is measured by the
difference between forward and
reversed current
Amperometry Current is measured resulting from the Detection of serum glucose, cancer Inexpensive, fast reproducible, Require redox elements to ng/mL- pg/mL [50,52]
oxidation or reduction of electroactive biomarkers, cancer cells, nucleic acid, reusable, suitable for enzymes' improve the current
material at a constant potential pesticides, and food placement, and appropriate for mass production. Poor resolution due
production even without calibration to matrix effect.
Impedimetry (EIS) The measurement of the current Detection of cancer biomarkers (nucleic Cost-effective, higher signal-to-noise Requirement of the bulky mg/mL- pg/mL [50e52]
6

response to the application of a
constant AC potential.
Conductometry The change in conductivity/current
flow of a solution is measured due to
the change in ionic species
concentration. This can be attributed to
the concentration of analytes,
Biorecognition elements, ion
concentration, substrate to product
conversion, etc.
Potentiometry Measures the potential difference
between working and reference
electrodes.
acid, and protein biomarkers), cancer
cells, DNA hybridization, direct
observation of antigen-antibody
reaction, and enzyme reaction,
Detection of biological receptors,
enzymatic reactions, environmental
pollutants, drugs, and food-borne
pathogens
Detection of cancer cells, and enzymatic
activity
ratio, label-free detection, fast response, readout device
low excitation voltage, insensitive to
environmental conditions, real-time,
and on-site detection
Inexpensive, insensitive to light, no Poor selectivity mg/mL- pg/mL [43,51]
requirement of the reference electrode,
reusable, easy miniaturization, and less
power consumption due to voltage
reduction
Inexpensive, simple, fast reproducible, Sensitive to environment and Up to pg/mL [50e52]
reusable, and suitable for enzymes' temperature
placement

Abbreviations: CV: cyclic voltammetry; CA153: cancer antigen153; DPV: differential pulse voltammetry; EIS: electrochemical impedance spectroscopy; HER2: Human epidermal growth factor receptor-2; IL: interleukin; LSV:

Materials Today Chemistry 26 (2022) 101129

linear sweep voltammetry; PSA: prostate-specific antigen; SWV: square wave voltammetry.
 Table 4

P. Lakhera, V. Chaudhary, A. Jha et al.

Commercial screen printed and glassy carbon electrodes in different configurations [38,74].

Sr. No Company name Electrode type Functionalization Image Dimension/specification Company link

1 Metrohm Available in different Carbon/gold/platinum, silver/ 3.4 x 1.0  0.05 cm. Reference www.dropsens.com/en
DropSens types (2, 3, carbon nanotubes electrode and electric contacts
multielectrode) made of silver

2 Pine Research Instrumentation
3 electrode system
Carbon, ceramic/gold platinum
Carbon working electrode and www.pineinst.com/echem/
counter electrode, and a silver/
silver chloride reference
electrode. 2/4/5 mm working
electrode

3 BVT technologies 3/8 electrode system Pure gold 38.10 mmx 7.26 mm x http://www.bvt.cz/
pure platinum, pure silver, 0.63 mm.
graphite, carbonreference 8 electrode system has 1
electrode common reference electrode
material
Ag/AgCl/Ag covered by AgCl
4 Rusens Ltd 3 electrode system Carbon paste/silver 10 mm  28 mm x 0.35 mm. http://www.rusens.com/
Polyethylene terephthalate.
Resistance: 10e30 U.

5 Gamry Instruments 3 electrode system Carbon/platinum 1 mm thick, 5 cm long https://www.gamry.com/cells-and-

7

1 cm wide, Alumina Substrate, accessories/electrodes/screen-

working electrode ¼ 12 mm2 printed-electrodes/
Area

6 Gwent Group 3 electrode system Ceramic carbon/platinum/gold Substrate 96% Alumina, 10.12 x www.gwent.org/gem_standard_
51.00  0.50 mm Pitch of electrodes.html
individual electrodes -
2.54 mm, working electrode
-2mm
7 Palm Sense 3 electrode system Platinum/gold/carbon/silver/ Alumina substrate,working https://www.palmsens.com/
silver chloride electrode product-select/
- 1 mm
Silver as reference electrode

Materials Today Chemistry 26 (2022) 101129

8 Zimmer and peacock
3 electrode system
material
Gold/carbon/graphene 30mm  10mm  0.635 mm,
working electrode: 2.0884 cm,
reference electrode: 2.1019 cm,
counter electrode ¼ 2.1049 cm
www.zimmerpeacocktech.com/
2019/02/10/manufacturer-of-
screen-printed-electrodes

9 Zensor 3 electrode system Graphite/gold/graphene/ Diameter of working www.nanochemazone.com/

carbon nanotube/platinum/ electrode:2 mm, 3 mm, 4 mm, product/zensor-screen-printed-
silver (customization possible) 5 mm,reference electrode electrodes
¼ 0.5  1 mm,

(continued on next page)

 Table 4 (continued )

P. Lakhera, V. Chaudhary, A. Jha et al.

Sr. No Company name Electrode type Functionalization Image Dimension/specification Company link

10 Basi Research Products 1 electrode system Electrode/gold/carbon paste 7.5 cm length x 6 mm https://www.basinc.com/products/
ec/sve

11 ALS Co. ltd 1 electrode system Customized Glassy carbon electrode/Pt/Au/ https://www.als-japan.com/1408.
10mm/6mm/3mm/4 mm html

12 Pine Research 3 electrode system Customized Glassy carbon electrode 3.0 mm https://pineresearch.com/shop/
disk. Available with Ag/AgCl products/cells-and-glassware/
reference electrode, a platinum standard-volume/web92-gc/
counter electrode, and a set of
polytetrafluoroethylene
stoppers

13 Ossila enabling Material 1 electrode system Customized 2/3/4 mm diameter https://www.ossila.com/products/

Science platinum/gold/graphite working-electrode

14 Wuhan Corrtest Instruments 1 electrode system Non-customized Glassy carbon and www.corrtest.en.made-in-china.
Corp., Ltd polytetrafluoroethylene com
8

3mm/2mm/1mm/4mm/5mm/
6 mm
Teflon diameter- 6 mm
Brass rod- 2 mm

15 Metrohm 1 electrode system Non-customized Glassy carbon, length ¼ 76 mm, https://www.metrohm.com/en-in/

diameter ¼ 2 products/61248040

16 Corrtest Instruments Corp. 1 electrode system Customized Glassy carbon 2mm/3mm/ http://www.csechem.com/
4mm/5mm/6 mm, Teflon electrochemical-accessories/
sleeve ¼ 6mm/8mm/10mm/

Materials Today Chemistry 26 (2022) 101129

working-electrode/glassy-carbon-
12 mm working-electrode.html
P. Lakhera, V. Chaudhary, A. Jha et al. Materials Today Chemistry 26 (2022) 101129

Fig. 4. Conventional electrodes used for electrochemical biosensing. a) screen-printed electrode; b) glassy carbon electrode.

functionalization with various NMs without compromising their
inherent properties [61]. GCE is an intertwining ribbon-like mate-
rial that exhibits ordered morphology microscopically with a
mixture of the edge plane and the basal plane of carbon [62].
However, the electrochemistry of GCE is affected by its cleanliness
and surface chemistry [63]. Therefore, the surface of GCE should be
maintained and activated for quick electron transfer kinetics which
can be carried out by washing, mechanical polishing, plasma
treatment, ultrasonication, vacuum heat treatment, and thermal
and laser activation [61].
The sensitivity, specificity, and EC efficiency of both electrodes
can be enhanced by using the modified electrodes with various
NMs like graphene (Gr), carbon nanodots (CNDs), quantum dots
(QDs), carbon nanotubes (CNTs), nanofibers, metallic/polymeric
nanoparticles (NPs), etc [56,64e68]. Further, the analyte-specific
part of the electrodes can also be modified with various enzymes,
nucleic acid, aptamer, and antibodies resulting in enzyme-, geno-,
apta-, and immunosensors respectively (Fig. 5) [69]. These
biomolecules can be immobilized on the WEs surface by various
techniques such as microencapsulation, adsorption, crosslinking,

Fig. 5. Fabrication and application of modified screen-printed electrode (SPE), and glassy carbon electrode (GCE) with different doping agents. a) surface modification of both
electrodes with various nanomaterials (1: carbon nanotubes, 2: quantum dots, 3: porous silica, 4: polymers, 5: carbon nanodots, 6: nanofibers, 7: metallic/polymeric nanoparticles,
8: graphene), and analyte-specific part coupled with various biorecognition molecules; b) sample analysis; c) electrochemical measurement of analytes of interest.

9
P. Lakhera, V. Chaudhary, A. Jha et al.
entrapment, or covalent attachment [70]. The modified SPEs with
different agents expedite the electron transfer rate on the electrode
surface with increased output signals that capture the analytes of
interest [71]. The modified GCEs possess increased surface area
[72], corrosion resistance, and high thermal and EC stability [73].
Thus, the modified SPEs and GCEs as WEs in EC biosensors, enable
the simple, inexpensive, ultrasensitive, and rapid determination of
various substances.
5. SPEs and GCEs-based electrochemical biosensors for the
detection of breast cancer biomarkers
BC biomarkers are present at very low concentrations in normal
cells, but the concentration level increases upon the progression of
BC. These biomarkers can be detected at an early stage in saliva,
urine or serum, or stool by using the EC techniques owing to their
simplicity, sensitivity, specificity, convenience, fast detection, and
minimum power consumption [75]. This section summarizes the
principle and application of various SPEs and GCEs-based (micro-
electrode/modified electrode) EC biosensors for the detection of
diverse BC biomarkers (Table 5).
5.1. Detection of genomic biomarkers
Genomic biomarkers used for the diagnosis of BC employ the
detection of specific changes in certain genes like amplification,
deletion, or mutation of proto-oncogenes, tumor suppressor genes,
cell-cycle regulator genes, etc. Various genetic biomarkers such as
ATM, CYP1A1, CHEK2, FGFR2, RAD1, PTEN, PALB BRIP1 2, TP53,
TGFB1, MAP3K1, BRCA1, and BRCA2 are used for the diagnosis of BC
[48,182]. The antioncogenes BRCA1 and BRCA2 have widely
accepted DNA biomarkers expressed in normal cells and are
responsible for DNA repair. These tumor suppressor genes control
and regulate the cell cycle and cell division [1,5,10]. Mutations or
other malfunctions in the BRCA1 genes are responsible for
increased risk of BC (60e80%), ovarian, and prostate cancer and are
responsible for ~21e40% of inherited BC cases [10,183,184]. More-
over, the specific mutation in BRCA1 5382insC increases the 10-
time risk of BC [22]. The BC can be diagnosed by the genome
screening of BRCA1 and BRCA2 genes on chromosome numbers 13
and 17 respectively [48].
The principle of EC genomic biosensor (genosensor) involves
immobilization of sequence-specific probe or capture probe (BRE)
on the electrode surface and followed by the affinity reaction be-
tween the probes and target DNA. Further, the EC transducer
transforms the different EC (current, potential, impedance, etc.) or
non-EC properties (van der Waals interactions, conformal changes,
mass transportation, etc.) that happen during the biorecognition
process into detectable signals [35]. A typical genosensor comprises
an electrode, capture probe (a molecule immobilized on the elec-
trode surface, and used to recognize and bind to target DNA), re-
porter/detector probe (an element that causes the detectable signal
in response to the EC reaction), and the target DNA/RNA. Single-
stranded DNA (ss DNA; linear or hairpin), aptamer, RNA se-
quences, peptides, and DNA-related proteins are commonly used as
capture or reporter probes (Fig. 6) [35].
It is a prerequisite to designing high specificity capture/target
DNA for these types of sensors [75]. Other components like inter-
mediate linkers and electrode coatings are also used to magnify the
genosensor efficiency. Moreover, some sensor encompasses the
integrated form of capture and reporter probes as a single unit
resulting in magnified sensitivity. Additionally, the probes can also
be integrated with various NMs and labeled with different elec-
troactive species like specific enzymes (horseradish peroxidase
(HRP), glucose oxidase (GOx), beta-lactamase, urease, and urea,
10
Materials Today Chemistry 26 (2022) 101129
etc.), or redox indicators (ferricyanide/ferrocyanide, quinones/hy-
droquinones (HQ), anthracyclines, viologens, phenothiazines, qui-
noxaline derivatives, metal complexes, metal chelates, etc.)
resulting in augmented signal intensity, specificity, and improved
limit of detection (LOD) [185]. The labeling molecule quantifies the
t-DNA/miRNAs in the sample either by direct or competitive
detection [75]. Direct labeling detection is based on the labeled
capture hybridized with the targets giving rise to a change in EC
signal response due to the variation in distance between the
labeled molecule and electrode surface in the electroactive species,
which is proportional to target concentration. The competitive
approach used for the detection of targets is based on the elimi-
nation of labeled probes from the electrode surface through
displacement reactions, or by using various splitting agents like
endonuclease, duplex-specific nuclease (DSN), or calcium ions
[186].
EC genosensor involves direct and indirect/sandwich hybridi-
zation techniques using label-free or labeled reporter probes based
on conformational changes in target DNA (Fig. 6) [35]. To further
explain direct assay encompasses the hybridization reaction be-
tween the capture probes immobilized on the electrode surface and
target probes and the signal can be collected by amperometry/DPV/
EIS (Fig. 6a and b). Mousavisani et al. developed a simple, stable,
reproducible, and label-free SPE-based impedimetric DNA
biosensor. Initially, a screen-printed carbon electrode (SPCE) was
modified with diazonium salt and followed by activation of the
existing carboxyl group by placing droplets of N-ethyl-N’-(3
dimethylamino propyl) carbodiimide hydrochloride (EDC) and N-
hydroxysuccinimide (NHS). Further, the activated electrode was
covered with aminated-DNA and followed by drop-casting of
ethanolamine to deactivate the unreacted carboxyl group. The
developed biosensor was evaluated to study the DNA damage by
using EIS. The fabricated biosensor can accurately detect DNA
damage with magnified stability, and reproducibility. Moreover, the
biosensor can also be used to investigate the antioxidant effect of
glutathione in reducing DNA damage [76].
Sandwich hybridization is the most extensively used technique
that involves initially binding surface-bound capture DNA (DNA-c)
to the specific part of target DNA (DNA-t; Fig. 6a and b) and fol-
lowed by hybridization of a reporter probe (DNA-r; usually labeled
with electroactive molecule) to the unhybridized part of DNA (Fig. 6
c) resulting in a sandwich of c-DNA|t-DNA|r-DNA. Further, the hy-
bridization reactions into detectable signals can be monitored by
using various EC transducers (Fig. 6 d). This method has been
explored with diverse modifications at either capturing step or the
recognition step. In a study, selective biosensing of BRCA1 mutation
was diagnosed by a carbon dots (CDs) modified screen-printed gold
electrode (SPGE) disposable genosensor by using Thionine (Th) as a
redox indicator. The biosensor was developed by firstly drop-
casting of CDs on the surface of SPGEs and followed by immobili-
zation of capture DNA (PROBEBRCA1) on the modified SPGE (PRO-
BEBRCA1/CDs/SPGE). Further, PROBEBRCA1/CDs/SPGE was hybridized
with the sample of gen BRCA1 comprising wild (WTBRCA1), or
mutated (MUTBRCA1) types leading to the form WTBRCA1/PRO-
BEBRCA1/CDs/SPGE, and MUTBRCA1/PROBEBRCA1/CDs/SPGE, respec-
tively. After the hybridization, Th was accumulated on the electrode
surface leading to the recognition of the target single-stranded DNA
(DNAss) using DPV. The results revealed that the developed
biosensor was efficient to discriminate WTBRCA1 and MUTBRCA1 with
a detection limit of 55.0 pg/mL [77]. Recently, Feng et al. developed
an SPE-based genosensor by using a tetrahedral-structured probe
(TSP) and poly-adenine mediated Au NPs for the magnified detec-
tion of BRCA1 by using CV and amperometry. The sensor was
fabricated by firstly electrodepositing the AuNPs on SPEs and
further functionalizing with TSP. Secondly, a stable sandwich
 Table 5

P. Lakhera, V. Chaudhary, A. Jha et al.

Key characteristics of SPE- and GCE-based electrochemical biosensors for the detection of various breast cancer biomarkers.

Biomarkers Electrode Surface functionalization of Method for electrode Advantage of functionalization Detection method Limit of detection Linear range Ref
type electrodes functionalization

Genomic biomarkers
BRCA1 SPCE Diazonium salt Covalent High, and long-term stability EIS e e [76]
SPGE CDs Drop casting Magnified electron transfer DPV 55.0 pg/mL - [77]
SPGE Au NPs Electrodeposition Fast electron conduction, easy to CV 0.1 fM 1 fM-1 nM [78]
label, and biocompatible
GCE Gr Covalent immobilization Good biocompatibility, large surface CV/ 50 aM 100 aM-1 nM [79]
area to volume ratio, high thermal, Chronoamperometry
conductivity, and electron mobility at
room temperature
GCE PEG/Au-NPs Drop coating Easy fabrication of biosensor along EIS 1.72 fM 50.0 fM-1.0 nM [80]
with, magnified sensitivity,
reproducibility, and antifouling
property
GCE PANI/PEG Galvanostatic technique Large surface area along with DPV 0.0038 pM 0.01 pM-1 nM [81]
magnified antifouling, and
conductivity
GCE PEDOT Electrodeposition Excellent conductivity, DPV 0.03 fM 1.0  1016M-1.0  1010M [82]
biocompatibility, stability, and
antifouling property
GCE GO/P3CA Amide bond Excellent conductivity, high surface CV, DPV, and EIS 3 fM 10 fM-0.1 mM [83]
area enabling more DNA
immobilization
GCE P[DA-b-CD/CTAB])-AgNPs Electrodeposition Excellent conductivity, and more DPV, and SWV 0.003 pg/mL 0.01565e10 pg/mL and [84]
immobilization of primary antibodies 0.625e20 pg/mL
due to large surface area respectively
3.01  1016 M 1.0  1015-1.0  107 M
11

GCE 3D-rGO/PANI Drop coating Reduced charge transfer resistance, CV, DPV, and EIS [85]
and magnified EC activity
15 15 10
GCE MWCNT/MOF@Fe3O4 NPs Drop coating Excellent conductivity, DPV 0.57  10 M 1  10 - 1  10 M [86]
electrocatalytic properties, and
magnified surface area
Transcriptomic biomarkers
miRNA-21 and SPdCE e e e Amperometry 0.6 nM 2.0e10.0 nM [87]
miRNA-205
miRNA-34a SPE CNF e high electrocatalytic effect and high DPV 10.98 mg/mL 25e100 mg/mL [88]
conductivity
miRNA-155 SPGE e e e EIS 5.7 aM 10 aM-1.0 nM [89]
miRNA-155 SPE MBs Electrodeposition Label-free detection of miRNA DPV 29 pmol/L 81 pmol/L-1.2 nmol/L [90]
miRNA-21 SPCE MBs functionalized with His-Tag- e Label-free detection of miRNA Amperometry 0.91 nM 3.0e100 nM [91]
ZFP
miRNA-21 SPCE Au-NPs/rGO Electrodeposition Increased surface area for loading, DPV 5 fM 10 fM-2 pM [92]
efficient electrical conductivity,

Materials Today Chemistry 26 (2022) 101129

biocompatibility, and easy
functionalization
miRNA-21 SPCE Gr/PPy/Au-NPs Drop casting Magnified distribution of DNA probe DPV 0.020 fM 1.0 fM-1.0 nM [93]
on the sensing surface leads to
improve the sensitivity
miRNA-155, miRNA- SPCE Au-NPs/Gr/QDs/GO Drop casting Improved electron transfer ability, CV, SWV, and EIS 0.33, 0.04, and 0.28 fM 0.001e1000 pM [94]
21, and miRNA- high surface area, and easy
210 immobilization of biomolecule
enabling improved analytical
performance
miRNA-21 GCE MWCNT Drop casting Excellent electrocatalytic and DPV 84.3 fM 0.1e500.0 pM [95]
conducting properties
(continued on next page)
 Table 5 (continued )

P. Lakhera, V. Chaudhary, A. Jha et al.

Biomarkers Electrode Surface functionalization of Method for electrode Advantage of functionalization Detection method Limit of detection Linear range Ref
type electrodes functionalization

miR-199a-5p GCE GNRs/GO Drop casting The composite provides enhanced EIS 4.5 fM 15 fM- 148 pM [96]
resistance to nucleases and improved
thermostability
MiRNA-24 GCE Polyamidoamine dendrimer Covalent grafting Short time, simple operation, and EIS 3.4 fM 1014M-108M [97]
functionalized PPy immobilized large quantity of DNA
miRNA-155 GCE GNRs/GO sheets Drop casting Increased electrical and thermal DPV 0.6 fM 2.0 fM-8.0 pM [98]
conductivity along with improved
mechanical flexibility and
biocompatibility
miRNA-21 GCE ZrO2 - rGO Drop casting Increased electrical and thermal EIS 4.3 fM 10 fM-100 pM [99]
conductivity along with improved
mechanical flexibility and
biocompatibility
miRNA-21 GCE MBs Drop casting Easily discriminate unwanted EIS 60 aM 0.5e40 fM [100]
constituents
miRNA-21 GCE Au-NPs/CNNS Drop casting Improved electron transfer ability, SWV 2.9 fM 10 fM-1 nM [101]
high surface area, and easy
immobilization of biomolecule
enabling improved analytical
performance
Proteomic biomarkers
Immunosensors
HER2 SPCE Au-NPs Physical adsorption Facilitation of rapid electron transfer EIS 0.01 ng/mL 0.01e100 ng/mL [102]
and providing a biocompatible
surface for the immobilization of
antibody fragments enabling
12

enhanced antigen-binding efficiency

SPCE Zwitterionic hydrogel Drop casting Antifouling properties EIS 77 fM e [103]
SPCE Au Drop casting Biocompatible, non-toxic, chemically CV 34.9 pg/mL 0.001e0.5 ng/mL [104]
inert, improved electrical
conductivity, large surface area,
oxygen transferability, and minor
swelling
SPE e e e CV 4 ng/mL 5 ng/mL-20 ng/mL [105]
SPCE e e e SWV 0.28 ng/mL 1e100 ng/mL [106]
SPCE e e e LSV 2.8 ng/mL 5e50 ng/mL, and 50 [107]
e100 ng/mL
SPCE e e e DPASV 0.29 ng/mL 0.50e50 ng/mL [108]
SPCE e e e DPASV 2.1 ng/mL 10e150 ng/mL [109]
GCE e e e DPV 2.0  105 ng/mL 5.0  104-50.0 ng/mL [110]
GCE Fe3O4@ TMU-21 encapsulated in Drop casting Enhanced electrocatalytic activity, EIS and CV 0.3 pg/mL 1.0 pg/mL-100 ng/mL [111]
MOF and MWCNT large surface area, and porosity

Materials Today Chemistry 26 (2022) 101129

GCE SNGQDs@AuNPs, CoPeBNF, and CoP Drop casting Enhanced electrocatalytic activity, EIS 0.0327 ng/mL, e [112]
eBNF/SNGQDs@AuNPs large surface area, and porosity 0.0454 ng/mL, and
0.1072 ng/mL
GCE WO3/poly-glutamic acid Electrochemical grafting Biocompatible DPV 1 fg/mL 1 ng/mL-1 fg/mL [113]
CEA SPE AuNPs/rGO Electrochemical deposition Enhanced electron transfer, large CV 0.28 ng/mL and 0.5e50 ng/mL, and 250 [114]
surface area 181.5 ng/mL e2000 ng/mL
SPCE Au NPs Elecrodeposition Efficient electron transfer, enhanced CV, EIS 0.05 ng/mL and 0.03 ng/ 1e10 ng/mL, and 0.5e7 ng/ [115]
conductivity, easy functionalization, mL, and 0.01 ng/mL mL
and good biocompatibility
SPE Ag NPs/rGO Elecrodeposition Efficient electron transfer, enhanced CV 0.035 mg/mL 0.05e0.50 mg/mL [116]
conductivity, easy functionalization,
and good biocompatibility
 P. Lakhera, V. Chaudhary, A. Jha et al.
SPE rGO-TEPA/Au-MNPs Electrodeposition Magnified electrical conductivity, SWV 1.42 pg/mL 5.0 pg/mL-200.0 ng/mL [117]
improved stability, and augmented
biorecognition efficiency
GCE APTES crosslinked Gr sheets Drop casting Biocompatible, efficient electron EIS 0.2 pg/mL 0.001e20 ng/mL [118]
transfer, improved thermal
conductivity and mechanical
stiffness, and better stability
GCE (SnO2)/rGO and AuNPs Drop casting Large specific surface area, improved EIS 0.17 pg/mL 0.5 pg/mL - 25 ng/mL [119]
electrical conductibility resulting in
magnified loading of antibodies
GCE PdeIr NPs Drop casting Efficient electron transfer, enhanced EIS 0.017 ng 0.05e50 ng/mL [120]
conductivity, easy functionalization,
and good biocompatibility
GCE PDA-Pb2þ/chitosan-Au NPs Drop casting Excellent electrical conductivity, SWV 0.26 fg/mL 1 fg/mL-100 ng/mL [121]
remarkable adsorption capacity
GCE rGO-Au NPs Drop casting Excellent conductivity and large SWV 5.3 pg/mL 50 pg/mL- 650 pg/mL [122]
surface area
MGCE Ag NPs Drop casting Excellent elecroconductivity DPV 0.03 pg/mL 0.0001e20 ng/mL [123]
GCE AueAg/rGO@PDA Drop casting Dual signal amplification due to CV 0.286 pg/mL 0.001 ng/mL- 80 ng/mL [124]
excellent elecroconductivity
GCE TGO/AuNPs @strp Drop casting Large specific surface area, improved DPV 75 fg/mL 100 fg/mL to 5 pg/mL [125]
electrical conductibility resulting in
magnified loading of antibodies
GCE rGO/chitosan/AuNPs Layer by layer deposition Large specific surface area, improved DPV 0.1471 pg/mL 0.3 ng/mL-30 ng/mL [126]
electrical conductibility resulting in
magnified loading of antibodies
GCE 2D-ReS2 nanosheets Drop casting excellent optical electronic, EIS 0.468 pg/mL 0.0005e10.0 ng/mL [127]
vibrational characteristics, and large
surface area
GCE 3DPt/HGO Drop casting Large specific surface area, improved DPV 0.0006 ng/mL 0.001e150 ng/mL [128]
13

electrical conductibility resulting in

magnified loading of antibodies
GCE rGO Drop casting Excellent electrical conductivity, non- CV, EIS 0.05 ng/mL 0.1e5 ng/mL [129]
toxicity, good biocompatibility, and
high surface area
MUC1 SPCE GO-COOH Drop casting Inherent peroxidase-like activity, DPV 0.04 U/mL 0.1U/mL- 2 U/mL [130]
excellent conductive surface
CA 27-29 GCE Au/MoS2/rGO NCs Drop casting Magnified electrochemical Amperometry 0.08 U/mL 0.1e100 U/mL [131]
conductivity
CA 15-3 SPE PPy nanowire and poly (1,5 Electropolymerization Large surface to volume ratio and EIS, CV 0.02 U/mL 0.05e20 U/mL [132]
diaminonaphthalene). magnified conductivity
SPGE e e e EIS, CV, SWV 0.05 U/mL 0.25e10.00 U/mL [133]
SPCE CuS NPs-rGO Drop casting Augmented electrocatalytic activity DPV 0.3 U/mL 1e150 U/mL [134]
towards oxidation of catechol
SPGE CoS2-Gr-AuNPs Drop casting Augmented electrocatalytic activity DPV 0.03 U/mL 0.1e150 U/mL [135]
towards the oxidation of catechol

Materials Today Chemistry 26 (2022) 101129

along with a large surface area
resulting in enhanced immobilization
of antibodies
SPCE Au-rGO Drop casting Excellent electrical conductivity, non- EIS 0.08 fg/mL 0.1 fg/mL-1 mg/mL [136]
toxicity, good biocompatibility, and
high surface area
SPGE MSA SAM Provide narrow carboxylic-acid- DPV 0.95 U/mL 1.0e1000 U/mL [137]
terminated monolayer
GCE Nitrogen-doped Gr sheets Drop casting Excellent electrical conductivity and DPV 0.017 U/mL 0.1e20 U/mL [138]
large surface area
GCE AuPd NCN Drop casting Excellent electrical conductivity, large Chronoamperometry 0.35 fg/mL 0.001 pg/mL-100 ng/mL [139]
surface area, and improved structural
stability
(continued on next page)
 Table 5 (continued )

P. Lakhera, V. Chaudhary, A. Jha et al.

Biomarkers Electrode Surface functionalization of Method for electrode Advantage of functionalization Detection method Limit of detection Linear range Ref
type electrodes functionalization

GCE rGO/PDA and NH2 functionalized Electropolymerization Excellent electrical conductivity and SWV 0.002 U/mL 0.002e125 U/mL [140]
mesoporous Si conjugated by Au NPs large surface area
GCE GAs and Pb-CD Drop casting and Excellent electrical conductivity and DPV, EIS 0.03 mU/mL 0.1 mU/mL-100 U/mL [141]
electropolymerization large surface area
GCE Cysteamine A/Au NSs/GQDs Electrodeposition Large surface area for the enhanced SWV 0.11 U/mL 0.16e125 U/mL [142]
immobilization of CA 15-3
GCE Gr ink conjugated with anti-CA15-3 Drop casting Excellent electrical conductivity DPV 15 U/mL 15e250 U/mL [143]
GCE Au NPs Electrodeposition Efficient electron transfer, enhanced EIS 5.06 mU/mL 10e100 U/mL [144]
conductivity, easy functionalization,
and good biocompatibility
ER SPCE e e e Amperometry 19 pg/mL 63e200 pg/mL [145]
PR SPCE e e e Amperometry 22 pg/mL 73e1500 pg/mL [146]
Aptasensors
HER2 SPE PLL Electrostatic adsorption Provide support for aptamer DPV 3.0 ng/mL 10e60 ng/mL [147]
immobilization
SPGE Thiolated DNA aptamer and MCH SAM Provide a bridge between the surface EIS 172 pg/mL 1 pg/mL-1 mg/mL [12]
irregularities and reduce the
adsorption of non-specific proteins
SPCE Au -NPs Drop casting Provide support for aptamer CV, DPV, and EIS 0.001 ng/mL 0.001e100 ng/mL [148]
immobilization
GCE rGO-chitosan film Drop casting Provide amine groups for aptamer DPV 0.21 ng/mL 0.5e2 ng/mL [149]
binding, stability, and large surface
area
GCE ErGO- SWCNT and AuNPs Drop casting (ErGO) and Better electrocatalytic property and CV, DPV, and EIS 50 fg/mL 0.1 pg/mL-1 ng/mL [32]
electrodeposition (AuNPs) magnified immobilization of
biorecognition molecule
14

CEA GCE (MoSe2)/Gr/Au Drop casting (MoSe2/Gr) and Magnified electron transfer DPV 0.03 pg/mL 0.1 pg/mL-100 ng/mL [150]
electrodeposition (AuNPs)
GCE GO Drop casting Better electrocatalytic property SWV 0.05 pg/mL 0.0001 pg/mL-10 ng/mL [151]
GCE AuNPs @ NH2-functionalized Drop casting Excellent electrical conductivity and EIS 9.8  104 ng/mL 1.0 £ 103-100.0 ng/mL [152]
mesoporous Si film large surface area
GCE GO Drop casting Excellent electrical conductivity and SWV 0.1 pg/mL 0.5 pg/mL- 1 ng/mL [153]
large surface area
GCE HAuCl4 Electrodeposition Large surface area EIS 0.023 pg/mL 0.05 pg/mL- 20 ng/mL [154]
MUC1 SPCE CNT Dip coating Excellent conductivity, EIS 0.02 U/mL 0.1e2 U/mL [155]
electrocatalytic properties, and
magnified surface area
GCE rGO/Au NPs Drop casting Excellent electrochemical CV and EIS 0.25 pM 1 pM-1 mM [156]
performance and support for aptamer
immobilization
GCE Au NPs Electrodeposition Excellent conductivity, SWV 0.33 pM 1.0 pM 10 mM [157]
electrocatalytic properties, and

Materials Today Chemistry 26 (2022) 101129

magnified surface area
ER SPGE e e e DPV 0.001 ng/mL 0.001e1000 pg/mL [158]
PR SPCE GQDseNiO-AuNFs/f-MWCNTs Drop casting Efficient immobilization matrix due DPV 1.86 pM 0.01e1000 nM [159]
to COOH group
VEGF SPCE PANI/CNT Drop casting Large surface area and excellent DPV 0.4 pg/mL 0.5e10.0  106 pg/mL [160]
electrocatalytic properties
GCE BSA/Au NCs Drop casting Easy functionalization, large specific DPV, and EIS 0.32 and 0.48 pM 1e125 pM, and 2.5e250 [161]
surface area, efficient conductivity, pM
and low toxicity
GCE PLL Electropolymerization Provide support for aptamer CV 4.6 pmol/L 6.0e20 pmol/L [162]
immobilization
Circulating tumor cells
EpCAM SPE Si NPs and IL Drop casting Large surface area and enhanced DPV 1 cells/mL 5-10000000 cells/mL [163]
conductivity
 P. Lakhera, V. Chaudhary, A. Jha et al.
GCE Au-NPs Drop casting Increased electron conductivity EIS, DPV 50 cells/mL 80- 10000000 cells/mL [164]
SKBR3 SPE rGO Drop casting Biocompatibility, large surface area, DPV 21 cells/mL 500e30000 cells/mL [165]
easy functionalization, and low-cost
processing technology
GCE e e e DPV 1 cell/mL 1-80 cells/mL [166]
MCF7 MGCE rGO/MoS2 Drop casting Large surface area, excellent electron DPV 6 cells/mL 15-45 cells/mL [167]
conductivity, and good
biocompatibility
GCE rGO/AuNPs e Large surface area, excellent electron CV, DPV 27 cells/mL 50-7000 cells/mL [168]
conductivity, and good
biocompatibility
GCE Au nanocages/MWCNT-NH2 Drop casting Large surface area, excellent electron DPV 80 cells/mL 100-1000000 cells/mL [169]
conductivity, and good
biocompatibility
GCE PANI Electrodeposition Excellent electron conductivity and DPV 20 cells/mL 50-1000000 cells/mL [170]
good biocompatibility
GCE TiO2 nanotube-rGO Drop casting Large surface area, excellent electron EIS 40 cells/mL 1000-10000000 cells/mL [171]
conductivity, and good
biocompatibility
GCE rGO-chitosan-Au NPs composite Drop casting Excellent electron conductivity and EIS 4 cells/mL 10-1000000 cells/mL [172]
large surface area
GCE VS2/Au NPs Drop casting Large surface area and good DPV 5 cells/mL 10-100000 cells/mL [173]
mechanical strength
MDA-MB-231 GCE Mannose-C2NH2 Electrografting e CV 10 cells/mL 10- 100000 cells/mL [174]
GCE HA-BSA-AuNPs Drop casting Large surface area, excellent electron EIS 128 cells/mL 200- 300000 cells/mL [175]
conductivity, and good
biocompatibility
Multiple targets
CA 15-3, and HER2- SPdCE AuNPs In-situ electrodeposition Large surface area, excellent electron LSV CA 15-3: 5.0 U/mL; CA 15-3: 0e70 U/mL; [176]
ECD conductivity, and good HER2-ECD: 2.9 ng/mL HER2-ECD: 0e50 ng/mL
15

biocompatibility
TNF, and RANKL SPdCE e e e Amperometry TNF: 3.0 pg/mL TNF: 9.9e1000 pg/mL; [177]
RANKL: 2.6 pg/mL; RANKL: 8.6e1000 pg/mL
MUC1, and MCF-7 GCE e e e SWV MUC1: 0.5 nM; MUC1: 1e500 nM; [178]
MCF-7: 50 cells/mL MCF-7: 50e5000 cells/mL
GCE Poly (glutamic acid)/MWCNT NCs Drop casting Large surface area and durability DPV MUC1: 3 ng/mL; MUC1: 5e200 ng/mL; [179]
MCF-7: 25 cells/mL MCF-7: 100
e10000000 cells/mL
CEA, and CA 15-3 GCE Au NPs/3-D Gr hydrogel e Fast electron transfer and large DPV CEA: 11.2 pg/mL; CEA: 5.0  102-60.0 ng/ [180]
surface area for the effective CA 15-3: 1.3  102 U/ mL;
immobilization of more aptamer mL CA 15-3: 5.0  102 -100.0
U/mL
miRNA21, and CA SPGE MoSe2/GO-NCs Large surface area, excellent electron DPV miRNA-21:1.2 fM; [181]
15-3 conductivity, and good CA 15-3: 0.14 U/mL
biocompatibility

Materials Today Chemistry 26 (2022) 101129

Abbreviations: 2D-ReS2: two-dimension rhenium disulfate; 3D:3-dimensional; 3DPt/HGO:3-dimensional graphene oxide-supported platinum metal nanoparticles; APTES: 3-aminopropyltriethoxysilane; Au NPs@ strp: gold
nanoparticles coated with streptavidin; Au: gold; BRCA1: breast cancer types A1; BSA: bovine serum albumin; CA: cancer antigen; cDNA: complementary DNA; CDs: carbon nanodots; CEA: carcinoembryonic antigen; CNNS:
carbon nitride nanosheets; CNT: carbon nanotube; CoPeBNF: cobalt porphyrin binuclear framework; CTC: circulating tumor cells; CuS: copper sulfide; CoS2: cobalt sulfide; CV: cyclic voltammetry; DNA: deoxyribonucleic acid;
DPASV: differential pulse anodic stripping voltammetry; DPV: differential pulse voltammetry; EIS: electrochemical impedance spectroscopy; ErGO: electrochemical reduced graphene oxide; EpCAM: epithelial cell adhesion
molecule; ER: estrogen receptor; GAs: graphene aerogel; GCE: glassy carbon electrode; GNRs: gold nanorods; GO: graphene oxide; Gr: graphene; HA: hyaluronic acid; HAuCl4: chloroauric acid; HCT: herceptin; HER2: human
epidermal growth factor receptors-2; His-Tag-ZFP: His-Tag-Zinc finger protein; IL: ionic liquid; Ir: iridium; LSV: linear sweep voltammetry; MBs: magnetic beads; MCH: 1-mercapto-6-hexanol; MGCE: magnetic glassy carbon
electrode; MIP: molecularly printed polymer; miRNA: micro-RNA; MOF: metal organic framework; MoS2: molybdenum di sulfide; MoSe2: molybdenum selenide; MSA: mercaptosuccinic acid; MWCNT: multiwalled carbon
nanotube; NCN: nanochain network; NCs: nanocomposites; NH2-GSs: amine functionalized graphene sheets; NPs: nanoparticles; P[DA-b-CD/CTAB]): poly (dopamine-beta cyclodextrine-Cetyl trimethylammonium bromide);
P3CA: pyrrole -3-carboxylic acid; PANI: polyaniline; Pd: palladium; PDA-Pb2þ: polydopamine-Pb2þ; PEDOT: poly(3,4-ethylenedioxythiophene); PEG: polyethylene glycol; PLL: Poly-L-Lysine; Poly A: poly-adenine; PPy: pol-
ypyrrole; PR: progesterone receptor; Pt: platinum; Pb-CD: polymeric b-cyclodextrin; QDs: quantum dots; RANKL: Receptor Activator of Nuclear Factor-kB Ligand; rGO: reduced graphene oxide; SAM: self-assembled monolayer;
Si: silica; SKBR3: SloaneKettering breast cancer; SNGQDs@AuNPs: gold nanoparticles functionalized graphene quantum dots; SnO2: stannic oxide; SPCE: screen printed carbon electrode; SPdCE: dual screen-printed carbon
electrode; SPE: screen printed electrode; SPGE: screen-printed gold electrode; SWCNT: single walled carbon nanotube; SWV: square wave voltammetry; TEPA: tetraethylene pentaamine; TGO: thiolated graphene oxide; TiO2:
titanium di oxide; TNF: Tumor Necrosis Factor-alpha; tPA: Human tissue plasminogen activator; TSP: tetrahedral-structured probe; VEGF: vascular endothelial growth factor; VS2: vanadium disulfide; WO3: tungsten trioxide;
ZrO2: Zirconium dioxide.
P. Lakhera, V. Chaudhary, A. Jha et al.
system was formed by the capture DNA, target DNA, and reporter
DNA (labeled with Au NPs) (Fig. 7). The genosensing principle was
based on the EC signals of the redox reaction in the presence of
3,3ˊ,5,5ˊ-tetramethylbenzidine (TMB). The developed biosensor
exhibited high sensitivity and specificity with a linear range (LR)
from 1 fM to 1 nM, and a LOD of 0.1 fM. The results revealed that the
DNAs were stable on the surface of the biosensor due to the
sandwich system resulting in increased specificity against the non-
complementary sequence and blank [78].
In another study, an ultrasensitive DNA biosensor was devel-
oped for the attomolar (aM) detection of BRCA1 by using Gr-
modified GCE (GCE/Gr). In this sensor, DNA-c was initially immo-
bilized on the GCE/Gr surface via p-p interaction and followed by
hybridization to the specific part of DNA-t resulting in the forma-
tion of the GCE/Gr/DNA-c|DNA-t complex. Further, the unhybri-
dized part of this complex was conjugated to the Au-NPs labeled
DNA-r (DNA-r-Au-NPs) resulting in a sandwich of GCE/Gr/DNA-c|
DNA-t|DNA-r-Au-NPs. The analytical signals originated from the EC
oxidation of Au-NPs in presence of perchloric acid was measured by
CV with a magnified LOD (1 aM). Moreover, the genosensor could
detect up to 50 aM DNA-t even with a low amount of sample (6 mL)
[79].
Wang et al. fabricated low fouling GCEs-based genosensor for
the detection of BRCA1 mutations. It was a modified GCEs-based
impedimetric hybrid genosensor where the electrode surface was
modified by cross-linked hydrated polyethylene glycol (PEG) and
self-assembly of Au-NPs resulting in PEG/AuNPs/GCEs. The capture
probe (thiol functionalized oligonucleotides, S1) was covalently
attached to the modified electrode surface resulting in the forma-
tion of S1/PEG/AuNPs/GCEs. Further, the hybridization experiment
was performed by immersing the S1/PEG/AuNPs/GCEs into
different concentrations of target analytes for 2 h followed by
rinsing with phosphate buffer saline to remove the non-specific
bound target analytes. The interfacial change and quantification
of BRCA1 biomarkers were carried out by EIS. The results revealed
that the developed hybrid GCE-based biosensor was highly sensi-
tive (LOD of 1.72 fM) and highly effective (LR from 50.0 fM to
1.0 nM) for the detection of BRCA1 due to the surface modification
of PEG and Au- NPs. Moreover, the developed biosensor was
biocompatible, sufficient to screen the DNA mismatch without any
complex chemistry, and quantify a broad range of serum bio-
markers [80]. Hui et al. developed another, antifouling GCEs-based
genosensor by grafting pegylated polyaniline fiber (PANI) on the
surface of GCEs leading to an increase in the surface area, con-
ductivity, and antifouling property. The detection of BRCA1 was
based on the hybridization reaction between the target BRCA1 and
immobilized capture DNA, using methylene blue (MB) as an EC
indicator. The DPV results revealed that the reduction current of MB
was decreased after the hybridization reaction. The developed
biosensor was highly sensitive to the BRCA1 with an LR from 0.01
pM 1 nM and LOD of 0.0038 pM. Moreover, the developed
biosensor could easily quantify BRCA1 in complex human serum
[81]. In another study, Wang et al. fabricated PEDOT (poly (3,4-
ethylene dioxythiophene) modified GCEs-based antifouling and
highly sensitive EC biosensor for the detection of BRCA1 gene
mutation by using DPV. The biosensing results revealed that the
DPV peak current was inversely proportional to the target DNA
concentration. Further, the target DNA analysis was similar to
another affinity biosensor and can detect the sample with a higher
target concentration. However, for the detection at a lower target
concentration, the biosensor needs to be revived to dissociate the
bound targets. The developed biosensor was highly selective and
ultrasensitive (LR:1.0  1016M-1.0  1010 M; LOD: 0.03 fM), and
able to detect BRCA1 in human plasma (5% v/v) with low fouling
16
Materials Today Chemistry 26 (2022) 101129
and magnified accuracy due to the integration of conduction
polymer (PEDOT) and antifouling zwitterionic peptide [82].
Shahrokhian et al. developed a DNA biosensor by using pyrrole-
3 carboxylic acid-coated reduced graphene oxide (rGO) modified
GCE (PP3CA/ERGO/GCE). The EC behavior of the redox probe was
determined by CV, DPV, and EIS resulting in the quantitative
determination of BRCA1 in the LR of 10 fMe0.1 mM with a LOD as
low as 3 fM. The results revealed that the PP3CA/ERGO/GCE
possessed more active sites in contrast to polymer-coated GCE.
Moreover, the fabricated ultrasensitive genosensor exhibited a
perfect differentiation between the complementary, non-
complementary, and mismatched DNA sequences due to the
polymeric and Gr coating on the GCEs surface. Additionally, the
modified GCE was efficient to determine the trace amount of DNA-t
in plasma samples [83]. Hasanzadeh et al. developed an ultrasen-
sitive immunosensor for the detection of BRCA1 based on modified
GCE with poly (dopamine-beta cyclodextrin-Cetyl trimethy-
lammonium bromide) doped with AgNPs (P[DA-b-CD/CTAB])-
AgNPs and functionalized mesoporous silica. The modified elec-
trodes exhibited augmented surface area and magnified conduc-
tivity leading to an increase in the immobilization of HRP-labeled
antibody (HRP-Ab2). The developed immunosensor exhibited
magnified sensitivity and specificity for the detection of BRCA1
with a LOD of 0.003 pg/mL, and an LR from 0.01 to 10 pg/mL and
0.62e20 pg/mL by DPV, and SWV respectively [84]. Recently, a
label-free EC biosensor based on three-dimensional rGO (3D-rGO)
and PANI-modified GCEs (3D-rGO-PANI/GCE) was developed for
the detection of BRCA1. The results revealed that PANI and 3D-rGO
exhibited a synergistic effect leading to reduce charge transfer
resistance and magnified EC activity of modified electrode. EC
analysis of BRCA1 was carried out by immobilization of capture
probe (ss DNA) on the surface of the modified electrode, and further
hybridized with the DNA-t. The EC evaluation of the functionalized
electrode was carried out by CV, DPV, and EIS. The fabricated
biosensor was highly sensitive (1.0  1015-1.0  107 M), and
selective with a LOD of 3.01  1016 M. Moreover, no significant
interference was observed in the biosensor selectivity neither in
the complex real blood samples nor the simple matrix [85]. Very
recently, a novel EC sandwich-type genosensor was developed to
detect the BRCA1 gene using modified GCE with the metal-organic
framework (MOF) with @ iron oxide (Fe3O4) NPs core, and
multiwalled carbon nanotube (MWCNT). The genosensor was
fabricated by the immobilization of the capture probe (cDNA) on
the modified electrode followed by casting the target analyte and
further hybridization with the redox-cycling ferrocene labeled
reporter label probe (r-Fc-DNA). The target was analyzed by
r-Fc-DNA via the electro-catalytic activity of Ferri/Ferrocyanide
resulting in an enhancement of the r-Fc-DNA oxidation peak cur-
rent (Fig. 8). The developed genosensor was stable, reproducible,
and ultrasensitive with an LR from 1  1015- 1  1010 M and a
LOD of 0.57  1015 M [86].
5.2. Detection of transcriptomic biomarkers
Messenger RNA (mRNA) transcribed from the DNA is widely used
as a biomarker owing to its ability to reveal the functional descrip-
tion of proteins. mRNAs are single-stranded RNA (ssRNA) structures
resembling ssDNA where thiamine is replaced by uracil, hence
having a similar detection principle (Fig. 6). The complete set of RNA
comprises mRNA, microRNA (miRNA), extracellular RNA (exRNA),
and circulatory RNA (cirRNA) is used for the early detection of
different cancers [5,48]. Among these biomarkers, miRNAs are the
most extensively non-invasive biomarkers for the detection of
various diseases such as cardiovascular, diabetes, viral infections,
P. Lakhera, V. Chaudhary, A. Jha et al. Materials Today Chemistry 26 (2022) 101129

Fig. 6. Working principle of the electrochemical genomic biosensor by using modified screen-printed electrode (SPE)/glassy carbon electrode (GCE). a) immobilization of capture
probe on the working electrode via functional groups; b) specific hybridization reaction between the capture probe and target probe; c) hybrid target DNA with capture probe and
labeled reporter probe; d) electrochemical transduction of affinity reactions into detectable signals.

rheumatoid, neurological disorders, and human cancers
[1,35,187,188]. miRNAs are non-coding endogenous RNA molecules
with a length of approximately 18e25 nucleotides [5,186] that play
an important role in various biological processes like cell prolifera-
tion, apoptosis, differentiation, metabolism, and tumorigenesis [5].
Typically, these are present in the range of 200 aM to 20 pM [1,189]
but abnormal expression (upregulation or downregulation) is
associated with cancer, therefore can be used as a diagnostic
biomarker. The miRNAs up-regulated in tumor cells are miR-16,
miR-21, miR-27a, miR29b, miR- 29c, miR- 93, miR-150, miR-155,
miR-191, miR-200c, miR-210, miR- 213, miR- 222, and miR- 451.
Similarly, the downregulated miRNAs are Let7a-2, miR- 10b, miR-
100, miR-125b, miR-145, miR-193, miR-205, and miR- 497 [10].
Among these miRNA, miR-21 is the widely used stable target
biomarker for the early detection of BC owing to its higher sensitivity
(87.6%), and specificity (87.3%) but possesses certain limitations like
presence in other cancers, sequence homology with related RNAs,
and low serum concentration [10,190]. EC nucleic acid-based

Fig. 7. Development and principle of electrochemical genosensor for the detection of BRCA1 based on carbon dots modified screen-printed gold electrode using thionine as a redox
indicator adapted from Ref. [78], MDPI, 2020.

17
P. Lakhera, V. Chaudhary, A. Jha et al.
biosensors (labeled or label-free) provide effective alternatives to
quantify miRNA and can overcome the limitation of various con-
ventional strategies like In Situ Hybridization (ISH) microarrays,
Reverse Transcriptase PCR (RT-PCR), and Northern Blotting [22,186].
Recently, various EC miRNAs biosensing approaches have been re-
ported by using modified SPEs and GCEs (Table 5). Regarding the
application of SPEs for the detection of miRNAs, Rodríguez et al.
developed A p19-based disposable amperometric magnetic
biosensor by using SPCE for the simultaneous detection of miR-21
and miR-205. The fabricated biosensor comprises antimiR-21 and
antimiR-205 probes, chitin-modified magnetic beads (Chitin-MBs),
the p19 viral protein as a capture probe and immobilized on the dual
SPCE (SPdCE). In this biosensor, SPdCEs were responsible for the
simultaneous detection of two BC by using amperometric tech-
niques with the hydrogen peroxide (H2O2)/HQ system. The devel-
oped biosensor was able to detect both the t-miRNAs in less than 2h
together with magnified sensitivity and selectivity with an LR from
2.0 to 10.0 nM and LOD of 0.6 nM. Further, the application of this
approach was analyzed by screening the endogenous levels of both
t-miRNAs in total RNA [87]. Erdem et al. developed an indicator-free
voltammetric miRNA biosensor by using carbon nanofiber modified
SPEs (CNF-SPEs) for the detection of miRNA-34a. This biosensor was
composed of amino linked DNA capture probe that was immobilized
on the CNF-SPEs. The detection of hybridization between the cap-
ture probe and the miRNA-34a target was investigated by DPV. The
results revealed that the CNF-modified SPEs were reproducible and
stable with an enhanced guanine signal (18-fold) in contrast to the
conventional electrode owing to the high conductivity and
increased electrocatalytic effect of CNF. Moreover, the LOD of t-
miRNA was found to be 10.98 mg/mL in the LR of 25e100 mg/mL [88].
In another study, Cardoso et al. developed a simple ultrasensi-
tive EC biosensor for the detection of the attomolar concentration
of miRNA-155 in BC. The biosensor was developed by immobili-
zation of anti-miRNA-155 (capture probe) on the SPGEs followed by
blocking the non-specific binding area with mercaptosuccinic acid
(MSA). The hybridization between the capture probe and the t-
miRNA-155 was analyzed by EIS. The developed biosensor was
simple and can detect the miRNA-155 from 10 aM to 1.0 nM with a
LOD of 5.7 aM in human serum samples. Moreover, this strategy can
simultaneously quantify multiple miRNA-155 in biological fluids
[89]. In another study, Mohammadi et al. developed a SPEs-based
label-free miRNA biosensor for the detection of miRNA-155. The
biosensing was based on sandwich hybridization of streptavidin
MBs integrated with a biotinylated capture probe with a portion of
the microRNA and monitored by DPV. The developed biosensor was
highly sensitive with a LOD of 29 pmol/L and LR from 81 pmol/L to
1.2 nmol/L [90].
Povedano et al., reported a simple amperometric biosensor for
the screening of miRNA using SPCE. The biosensor comprised an
MBs functionalized His-Tag-Zinc finger protein (His-Tag-ZFP) cap-
ture probe that binds preferably to RNA hybrids and a biotinylated
synthetic complementary RNA detector probe (b-RNA-Dp). Further,
StrepeHRP conjugation allowed amperometric detection using the
H2O2/HQ system upon capturing the magnetic bioconjugates at
SPCEs. The developed biosensor was sensitive (LOD:0.91 nM;
LR:3.0e100 nM) and can detect the t-miRNAs within 2 h. Moreover,
the biosensor can also analyze miRNA-21 from total RNA extracted
from adenocarcinoma and epithelial non-tumorigenic breast cells
without target amplification, concentration beforehand, or reverse
transcription steps [91]. In another study, Zouari et al. fabricated an
enzyme-free voltammetric biosensor based on Au-NPs and rGO-
modified SPCE for the detection of miRNA-21. The biosensing
assay was based on the sandwich-type hybridization of a capture
probe (thiolated DNA) and a ferrocene-capped AuNPs conjugated
with streptavidin (FcAuNPsStrep) labeled biotinylated detector
18
Materials Today Chemistry 26 (2022) 101129
probe with a target analyte and further monitored by DPV using Fc
as redox reporter (Fig. 9). This enzyme-free biosensor was able to
detect the target analyte with a LOD of 5 fM and an LR from 10 fM-2
pM. Moreover, the biosensor can directly detect the t-miRNA-21
from the diluted samples from BC patients with 2-month storage
[92].
Recently, a highly sensitive EC biosensor based on modified SPEs
was fabricated for the early screening of miRNA by using DPV. The
biosensor was composed of modified SPCE with Gr, polypyrrole
(PPy), and Au-NPs leading to ameliorating the electron transfer
properties and augmenting the intercalation of MB for signal
amplification. The fabricated biosensor was operated by observing
the MB signal response because of the number of hybridization
products between miRNA-21 and DNA-21 probes immobilized on
the electrode surface. The DPV results revealed that the increase in
peak current was directly proportional to the concentration of the
target. The fabricated biosensor was reproducible, selective, and
highly stable possessing an LR of 1.0 fM to 1.0 nM with a LOD of
0.020 fM and can detect the miRNA [93]. In another study, the same
group fabricated a label-free EC biosensor-based SPCE modified
with Au-NPs/Gr QDs/GO for the detection of miRNA-155, miRNA-
21, and miRNA-210. Different redox indicators like MB, anthraqui-
none (AQ), and polydopamine (PDA) were used as signal indicators
for anchoring capture probes, which hybridized with the comple-
mentary miRNA-155, miRNA-21, and miRNA-210, respectively.
Quantification of miRNA-155, miRNA-21, and miRNA-210 was car-
ried out by measuring decreases in the MB, AQ, and PDA EC signals
respectively. Further, the three well-separated peaks were moni-
tored by SWV. The fabricated biosensor was ultrasensitive and can
easily detect the miRNA-155, miRNA-21, and miRNA-210 simulta-
neously with an LR from 0.001 to 1000 pM and LOD of 0.33, 0.04,
and 0.28 fM respectively [94].
In the recent past, EC biosensors based on modified GCEs, and
label-free/labeled probes played an important role in the detection
of miRNA. Rafiee-Pour et al. developed a label-free EC biosensor
based on MWCNT-modified GCE using MB as a redox indicator. The
modified electrode was immobilized with ss-DNA and incubated
with the t-miRNA-21. EIS and CV were used to analyze the hy-
bridization reaction whereas, DPV was used to measure the MB
oxidation peak current. The results revealed that after hybridization
peak current was increased. This strategy can successfully detect
the miRNA in an LR from 0.1 to 500.0 pM with a relatively LOD of
84.3 fM [95]. In another study, the EC biosensor based on GCE
modified with GO and gold nanorods (GNRs) was developed for the
detection of miR-199a-5p in serum. The sensor was developed by
immobilization of the thiolated oligonucleotide probe on the
modified electrode surface and followed by blocking of unspecific
binding by 6-mercapto-1-hexanol solution. The developed
biosensor was highly sensitive and can evaluate a low concentra-
tion of serum miR-199a-5p with an LR from 15 fM to 148 pM and
LOD of 4.5 fM [96]. Recently, a label-free miRNA biosensor was
developed by using modified GCE with polyamidoamine dendrimer
functionalized PPy nanowires using EIS. The fabricated biosensor
was ultrasensitive with an LR from 1014M-108M and LOD of
0.34  1014 M. Moreover, the biosensor exhibited ~3.12-time
higher sensitivity in contrast to the PPy-modified biosensor [97].
Direct labeling detection is based on the labeled capture hy-
bridized with the t-miRNAs giving rise to a change in EC signal
response, which is proportional to t-miRNAs concentration [186].
Azimzadeh et al. developed another miRNA biosensor based on
modified GCE with GNRs attached to the GO sheet surface. Early
diagnosis of BC was carried out by the quantification of miRNA-155
by using ss-probe and Oracet blue (OB, as an EC indicator), and
signals were measured by DPV. The developed biosensor was able
to discriminate between the complementary t-miRNA-155, single-
P. Lakhera, V. Chaudhary, A. Jha et al. Materials Today Chemistry 26 (2022) 101129

Fig. 8. Fabrication principle of electrochemical sandwich-type genosensor for BRCA1 based on glassy carbon electrode modified with the metal-organic framework (MOF) with @
iron oxide nanoparticles (Fe3O4 NPs) core, and multiwalled carbon nanotube (MWCNT). Reprinted with permission from Ref. [86].

and three-base mismatch, and non-complementary miRNA along
with high specificity and sensitivity (LR: 2.0 fM-8.0 pM; LOD:
0.6 fM). Moreover, the biosensor was stable, reproducible, and
exhibited real plasma sample analysis [98].
Another approach used for the detection of t-miRNAs is based on
the elimination of labeled probes from the electrode surface after the
hybridization with the target miRNA via displacement reaction [99]
or using different splitting agents [100]. Regarding this, an impedi-
metric miRNA biosensor based on ZrO2erGO modified GCE
integrated with a catalytic hairpin assembly (CHA) was developed
for the detection of miRNA-21. The CHA assembly comprised two
hairpin DNA molecules; H1 and H2). H1 modified with the amine
group was covalently attached to the modified GCE surface via
amine coupling chemistry. It was observed that there was no hy-
bridization reaction between H1 and H2 in the absence of the
miRNA-21 but in the presence of the target, a miRNA hybridization
reaction took place between the target analyte and H2 structure
resulting opening of the H2 closed structure. Later on, H1 was

Fig. 9. Sandwich-type detection of miRNA based on gold nanoparticles (Au-NPs) and reduced graphene oxide (rGO) modified screen-printed carbon electrode (SPCE) using DPV.
Sandwich hybridization involves thiolated DNA as a capture probe (SH-DNAcp) and ferrocene-capped AuNPs conjugated with streptavidin (FcAuNPsStrep) labeled biotinylated
detector probe. Reprinted with permission from Ref. [92].

19
P. Lakhera, V. Chaudhary, A. Jha et al.
hybridized with the unfolded H2 and t-miRNAs were released due to
the displacement reaction. Further, H2 was immobilized onto the
electrode surface, and the released t-miRNA initiated another next
cycle. The developed biosensor was able to determine the target
analyte in the LR from 10 fM to 100 pM with a LOD of 4.3 fM [99]. In
another study, Cheng et al. developed an immobilization-free
impedimetric biosensor by using MBs modified GCEs based on
duplex-specific nuclease-assisted target recycling (DSNATR) for the
detection of miRNA-21. The capture probe was not hydrolyzed in the
absence of target RNA owing to the less activity of DSN while forming
DNA-RNA heteroduplex in presence of target RNA. Further, DSN
hydrolyzed the target binding portion of the capture probe resulting
in free target RNA to hybridized with another new capture probe and
instigating the second hydrolysis cycle. The fabricated biosensor was
a promising approach for the early diagnosis of BC along with a LR
from 0.5 to 40 fM and LOD of 60 aM. Moreover, the approach
exhibited very little cross-hybridization between similar miRNAs
[100]. Recently, Ma et al. developed an effective and simple EC
biosensor for the detection of miRNA-21 by using Au-NPs/carbon
nitride nanosheets (CNNS) modified GCEs, thiol modified aptamer
capture t-DNA probe (Fig. 10). Moreover, MB integrated on the 3’ of
capture t-DNA was utilized as an EC signal. Additionally, the selec-
tivity and sensitivity of the biosensor were magnified by DSN and
CHA assembly. The results revealed that the SWV signal was strong
in the absence of t-RNA, whereas the MB signals became poor in the
presence of t-RNA. The DSN can selectively split capture t-DNA of the
DNA-t-RNA hybrid and liberate the t-RNA repeatedly. The variation
in the current peak was related to the concentration of miRNA-21.
The developed biosensor was reproducible and stable with an LR
of 10 fM to 1 nM and LOD of 2.9 fM [101].
5.3. Detection of proteomic biomarkers
The proteomic biomarkers (soluble, glycoproteins, or membrane-
associated glycoproteins) are widely associated with BC development
Fig. 10. Development of an electrochemical biosensor for the detection of miRNA-21 based on glassy carbon electrode (GCE) modified with gold nanoparticles (Au-NPs)/carbon
nitride nanosheets (CNNS). Reprinted with permission from Ref. [101].
20
Materials Today Chemistry 26 (2022) 101129
and progression owing to their relative abundance in comparison to
DNA and RNAs. There are various proteomic biomarkers associated
with BC development like the human epidermal growth factor (HER)/
epidermal growth factor receptor (EGFR) family, carcinoembryonic
antigens (CEA), mucin 1 (MUC1), cancer or carbohydrate antigens (CA;
CA15-3, CA27-29, CA125), estrogen receptor (ER), and progesterone
receptor (PR) [10,48]. Other proteomic biomarkers are vascular
endothelial growth factor (VEGF), epithelial cell adhesion molecule
(EpCAM), Cyclin E, human epididymal protein 4 (HEP4), cathepsin D
inflammatory proteins (fibrinogen and soluble intracellular adhesion
molecule 1), cell-free DNA (cfDNA), urokinase-dependent plasmin-
ogen activator system (uPA), Thomsen-Friedenreich (TF), plasmin-
ogen activator inhibitor (PAI), amino acid transporter (ASCT2), folic
acid (FA), beta protein 1(BP 1), tenascin-C (TN-C), matrix metal-
lopeptidase 9 (MMP-9), murine double minute 2 (MDM-2), serine
hydroxymethyltransferase 2 (SHMT2), apurinic/apyrimidinic endo-
nuclease 1 (APE-1), Human tissue plasminogen activator (tPA), and a
cluster of differentiation 105 and 146 antigens (CD 105, CD146)
[1,5,37]. Among the various proteomic biomarkers, HER2, CA 15e3, CA
27e29, and CA125 are FDA-approved for clinical application [48,191].
EC sensing of proteomic biomarkers is usually carried out by using
antibodies (immunosensors) or aptamer (aptasensors) or a combi-
nation of both as a BRE. Therefore, this section is further divided ac-
cording to the BRE.
5.3.1. Electrochemical immunosensors for the detection of
proteomic biomarkers
This section summarizes the principle and recent examples of EC
immunosensors (ECIs) based on SPEs and GCEs coupled with various
NMs for the detection of extensively used proteomic biomarkers.
ECIs are robust analytical tools that detect the concentration of the
target analyte in a sample by measuring the signals in response to
specific binding between antibodies and antigens [36,37]. These
sensors are usually constructed via the immobilization of BRE (an-
tibodies or antigens) on the electrode surface and coupled to a
P. Lakhera, V. Chaudhary, A. Jha et al.
physical transducer that measures the change in voltage or currents
resulting from complementary binding species (Fig. 11) [51]. These
sensors possess the inherent specificity of immunoreactions and
intelligibility of commercially available various physical transducers
leading to magnifying their sensitivity [192]. Mainly two types of
ECIs potentiometry and amperometry are widely used for clinical
applications [37]. Recently, impedimetric and capacitive sensors
have also gained interest owing to their direct application for the
determination of antigen-antibody reaction without using separa-
tion step and other reagents [38].
The ECIs work either with direct (label-free) or indirect (labeled)
sensors. The basic principle of both is the same in which the target
analyte (usual antigens) is captured by antibodies and followed by
blocking of unreacted surface and biorecognition of the analyte
(Fig. 11). A label-free ECI can detect the physical/chemical changes
directly during the complex formation without labeling the anti-
gens and antibodies (Fig. 11 a), whereas a labeled immunosensor
encompasses the labeled antibodies/antigens and measures the
signals generated by the labels (enzymes/electroactive species/
fluorophores/NPs) (Fig. 11 b, c) [36,38]. Direct immunosensors are
simple, cost-effective, and can be used for real-time analysis but
suffers from non-specificity and poor sensitivity. Therefore, a
proper blocking agent (surfactant, bovine serum albumin, casein,
etc.) is required to overcome the limitations [51]. However, indirect
immunosensors are highly sensitive, reliable, and specific as a
comparison to direct immunosensors, but also face certain limita-
tions like labeling molecules may influence the antigen-antibody
binding efficiency, and the yield of the antigen-antibody complex
labeled with various biomolecule may be variable [36]. Moreover,
indirect immunosensors can be divided into non-competitive and
competitive immunosensors [51]. Non-competitive immuno-
sensors are also known as two-site ῾sandwich assays´ which are
usually applied for huge antigens that possess more than one
epitope. In this method, the target analyte is sandwiched between
the excess amount of primary antibodies (capture antibody
immobilized on electrode surface), and secondary antibodies (a
labeled antibody that binds to antigen-antibody complex) resulting
Fig. 11. Working principle of electrochemical immunosensor for the detection of various proteomic biomarkers by using different immunoassays. a) label-free immunoassay; b)
sandwiched immunoassay; c) competitive immunoassay.
21
Materials Today Chemistry 26 (2022) 101129
in a detectable signal which is directly proportional to the con-
centration of analyte (Fig. 11 b) [37,51,193]. To remove the unbound
components a washing step is mandatory after each incubation
process. Competitive immunoassays also known as limited reagent
assays (limited amount of antibodies) are used for small antigens
with one epitope in which sample/unknown analyte and labeled
analyte are mixed where both compete with each other for the
same binding site of antibodies. Briefly, the limited number of
capture antibodies are immobilized on the electrode surface and
then incubated with the sample mixture having a known concen-
tration of labeled antigens and sample analyte (Fig. 11 c). Quanti-
fication of sample analyte is carried out by the determination of
labeled antigens concentration those bind to the antibodies. The
signal intensity generated by the labeled analyte is inversely pro-
portional to the sample analyte concentration [51]. Herein, EC
detection of major proteomic BC biomarkers will be discussed by
using immune or aptasensors based on SPEs and GCEs.
5.3.1.1. Detection of human epidermal growth factor and epidermal
growth factor receptor. HER and EGFR are cell transmembrane
glycoprotein receptors belonging to the ErbB family. This family
mainly comprises four receptors: (i) EGFR/HER1; (ii) HER2/neu; (iii)
HER3; (iv) HER4. These receptors are mainly responsible for auto-
phosphorylation of tyrosine, activating tyrosine kinase, and insti-
gating cell proliferation, and differentiation after binding with its
ligands like transforming growth factor (TGFa) and epidermal
growth factor [1,48]. Among these receptors, HER2/neu is the most
widely accepted prognostic BC biomarker that is overexpressed in
20e30% of non-invasive BC and leads to growing BC more aggres-
sively [10,12]. It comprises different domains such as the extracel-
lular domain (ECD), a transmembrane domain, and an intracellular
tyrosine kinase domain [176]. HER2-ECD is a minimal-invasive and
clinically relevant biomarker for the diagnosis of BC [194,195]. The
normal serum concentration of HER2 is 2e15 ng/mL and 15e75 ng/
mL in HER2-positive BC patients. It has been reported that over-
expression of HER2 is responsible for brain metastasis, chemother-
apeutic resistance, and recurrence of the disease in BC. Moreover, its
P. Lakhera, V. Chaudhary, A. Jha et al.
overexpression is also associated with other different cancers such
as ovarian, stomach, and lung cancer [3].
Recently, various EC immune/aptasensors with different trans-
ducers have been developed for the detection of HER2 and HER2-
ECD by using label-free [102e104] or sandwich immunoassays
[105e109]. Sharma et al. developed a low-cost and ultra-sensitive
label-free impedimetric immunosensor based on AuNPs/SPCE for
the quantitative detection of HER-2. The AuNPs modified electrode
exhibited fast electron transfer and was biocompatible resulting in
increased surface for the immobilization of single-chain fragment
variable antibody fragments on the electrode surface. The devel-
oped immunosensor was ultrasensitive with an LR from 0.01 to
100 ng/mL and a LOD of 0.01 ng/mL [102]. Another label-free
impedimetric immunosensor based on zwitterionic hydrogel
modified SPCE for the detection of HER2 was developed by Cho-
cholova et al. The immunosensor was fabricated by the immobili-
zation of an anti-HER2 antibody on the electrode surface and
subsequently in situ glycoprofiling of HER2 was carried out by using
lectins. The developed biosensor was able to detect the 5 pg/mL of
target analyte with a LOD of 77 fM [103]. Recently, a label-free
voltammetric immunosensor was designed by the immobilization
of pegylated cerium oxide-antiHER2 conjugate on the surface of
Au-modified SPCE. The results revealed that the complex of HER2
with cerium oxide-anti-HER2 conjugate exhibited electron transfer
inhibition along with voltammetric peak current reduction, which
was proportional to HER2 concentration. The developed label-free
immunosensor showed consistent results with an LR from 0.001 to
0.5 ng/mL and a LOD of 34.9 pg/mL [104].
Moreover, sandwich-based immunoassays are also used for the
efficient detection of HER2 by using labels. Tallapragada et al.
designed an SPE (bare) based immunosensor for the detection of
HER2 by using sandwich ELISA. The immunosensor was fabricated
by the immobilization of mouse anti-human ErbB2 (primary anti-
body), followed by the incorporation of recombinant human HER2
antigen. Biotinylated goat anti-human ErbB2 was used as the sec-
ondary antibody which was linked to streptavidin-conjugated HRP.
Quantification of the target analyte was carried out by measuring
the redox reaction by CV. It was observed that the signal increased
linearly (LR: 5 ng/mL-20 ng/mL) with an increase in the HER2
concentration and a LOD of 4 ng/mL. Moreover, the developed
immunosensor was highly sensitive and biocompatible with the
ELISA [105]. In another study, Haiza Lah et al. developed sandwich
ECIs using SPCE immobilized with primary antibody (Ab1), and
lead sulfide QDs-conjugated secondary HER2-antibody (Ab2-PbS
QDs) for the detection of HER2. The developed immunosensor
exhibited magnified sensing by SWV with an LR from 1 to 100 ng/
mL and a LOD of 0.28 ng/mL due to the stripping signal of lead via
acid dissolution [106].
Recently, Freitas et al. developed a high-performance EC mag-
netic immunosensor for the detection of HER2-ECD in human
cancer cells by using SPCE. The biosensor was based on the sand-
wich immunoassay where MBs anchored with COOH (COOH-MBs)
were used as a capture antibody and biotinylated antibody labeled
with streptavidin alkaline phosphatase (SAP) as a detector antibody
(biotinylated Ab/SAP). In the presence of HER2-ECD, a sandwich
complex (COOH-MBs|HER2-ECD|biotinylated Ab/SAP) was formed
on the SPCE surface. After the addition of the enzymatic substrate,
the immunoreactions were measured by LSV. The developed
immunosensor was selective for the detection of the target analyte
with two LR (5e50 ng/mL and 50e100 ng/mL) and a LOD of 2.8 ng/
mL. Moreover, the biosensor was able to detect the CTC (SK-BR-3)
with a LOD of 3 cells/mL [107]. Further, the same group developed
another EC magnetic immunosensor for the detection of HER2-
ECD. The immunomagnetic biosensor was also based on a sand-
wich immunoassay where COOH-MBs were used as primary
22
Materials Today Chemistry 26 (2022) 101129
antibodies (immobilized on SPCE) and biotinylated antibodies
labeled with streptavidin-QDs (CdSe@ZnS) as a secondary antibody.
In the presence of the target analyte, a sandwich complex was
formed between the primary and secondary antibodies, and the
immunoreactions were measured by differential pulse anodic
stripping voltammetry (DPASV). The developed immunosensor was
effective for the detection of HER2-ECD with an LR from 0.50 to
50 ng/mL and LOD of 0.29 ng/mL. Additionally, the biosensor was
able to detect the CTC [108]. In another study, HER2 was also
detected by sandwich-type immunoassay based on bare SPCE. The
immunosensor was fabricated by the immobilization of the capture
antibody on the electrode surface and followed by blocking the free
surface with BSA. The affinity reaction between the capture anti-
bodies and target antigens was analyzed using a label (CdSe@ZnS
QDs) and further monitored by DPASV (Fig. 12). The developed
immunosensor responded linearly to HER2-ECD with an LR from 10
to 150 ng/mL and a LOD of 2.1 ng/mL [109].
Bare or modified GCE is also used to detect HER2 by using
label-free or sandwich immunoassays. Regarding this, a
sandwich-type immunosensor was developed using the nano-
magnetic platform using GCE. The nanomagnetic platform was
designed by immobilization of antiHER2 functionalized with
3-aminopropyltrimethoxysilane coated Fe3O4 NPs (antiHER2/
APTMS-Fe3O4) on a bare GCE surface followed by blocking of the
non-specific binding site of the electrode with bovine serum al-
bumin (BSA). Further, the various concentration of target antigen
(HER2) was placed on the modified electrode and followed by
placing the secondary antibody (antiHER2/Hyd@AuNPs-APTMS-
Fe3O4) containing Ag ions as a label conjugate. A linear relationship
between the DPV stripping signal of Ag and the log of HER2 con-
centrations was observed with an LR of 5.0  104 e50.0 ng/mL and
a LOD of 2.0  105 ng/mL [110]. In another study, a label-free
amperometric immunosensor was developed by using modified
GCE with MOF and MWCNT. The biosensor was designed by the
immobilization of capture antibody (HER2) on the modified elec-
trode surface and followed by blocking the non-specific binding
site of the electrode with BSA. The electrocatalytic activity of the
immunosensor was based on the reduction of H2O2. The ampero-
metric results revealed that in the presence of the target biomarker,
the decline of H2O2 was decreased due to the formation of AgeAb
complexes on the electrode. The immunosensor was able to detect
the target analyte within a range of 1.0 pg/mL-100 ng/mL and a LOD
of 0.3 pg/mL [111]. Recently, three label-free biosensors were
developed by the surface modification of GCE with Au functional-
ized Gr-QD (GQD) (SNGQDs@AuNPs), cobalt porphyrin binuclear
framework (CoPeBNF), and CoPeBNF/SNGQDs@AuNPs for the
detection of HER2. The biosensor was developed by the immobi-
lization of antibodies on the modified electrode surface and signals
were measured by EIS. The developed sensors were stable, repro-
ducible, and exhibited lower LOD of 0.0327 ng/mL, 0.0454 ng/mL
and 0.1072 ng/mL respectively [112]. In another recent study,
Nasrollahpour et al. synthesized an EC nano biosensor for the
detection of HER2 using WO3/poly-glutamic acid-modified GCE.
The developed biosensor was stable, specific, and stable with an LR
from 1 fg/mL to 1 ng/mL and a LOD of 1 fg/mL [113].
5.3.1.2. Detection of carcinoembryonic antigen. CEA is a glycopro-
tein secreted by gastrointestinal cells during embryo development
and before birth. It is usually present in lower concentrations unless
certain diseases such as pancreatitis, Crohn's disease, ulcerative
colitis, inflammatory bowel disease, and various type of cancers
(adenocarcinoma, gastric, ovarian, lung, colorectal, breast cancer,
etc.) are present. It is an FDA-approved biomarker for the moni-
toring of colon cancer. Moreover, elevated levels of CEA can also
differentiate between the different types of cancers like benign/
P. Lakhera, V. Chaudhary, A. Jha et al.
metastatic, and aneuploid/diploid. CEA is more prevalent in ductal
carcinoma in contrast to lobular carcinoma suggesting that CEA is
an early diagnostic marker [196]. However, the increased level of
CEA is not specific to BC since it can be observed in different can-
cers. Therefore, various biosensors like DPV, EIS, SWV, chro-
noamperometry, conductometry, fluorescence resonance energy
transfer (FRET), magnetic DNA nanoprobes, surface plasmon reso-
nance (SPR), Au interdigitated capacitor transducer, etc., have been
developed for the detection of CEA concentration in BC patients
suggesting that CEA may act as a promising diagnostic biomarker
for detection of BC [1,48]. Herein, a recently developed EC immu-
nosensor based on SPE and GCE for the detection of CEA will be
discussed (Table 5).
Chan et al. developed an immunosensor based on modified SPE
with AuNPs/rGO for the detection of CEA. The modified SPE
exhibited higher surface area, increased stability, and augmented
electron transfer kinetics. Moreover, modified SPE was compatible
with the sandwich ELISA system leading to enhancing the sensi-
tivity of the sensor toward the detection of CEA. The sandwich-type
immunosensor was fabricated by the immobilization of the pri-
mary antibody, target antigen, and secondary antibody onto the
modified electrode surface. The developed immunosensor was easy
to fabricate, cost-effective, and exhibited double EC responses for
the detection of CEA with an LR from 0.5 to 50 ng/mL and
250e2000 ng/mL and LOD of 0.28 ng/mL and 181.5 ng/mL,
respectively [114]. In another study, CEA was detected by a
sandwich-type immunosensor based on modified SPCE with
cysteamine/Au NPs via electrodeposition. The sensor was fabri-
cated by covalent coupling of different lectins (concanavalin A,
wheat germ agglutinin, and Lens culinaris agglutinin) on the
modified electrode surface and followed by coupling with the
target antigen and HRP labeled anti-CEA detection probe. It was
observed that the immunosensor produced a catalytic current
signal in the presence of H2O2 and HQ. The immunosensor was able
to differentiate CEA between healthy and cancer serum samples
with an LR from 1 ng/mL to 10 ng/mL with LOD of 0.05 ng/mL and
0.03 ng/mL using wheat-germ agglutinin and concanavalin A as
BRE, respectively, and in the range of 0.5 ng/mL to 7 ng/mL with
LOD of 0.01 ng/mL using Lens culinaris agglutinin as BRE [115].
Lee et al. also developed a sandwich-type disposable immu-
nosensor based on modified SPEs with AgNPs/rGO leading to
augmenting the surface area for increased electron transfer rate.
The immunosensor was developed by the immobilization of the
primary antibody on the modified electrode surface and followed
by coupling with the target antigen and HPR-labeled secondary
antibody. The developed immunosensor was simple, rapid, cost-
effective, and able to detect the CEA in the LR from 0.05 to
0.50 mg/mL with a LOD of 0.035 mg/mL [116]. Recently, Cao et al.
developed a novel immunosensor for the detection of CEA based
on Au and magnetic NPs (MNPs) anchored with rGO-
tetraethylenepentamine (rGO-TEPA/Au-MNPs) as a biosensing
platform and secondary antibody modified with Au@PtNPs as
detector probe. In the presence of EC substrate, o-phenylenedi-
amine (OPD) was oxidized to the 2, 20 -diaminoazobenzene
(electroactive material) owing to the catalytic effect of Au@PtNPs.
The SWV results revealed that the EC signals were proportional to
the CEA concentration analyte in the sample. The magnetic bio-
sensing platform exhibited magnified electrical conductivity
improved stability due to rGO-TEPA and improved biorecognition
efficiency owing to Au NPs in contrast to the MNPs. The sandwich
immunocomplex possessed excellent magnetism and could be
separated and immobilized on the SPE surface. The developed
biosensor was reliable, selective, and stable with an LR of 5.0 pg/
mL-200.0 ng/mL, and a LOD of 1.42 pg/mL [117].
23
Materials Today Chemistry 26 (2022) 101129
Interestingly, Li et al. developed an ultrasensitive sandwich-type
immunosensor for the detection of CEA based on a secondary anti-
body labeled with palladium (Pd)/Pt nanocages anchored on the
surface of amino-functionalized MWCNT (PdPt nanocages/MWCNT-
NH2) as a signal amplifier. The immunosensor was fabricated by the
modification of GCE with 3-aminopropyltriethoxysilane (APTES)
crosslinked Gr sheets (NH2-GSs) leading to increase sensitivity,
electron transfer, and can easily immobilize a large quantity of
capture antibody (anti-CEA). The resulting PdPt nanocages/MWCNT-
NH2 and modified GCE exhibited enhanced electrocatalytic activity
toward the reduction of H2O2 (Fig. 13). The fabricated sensor
exhibited enhanced sensitivity, stability, and augmented immobili-
zation of secondary antibodies owing to the high surface area of the
catalytic label [118].
Han et al. developed a sandwich-type non-enzymatic ECI for
CEA based on a nanocomposite (NC) of stannic oxide (SnO2)/rGO
and AuNPs modified GCE resulting in augmented surface area and
magnified electron transfer. Further, the modified GCE was immo-
bilized with the capture antibody. The capture CEA was labeled
with Pd NPs-vanadium pentoxide (V2O5)/MWCNTs and function-
alized with secondary antibodies. The developed composite
exhibited nexcellent catalytic activity towards the reduction of
H2O2. The developed immunosensor was sensitive for CEA with an
LR from 0.5 pg/mL-25 ng/mL and a LOD of 0.17 pg/mL [119]. A label-
free ECI for CEA was developed by the immobilization of an anti-
CEA antibody on the GCE modified with Pd-iridium (PdeIr) bime-
tallic NPs. The immunocomplex between the CEA antigen and CEA
antibody results in the blockage of catalytic activity of bimetallic
NPs to the reduction of H2O2, and a decrease in the response signal.
The developed immunosensor was simple with an LR from 0.05 to
50 ng/mL and a LOD of 0.017 ng/mL [120]. Another novel label-free
ultrasensitive amperometric immunosensor for CEA based on GCE
modified with PDA-Pb2þ (redox species) and chitosan-Au NPs
leading to enhancing the current signal, and immobilization of
capture antibody (anti-CEA). The immunosensor was fabricated by
the immobilization of capture antibody (anti-CEA) on the modified
electrode surface and followed by blocking the unbound electrode
surface with BSA and various concentrations of the target antigen
(CEA). The developed immunosensor was simple and ultrasensitive
with an LR from 1 fg/mL to 100 ng/mL and a LOD of 0.26 fg/mL [121].
Luo et al. fabricated a sandwich-type immunosensor for the
detection of CEA using SWCNTs@GQDs composites as a signal
amplifier. The composite not only amplifies the signal response but
also exhibited magnified electrocatalytic activity, and sensitivity.
Additionally, rGO-Au-NPs are used to modify the GCE surface
resulting in increased surface area, remarkable conductivity, and
dual amplification. The developed immunosensor exhibited excel-
lent specificity and sensitivity toward the detection of CEA with an
LR from 50 pg/mL to 650 pg/mL, and a LOD of 5.3 pg/mL [122].
Wang et al. designed an ultrasensitive sandwich-type immuno-
sensor based on silver/molybdenum disulfide/iron oxide composite
(Ag/MoS2@Fe3O4) and an analogous ELISA method for CEA detec-
tion. The composite was used as a label for secondary antibodies
leading to an increase the sensitivity and specificity owing to its
magnetic property. The immunosensor was fabricated by 96 well
microplates and the detection process was accomplished on mag-
netic GCE modified with Ag NPs. The DPV signals revealed that the
current responses were inversely proportional to the CEA concen-
tration. The developed immunosensor was stable, reproducible,
and selective with an LR from 0.0001 to 20 ng/mL and a LOD of
0.03 pg/mL [123].
In a study, CEA was also quantified by ECI based on modified GCE
with AueAg/rGO@PDA-NC as a dual signal amplification. The
developed sensor was stable sensitive, and specific with an LR from
P. Lakhera, V. Chaudhary, A. Jha et al. Materials Today Chemistry 26 (2022) 101129

Fig. 12. A sandwich-type electrochemical immunosensor for the detection of HER2 based on a bare screen-printed electrode and quantum dot as an electroactive label. Recreated
with permission from Ref. [109].

0.001 ng/mL to 80 ng/mL, and a LOD of 0.286 pg/mL [124]. In
another study, Nakhjavani et al. proposed an Au/Ag nanohybrid-
based sandwich-type ultrasensitive immunosensor for CEA detec-
tion using various strategies. Initially, GCE was modified with
thiolated-graphene oxide (TGO) to magnify the surface area,
followed by the introduction of Au NPs coated with streptavidin
(AuNPs@strp) resulting in enhanced immobilization of CEA bio-
tinylated monoclonal antibody (BmAB). Further, a sandwich system
was formed after the coupling of secondary antibodies conjugated
with streptavidin-coated Ag NP. Finally, HRP, HQ, and H2O2 were

Fig. 13. Fabrication and principle of an ultrasensitive sandwich-type electrochemical immunosensor for the detection of CEA based on 3-aminopropyltriethoxysilane crosslinked
graphene sheets (NH2-GSs) modified glassy carbon electrode. Recreated with permission from Ref. [118].

24
P. Lakhera, V. Chaudhary, A. Jha et al.
applied to amplify the EC signals, electron mediator, and sensing
analyte respectively. The EC signals were significantly increased in
the presence of HQ and H2O2. the tailored immunosensor exhibited
excellent performance in an LR from 100 fg/mL to 5 pg/mL with a
LOD of 75 fg/mL [125]. Also, CEA was detected by sandwich-type
immunoassay based on rGO/chitosan/AuNPs modified GCE as a
biosensing platform and polythionine-Au (PTheAu) composites as
a signal label. The modified electrode with rGO/chitosan/AuNPs
effectively augments electron transfer and magnifies the surface
area leading to enhance immobilization of the primary antibody.
The developed biosensor was biocompatible, sensitive, and specific
with an LR of 0.3 ng/mL to 30 ng/mL and LOD of 0.1471 pg/mL due to
the high conductivity of PTheAu [126].
Recently, a sandwich-type high-performance photo-
electrochemical immunosensor based on two-dimension rhenium
disulfite nanosheets (2D-ReS2) modified GCE as a biosensing plat-
form. The sensor was fabricated by immobilization of primary
antibody on the modified electrode and followed by the capture of
target CEA, and secondary antibody labeled with alkaline phos-
phatase. The label was able to catalyze vitamin C magnesium
phosphate to ascorbic acid that can donate electrons to capture
holes photoexcited from ReS2 nanosheets leading to generating
photocurrent signals for CEA detection. The developed immuno-
sensor was stable, selective, and specific with an LR from 0.0005 to
10.0 ng/mL and a LOD of 0.468 pg/mL [127]. In another recent study,
CEA was detected by a sandwich-type ECI based on 3-D GO-
supported Pt-NPs (3DPt/HGO) modified GCE as a sensing plat-
form, and a secondary antibody labeled with HRP-functionalized
Au NPs. The surface modification leads to enhancing the surface
area, electrical conductivity, and increased immobilization of pri-
mary antibodies. The developed immunosensor exhibited a linear
relationship in the range of 0.001e150 ng/mL and a LOD of
0.0006 ng/mL [128]. Very recently, Jozghorban et al. developed a
label-free immunosensor based on rGO-modified GCE for the
detection of CEA. The EC behavior of immunosensor and immu-
noreactions were analyzed by CV and EIS. The developed sensor
was able to detect the CEA in a human serum sample with an LR
from 0.1 to 5 ng/mL and a LOD of 0.05 ng/mL [129].
5.3.1.3. Detection of mucin proteins. Mucin (MUC) proteins are
tandem repeat structures with a high proportion of prolines,
threonines, and serines (known as the PTS domain). Its main role is
in the protection of cells from external infection but also plays a
part in signal transduction, apoptosis, transcriptional, and acety-
lation of histone proteins [48]. The human MUC family includes 21
members (MUC1-MUC21) which are further subdivided into
secreted (MUC2, MUC5AC, MUC5B, and MUC6) and trans-
membrane proteins (MUC1, MUC4, MUC13, and MUC16) [197]. The
elevated level of MUC glycoproteins has been used to diagnose the
different types of cancers. Among various MUC, MUC1, and CA
(CA27-29, CA15-3, and CA125) are the major biomarkers used for
BC diagnosis [48]. There are various techniques used for the
screening of these biomarkers but suffer from certain drawbacks
(Table 1). ECIs are a promising solution to overcome the limitations
owing to their rapid detection and direct readout of the signals
generated from antigen-antibody reactions. Rauf et al. fabricated a
disposable ECI based on SPCE modified with COOH-rich GO (GO-
COOH) for the detection of MUC1 using MB as a redox probe. The
authors explored that surface modification of SPCE enhanced the
conductivity of the electrodes leading to magnifying the sensitivity
of the immunosensor [130].
CA 27e29, and CA15e3 are the epitope of polymorphic
epithelial transmembrane MUC belonging to the MUC1 family.
These are usually overexpressed in colon cancer, lung cancer,
ovarian cancer, bladder cancer, endometrium cancer, and BC [48].
25
Materials Today Chemistry 26 (2022) 101129
These are some of the most important BC biomarkers but are not
specific or sensitive tools for cancer diagnosis. These are mainly
used to observe a person's response to BC treatment and to monitor
BC recurrence subsequent to surgery[44,198]. It can only be used as
a biomarker if the cancer cell is producing increased amounts of it.
The normal serum concentration of CA15-3 level is < 30 U/mL in a
healthy person which increased significantly (>100 U/mL) in 30% of
patients with BC or ovarian cancer and could be correlated with
tumor stage [49,199]. Various methods are used to detect the CA15-
3 but suffer from certain limitations (Table 1) and can not meet the
clinical demand for early detection of CA15-3 with ultralow con-
centrations. EC sensors are a promising solution to the conundrums
associated with the early diagnosis of BC.
For CA 27e29 detection, a label-free ultrasensitive ECI based on
GCE modified with Au/MoS2/rGO nanocomposites was developed.
The nanocomposites provided magnified EC sensitivity due to the
additive effect of AuNPs, MoS2, and rGO. Signal enhancement of the
amperometric detection was assessed by measuring the electro-
catalytic current response of H2O2 reduction via the immuno-
reaction between the CA 27e29 antigen and anti-CA 27e29 on the
modified electrode surface. The fabricated immunosensor was
reproducible, stable, sensitive, and specific for CA 27e29 with an LR
from 0.1 to 100 U/mL and a LOD of 0.08 U/mL [131].
For CA15-3 detection, Nguyen et al. developed an ECI based on
SPCE modified with a bilayer film of PPy nanowire and poly(1,5-
diaminonaphthalene). The combination of the bilayer was a
promising material for biosensing due to its large surface area. The
developed immunosensor was sensitive and specific for CA 15-3
with an LR from 0.05 to 20 U/mL and a LOD of 0.02 U/mL [132]. In
another study, Gomes et al. proposed an alternative EC device for
CA 15-3 diagnosis in liquid biopsies based on the combination of a
biomimetic film with EC detection. The biomimetic film was ach-
ieved by electro-polymerization of OPD around a CA 15-3 target
which was previously immobilized on the SPGE surface. Further,
the protein target was eliminated by trypsin enabling the vacant
sites for rebinding (Fig. 14). Furthermore, EC techniques were used
to monitor the rebinding of material with CA15-3. The fabricated
device exhibited successful characteristics for CA 15-3 detection
and a promising tool for BC screening in POC applications with an
LR from 0.25 to 10.00 U/mL and a LOD of 0.05 U/mL [133].
Amani et al. reported a label-free EC immunosensor for the
detection of CA15-3 based on SPCE modified with copper sulfide
(CuS)-rGO composite. The modified electrode exhibited magnifi-
cent activity towards the catechol oxidation as an EC probe. The
developed enzyme-free immunosensor was rapid, reproducible,
stable, selective, and sensitive with an LR from 1.0 to 150 U/mL and
a LOD of 0.3 U/mL [134]. Another label-free and enzyme-free
immunosensor for the detection of CA 15-3 based on SPCE modi-
fied with NC of cobalt sulfide (CoS2)-Gr-AuNPs was developed. In
the developed immunosensor, AuNPs act as the binding site of
antibodies, and CoS2-Gr was responsible for magnified electro-
catalytic activity toward the catechol oxidation and enhanced sur-
face area leading to improve the immobilization of CA 15-3
antibody. Quantification of the target analyte was based on the
hindrance of the electrocatalytic oxidation of the probe after
binding of a specific antibody (anti-CA 15-3) with the CA 15-3 an-
tigen. The developed immunosensor was accurate, stable, and
specific sensitive for CA 15-3 detection in human serum with an LR
from 0.1 to 150 U/mL and a LOD of 0.03 U/mL [135]. Recently, a
sandwich-type EC immunosensor was developed for CA 15-3 with
an LR of 0.1 fg/mL-1 mg/mL and LOD of 0.08 fg/mL. The immuno-
sensor was based on Au-rGO modified SPCE. The augmented
sensitivity was achieved by using secondary antibodies labeled
with HRP in the presence of H2O2 (signal magnifier), and HQ
(electron mediator) [136]. In another recent study, a novel, cost-
P. Lakhera, V. Chaudhary, A. Jha et al.
effective, and disposable immunosensor was fabricated for CA 15-3
detection. The immunosensor was fabricated through the deposi-
tion of a self-assembled monolayer (SAM) of MSA on SPGE resulting
in a narrow COOH-terminated monolayer. Further, the anti-CA 15-3
was conjugated with the COOH- groups present on the electrode
surface. The fabricated immunosensor was more sensitive and
specific for CA 15-3 detection with an LR from 1.0 to 1000 U/mL and
a LOD of 0.95 U/mL in contrast to CA 19-9, and CA 125 [137].
A label-free EC immunosensor for sensitive detection of CA15-3
based on GCE modified with nitrogen-doped Gr sheets (N-GS).
Surface modification of GCE was responsible for the augmented
sensitivity of the immunosensor due to the high conductivity of N-
GS. The proposed immunosensor was facile and applicable for the
detection of CA 15-3 with an LR from 0.1 to 20 U/mL, and a LOD of
0.017 U/mL [138]. An enzyme-free sandwich-type immunosensor
was developed based on GCE modified with AuPd nanochain
network (AuPd NCN). The modified electrode was further incu-
bated with a primary antibody (anti-CA 15-3) and subsequently
with BSA (blocking agent of a non-specific binding site). Further,
different concentrations of CA15-3 were placed onto the electrode
surface and followed by placing AuPd NCNs-secondary antibody
suspension. The detection of CA 15-3 was based on the electro-
catalytic activity of AuPd NCN via the H2O2 reduction. The devel-
oped immunosensor was reproducible, stable, and sensitive with
an LR from 0.001 pg/mL-100 ng/mL, and a LOD of 0.35 fg/mL [139].
Hasanzadeh et al. proposed a sandwich-type immunosensor based
on GCE modified with rGO/PDA and NH2 functionalized meso-
porous silica conjugated by Au NPs enabling magnified surface area
for primary antibodies immobilization and enhanced conductivity.
Under optimal conditions, the proposed immunosensor exhibited
magnificent analysis of CA 15-3 with an LR from 0.002 to 125 U/mL
and a LOD of 0.002 U/mL [140].
A novel ultrasensitive immunosensor for the CA 15-3 based on
GCE modified with polymeric b-cyclodextrin (Pb-CD) loaded 3-D
porous Gr aerogel (GAs), and hexacyanoferrate as an EC probe
was developed. The surface modification of the electrode was
responsible for enhanced surface area and outstanding conductiv-
ity resulting in large immobilization of antibodies. The developed
immunosensor was reproducible, stable, and selective for CA 15-3
with an LR from 0.1 mU/mL-100 U/mL, and a LOD of 0.03 mU/mL
along with a low working potential (0.2 V) due to the collaborative
effect of Pb-CD, and GAs [141]. Hasanzadeh et al. developed an
innovative immunosensor for ultrasensitive detection of CA 15-3 in
MCF-7 and human plasma using Au nanospears (NSs) electro-
chemically immobilized onto thiolated GQDs modified GCE was
developed. The hybrid interface (CysA/Au NSs/GQDs/GCE) fur-
nished a large surface area for the immobilization of CA 15-3 an-
tigens together with their stability. Under optimized conditions, the
developed immunosensor was stable, specific, and sensitive for CA
15-3 with an LR from 0.16 to 125 U/mL and a LOD of 0.11 U/mL due
to the increased immobilization of anti-CA 15-3 onto the modified
electrode [142].
A novel immunosensor based on GCE modified with Gr ink
conjugated with anti-CA15-3 (bioink) was developed for the
detection of CA 15-3. The conductivity of bioink was analyzed by
plotting various conductive patterns on paper using a rollerball
pen, and DPV was used to evaluate the EC behavior of the tailored
immunosensor. The fabricated immunosensor was sensitive and
selective for CA 15-3 assay in unprocessed human plasma samples
with an LR of 15e250 U/mL and a LOD of 15 U/mL [143]. Zhang et al.
proposed an impedimetric immunosensor based on GCE modified
with Au NPs using a cascade catalysis-initiated radical polymeri-
zation signal amplification strategy. The immunosensor was fabri-
cated by the immobilization of CA 15-3 antibodies on the modified
GCE surface followed by dripping of BSA to block the non-specific
26
Materials Today Chemistry 26 (2022) 101129
binding sites. Further, the modified electrode was incubated with
various concentrations of the standard solution of CA 15-3 antigen,
and subsequently, glucose, acetylacetone (ACAC), N-isopropyl
acrylamide (NIPAM), and N, N0 -methylene-bisacrylamide (MBA)
was placed on the electrode surface to instigate radical polymeri-
zation. Parallelly Cu-MOF-based immunoprobes were prepared
through the covalent conjugation of the secondary antibody and
GOx due to the magnified resistance and augmented catalytic ac-
tivity of Cu-MOF. In the presence of a target antigen, the immu-
noprobes were specifically bound to the modified electrode via
immunoreaction between antibody and antigen and triggered the
radical polymerization. Firstly, glucose was catalyzed in the pres-
ence GOx leading to generating H2O2, which further reacted with
ACAC through Cu-MOF (peroxidase mimics) catalysis to form ACAC
radicals for the polymerization of N-isopropyl acrylamide (NIPAM)
leading to synthesize a new polymer poly (N-isopropyl acrylamide)
(PNIPAM). The EIS results revealed an increase in resistance due to
the poor conductivity of PNIPAM and Cu-MOF. Due to the novel
signal amplification strategy, the developed immunosensor was
potentially sensitive for the CA 15-3 detection with an LR from 10 to
100 U/mL and a LOD of 5.06 mU/mL [144].
CA125 also known as MUC 16 is mainly associated with ovarian
cancer but also associated with non-Hodgkin's lymphoma, meso-
thelioma, uterus, cervix, pancreas, liver, colon, breast, and lung
cancers [56,200,201]. Moreover, various non-pathological condi-
tions like menstruation and pregnancy are also responsible for an
increased level of CA125 in a healthy population. Various studies
revealed that 90% of females have an increased level of CA125 in the
advanced stage of BC or ovarian cancer. However, 50% of cancerous
patients have a normal range of CA125. Therefore, it is considered a
valuable biomarker for cancer diagnosis as well as for cancer pro-
gression monitoring, and treatment [56,198,202,203]. Various SPE-
and GCE-based EC biosensors have been developed for CA 125 in
ovarian cancer but as per our knowledge from 2015 to date, no EC
biosensor based on these two electrodes has been developed for
the monitoring of CA 125 in BC.
5.3.1.4. Detection of other proteomic biomarkers. ER and PR re-
ceptors are proteomic biomarkers that belong to the superfamily of
steroid hormone nuclear receptors [204,205]. These receptors have
two types of isoforms ERa and Erb, and PR-A and PR-B, respectively.
Clinical and experimental studies demonstrate that these receptors
are very important in tumorigenesis, mammary gland develop-
ment, and BC development [6]. Moreover, it has been documented
that ER is responsible for ductal morphogenesis whereas PR is for
lobuloalveolar differentiation. However, the PR expression is highly
dependent on the presence of ER. Therefore, these two biomarkers
are used in prognosis and predicting response to hormone treat-
ment. Eletxigerra et al. developed an amperometric EC magneto-
immunosensor for the detection of ERa in human serum and cell
lysate samples to differentiate between ERa positive and negative
BC cells. The capture antibodies were immobilized onto MBs-COOH
and the captured target was sandwiched with biotinylated detector
antibodies labeled with a streptavidin-HRP conjugate. The EC
monitoring of the reactions was performed at SPCEs using H2O2 as
HRP substrate and HQ as electron transfer mediator. The developed
immunosensor was inexpensive, easy to operate, quick, simple, and
sensitive with an LR from 63 to 200 pg/mL and a LOD of 19 pg/mL
[145]. In another study, the same group developed an EC magne-
toimmunosensor for the detection of PR receptors based on SPCE
using functionalized MBs and specific antibodies. The developed
immunosensor was accurate for the detection of PR with an LR from
73 to 1500 pg/mL and a LOD of 22 pg/mL [146].
VEGF is an important angiogenesis regulator and a signaling
protein that stimulates vascular endothelial cell proliferation,
P. Lakhera, V. Chaudhary, A. Jha et al.
growth, and survival. In mammals, it possesses five members
(VEGFA, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor), and
different isoforms of VEGFA (VEGF121, VEGF121b, VEGF145,
VEGF165, VEGF165b, VEGF189, and VEGF206) [1]. Among all iso-
forms, VEGF165 is associated with the growth and metastasis of
various cancers. Although VEGF is an important biomarker for BC
diagnosis but also associated with various diseases like brain in-
juries, Parkinson's, rheumatoid arthritis, psoriasis, and other can-
cers. The normal serum concentration of VEGF is 1e177 pg/mL, and
it can increase up to 18e328 pg/mL in BC patients [3,49]. Palabiyik
et al. reported a sensitive label-free impedimetric immunosensor
based on SPCE modified with molecularly imprinted polymer (MIP)
for the detection of VEGF. The developed immunosensor was
reproducible and sensitive with an LR of 20e200 pg/mL and a LOD
of 0.08 pg/mL [206].
5.3.2. Electrochemical aptasensors for the detection of proteomic
biomarkers
EC aptasensors (aptamers used as a bioreceptor) based on SPE
and GCE are extensively used for the detection of BC biomarkers
due to their low cost, flexibility, stability, versatility, and excep-
tional binding affinity. Moreover, EC methods require lesser sample
quantity and display the capability of conceivable biosensors with
adequate stability and low detection limit leading to easy minia-
turization [207]. Aptamers are small nucleic acid ligands of ssDNA,
RNA, and peptide molecules screened by systematized exponen-
tially enriched ligands (SELEX) [208]. Aptamers or chemical anti-
bodies have been widely used as bioreceptor in various sensing
applications due to their sensitivity, specificity, cost-effectiveness,
easy synthesis, and stabilization [209]. Moreover, aptamers can
also selectively bind to the unique biomarkers that are overex-
pressed to the tumor cell surfaces. Based on the methods the EC
aptasensors are classified into the direct, sandwich-type,
displacement-type, and folding-type aptasensors. The direct assay
is based on the molecular recognition of the target biomarker using
the aptamer immobilized sensing interface. The sandwich-type
bioassays are similar to antibodies in immunosensors by using
the binary aptamer (Fig. 15, a). Further, an aptamer can be com-
bined with antibodies when secondary aptamer is not available
leading to form aptamer-antibody (Fig. 15, b) or an antibody-
aptamer (Fig. 15, c) sandwich assay. The displacement assay in-
volves the competition between the authentic target analyte and
labeled target analytes for the specific binding site on the biosensor
surface. The folding-type EC aptasensor is based on the binding of
electrode-bound DNA probes to cause folding [210]. Recently, EC
aptasensors are widely used in various applications like clinical
diagnosis, environment, and food monitoring. Here in this section,
we will summarize the application of EC aptasensors based on
modified SPEs/GCEs for the detection of various proteomic BC
biomarkers.
Bezerra et al. developed an aptasensor based on modified SPE
with Poly-L-Lysine (PLL) for the detection of HER2 in human serum.
The developed biosensor was reproducible, and stable with an LR
from 10 to 60 ng/mL and a LOD of 3.0 ng/mL. Moreover, the
developed aptasensor was sensitive and promising for the detec-
tion of serum HER2 with albumin consumption leading to the
molecular diagnosis of BC [147]. Recently, another SPGE-based DNA
aptasensors were developed for the detection of HER2 biomarkers
by using a mixed SAM of 1-mercapto-6-hexanol (MCH) with thio-
lated aptamer specific to HER2 and a ternary SAM with the same
aptamer and 1,6-hexanediol (HDT). Both biosensors were able to
detect the target analyte with an LR from 1 pg/mL-1 mg/mL. The
ternary SAM aptasensor exhibited non-specific attachment reduc-
tion to the electrode surface owing to the antifouling properties of
HDT. Additionally, this biosensor exhibited 172 pg/mL as LOD and
27
Materials Today Chemistry 26 (2022) 101129
4.12% sensitivity as compared with the SAM platform (LOD: 179 pg/
mL) [12]. In another recent study, Harahsheh et al. fabricated an EC
aptasensor based on Au-NPs modified SPCE for the selective
detection of HER2. The developed aptasensor was sensitive, and
fast with a 5 min binding time and exhibited an LR of 0.001e100 ng/
mL and a detection limit of 52.85 mA/ng/mL, and 0.001 ng/mL,
respectively [148]. In a study, an EC aptasensor was developed by
using rGO-chitosan film modified GCE, specific HER2 aptamer, and
HER2 proteins to recognize and detect HER2 in human serum. The
developed aptasensor exhibited magnified selectivity and sensi-
tivity (LR: 0.5e2 ng/mL; LOD: 0.21 ng/mL) for HER2 due to signal
amplification of MB and increased charged transfer kinetics of rGO-
chitosan film [149]. Rostambadi et al. developed an impedimetric
aptasensor based on GCE modified with AuNPs placed on single-
walled CNT (SWCNT) for the detection of HER2. The biosensor
was designed by the immobilization of an antiHER2 aptamer and
characterized by CV, DPV, and EIS. The developed aptasensor was
able to recognize HER2 with a LOD of 50 fg/mL and LR from 0.1 pg/
m to 1 ng/mL [32].
Interestingly, He et al. designed a sensitive EC aptasensor for the
detection of CEA based on molybdenum selenide (MoSe2)/Gr/Au-
modified GCE. The modified electrode was further conjugated with a
thiol-labeled aptamer against CEA. The developed aptasensor was
sensitive with an LR from 0.1 pg/mL to 100 ng/mL and a LOD of
0.03 pg/mL [150]. In another study, GCE (modified with graphene
oxide [GO]) based EC aptasensor was developed for the detection of
CEA. The aptasensor was based on the sandwich method with
sequential immobilization of anti-CEA antibody, CEA protein, and
GNR-aptamer onto GCE. The developed aptasensor was specific,
sensitive with an LR from 0.0001 pg/mL-10 ng/mL and LOD of
0.05 pg/mL [151]. Shekari et al. developed another GCE-based label-
free impedimetric aptasensor for the selective detection of CEA. The
aptasensor was developed by the immobilization of an NH2-modi-
fied CEA aptamer on the surface of GCE modified with AuNPs @
amine-functionalized mesoporous silica film. The developed apta-
sensor was sensitive and specific for the quantification of CEA with
an LR from 1.0  103 to 100.0 ng/mL and a LOD of 9.8  104 ng/mL
[152]. An aptasensor based on GO-modified GCE immobilized with
anti-CEA antibodies was developed for the detection of CEA. The
modified electrode was further incubated in CEA solution and fol-
lowed by immobilization of AuNPs-aptamer primer conjugate
resulting in the formation of anti-CEA/CEA/AuNPs-aptamer-primer
onto the GCE surface. The developed aptasensor exhibited
enhanced signal amplification due to the integration of the DNA-
generated technique with rolling circle amplification (RCA). The
developed aptasensor conjugated with RCA was highly sensitive
with an LR from 0.5 pg/mL to 1 ng/mL and a LOD of 0.1 pg/mL in
contrast to the aptasensor without RCA [153]. Interestingly, Zhou
et al. proposed an impedimetric aptasensor for CEA detection based
on a cascade catalysis signal amplification strategy. Initially, Cu-
based MOF encapsulated with Pt-NPs was prepared to achieve
Pt@CuMOFs with peroxidase-like activity. Then, G-rich CEA aptamer,
hemin, and GOx were immobilized onto the surface of Pt@CuMOFs.
In the presence of hemin, the G-rich CEA aptamer converted to
hemin@G-quadruplex (hGq) resulting in the Pt@CuMOFs-hGq-GOx
complex (signal transduction probe). The developed complex was
further immobilized to the modified GCE interface. After the intro-
duction of glucose and 3,3-diaminobenzidine (DAB), a cascade re-
action was instigated by GOx to catalyze the oxidation of glucose, in
situ generating H2O2. At the same time, the decomposition of
generated H2O2 was enhanced by Pt@CuMOFs and hGq along with
the oxidation of DAB and the production of insoluble precipitates
enabling the signal amplification. The developed aptasensor was
sensitive with an LR from 0.05 pg/mL to 20 ng/mL and a LOD of
0.023 pg/mL [154].
P. Lakhera, V. Chaudhary, A. Jha et al.
Nawaj et al. prepared an aptasensor for MUC1 using SPCE
functionalized with CNTs.The aptasensor was easy to fabricate
because there is no requirement for prior modification of SPCE
together with a small number of CNTs [155]. A 3-D Gr-based EC
aptasensor was developed for the selective detection of MUC1. The
aptasensor was fabricated by surface modification of GCE with rGO
modified Au-NPs and followed by immobilization of reduced
thiolated Fc-aptamer. Further, the developed aptasensor was
incubated with BSA to block the unspecific binding site. The
developed aptasensor was specific and sensitive for MUC1 with an
LR from 1 pM to 1 mM, and a LOD of 0.25 pM [156]. In another study,
a competitive EC aptasensor was developed for the detection of
MUC1. The aptasensor was developed by using cDNA-ferrocene/
Ti3C2 (MXene) modified GCE. The modified electrode was further
immobilized with MUC1 aptamer via the AueS bond. The devel-
oped aptasensor was very promising for the clinical diagnosis of BC
with an LR of 1.0 pMe10 mM and a LOD of 0.33 pM [157].
Ahirwar et al. developed an EC aptasensor for the rapid and
sensitive detection of ERa in human BC. The aptasensor was
developed by the immobilization of the thiolated aptamer on the
screen-printed graphite electrodes (SPGE) and the change in signals
was measured by DPV. It was observed that the modified electrode
exhibited an augmented flow of electrons, which starts to bring
down when more ERa binds to the aptamer-modified electrode. The
developed aptasensor was coated with aptamer [158]. A label-free
EC aptasensor was developed for the detection of PR (P4) via the
covalent immobilization of an amine-functionalized P4-specific
aptamer on the modified SPCE. SPCE was modified with
GQDseNiO-AuNFs/f-MWCNTs to develop an effective immobiliza-
tion matrix with enough COOH groups. Quantification of PR was
carried out by measuring the DPV response with increasing PR
concentration. The developed aptasensor exhibited great potential
for the detection of PR in a human serum sample with an LR from
0.01 to 1000 nM and a LOD of 1.86 pM [159].
Recently, Park et al. developed a highly sensitive EC aptasensor
for the detection of VEGF165 based on polyaniline/CNT nano-
composite (PANI/CNT) modified SPCE with an anti-VEGF165 RNA
aptamer. The developed aptasensor was sensitive to detect the
cancer biomarker with an LR from 0.5 to 10.0  106 pg/mL and a LOD
of 0.4 pg/mL [160]. A label-free EC aptasensor for VGEF165 based on
the ῾signal off´ and ῾signal on´ mechanism using an anti-VEGF165
aptamer immobilized BSA-Au nanoclusters/ionic liquid/GCE. The
LR of the developed aptasensor using the ῾signal off´ and ῾signal on´
strategies were 1e125 pM and 2.5e250 pM with corresponding
LODs of 0.32 and 0.48 pM, respectively [161]. In another study,
detection of VEGF was carried out by using GCE electropolymerized
with PLL. Further, the modified electrode was functionalized with L
amino-aptamer 13 solutions followed by L aptamer12-
functionalized Ag/Pt bimetallic nanoclusters. The developed label-
free aptasensor was simple, cost-effective, highly specific for VEGF,
and able to detect VEGF concentration in human serum of BC pa-
tients with an LR from 6.0 to 20 pmol/L and LOD of 4.6 pmol/L [162].
5.4. Detection of circulating tumor cells
Circulating tumor cells (CTCs) are primary cancer biomarkers
that play a pivotal role in the early diagnosis and prognosis of
cancer and are an important indicator of tumor metastasis. These
cells (single or cluster) are liberated from the tumors and circulated
in the blood at low concentrations of up to 10 cells/mL, as compared
with erythrocytes (109/mL), and leucocytes (106/mL) in total blood.
Based on the size, morphological, and biochemical aspects CTCs are
significantly different from normal cells leading to the easy detec-
tion of cancer and its. Moreover, these cells also predict the nature
of cancer and could be used to recognize the chemotherapeutics
28
Materials Today Chemistry 26 (2022) 101129
response to personalized anticancer therapy mechanisms. How-
ever, their detection is challenging due to their lower concentration
and has not been detected by the affinity-based approach [5,10].
There are various techniques such as optical, EC, acoustic wave,
field-effect transistor, photothermal, and quartz crystal microbal-
ance to detect CTCs. Among all techniques, EC cytosensing features
magnified sensitivity, rapid response, and easy fabrication. More-
over, these cytosensors can also be used clinically for POC diagnosis
due to their miniaturization. The working principle of cytosensors
is similar to immunosensors (Fig. 11) and aptasensors (Fig. 15). EC
cytosensors quantify the cells via transforming the electrical output
through the reaction between the biorecognition probes and CTCs.
Based on the BRE EC cytosensors are further categorized into
antibody-based cytosensors and aptamer-based cytosensors.
Antibody-based cytosensors are more selective but possess certain
limitations such as the production of purified antibodies is a time-
consuming and expensive process, and antibodies are sensitive to
high temperatures and harsh chemicals. Aptamer-based cyto-
sensors are advantageous as compared to antibody-based due to
their small size, cost-effectiveness, high purity, magnified binding
affinity, low toxicity, augmented stability, easy functionalization,
simple synthesis without immunogenicity, and reusability [7]. This
section summarizes the SPE-/GCE-based EC cytosensors for the
diagnosis of CTCs.
Epithelial cell adhesion molecule (EpCAM) is a transmembrane
glycoprotein and a potent biomarker for BC metastasis. This
biomarker is responsible for cell proliferation, adhesion, and
migration. Clinically EpCAM is detected by the CellSearch method
which is based on the conjugation of EpCAM-specific antibodies
with ferrofluid leading to magnetic separation. However, this
method can only detect the high EpCAM-expressing cells. Recently,
Hashkavayi et al. developed an ultrasensitive, EC aptasensor based
on GCE modified with thiol functionalized silica NPs and ionic
liquid for the detection of EpCAM. Further, the modified electrode
was functionalized with gold nanostar structures (GNSTs) using a
dual signal amplification strategy. The developed aptasensor was
stable and sensitive with an LR from 5-107 and LOD of 1 cells/mL
[163]. In another recent study, Zhang et al. developed an ultra-
sensitive multifunctional EC cytosensor for rapid capture, specific
detection, and release of the captured EpCAM based on GCE
modified with Au NPs. The cytosensor was developed by the
immobilization of anti EpCAM onto the modified electrode fol-
lowed by BSA incubation to block the non-specific binding sites.
The capture efficiency of the developed device was monitored by
EIS and DPV. The analytical results revealed that under optimal
conditions the capture response changed linearly with an LR from
8.0  10 e1  107 cells/mL, and a LOD of 50 cells/mL. After the
capture of EpCAM glycine HCl was used to destroy the antigen-
antibody complex to liberate the captured cells without disturb-
ing their viability. The developed cytosensor was reproducible,
accurate, and stable with a fine recovery [164].
SloaneKettering breast cancer (SKBR3) is one of the important
BC cells that possess the HER2 antigen on its plasma membrane.
These cells have been used to overcome Herceptin (HCT) treatment
resistance to HER2-overexpressing BC. Tabrizi et al. developed a
sandwich-type cytosensor for SKBR3 using rGO modified SPE as a
platform for the immobilization of primary HCT antibody (Anti-
HCT). In this study, various rGO-tetrasodium 1, 3, 6, 8-pyrene-tetra
sulfonic acid/metal hexacyanoferrates (rGO-TPA/MHCFnano)
nanocomposites (rGO-TPA/FeHCF, rGO-TPA/NiHCF, rGO-TPA/CoHCF,
and rGO-TPA/CuHCF) were used as a signal tag for secondary anti-
HCT. The results revealed that the cytosensor using rGO-TPA/
FeHCF-labeled secondary anti-HCT exhibited higher sensitivity
(30000 cells/mL) in contrast to the other rGO-TPA/MHCF nano--
labeled secondary anti-HCT antibodies due to enhanced
P. Lakhera, V. Chaudhary, A. Jha et al.
Fig. 14. Detection of CA 15-3 by electropolymerization of o-phenylenediamine on a screen-printed gold electrode (SPGE). Reproduced from Ref. [133] PLOS one, 2018.
immobilization of primary and secondary anti-HCT. The proposed
cytosensor was stable, selective, and sensitive for SKBR3 with a LOD
of 21 cells/mL [165]. Recently, a rapid immunosensor was
developed for the detection of SKBR3 based on GCE modified with
HCT-conjugated Gr with specific anti-HER2. The developed
biosensor was simple, inexpensive, and sensitive for SKBR3 even at
lower concentrations (1 cell), which can be used for real sample
analysis [166].
MCF-7 is a typical BC cell line with progesterone, estrogen, and
glucocorticoid receptors that are used for in vitro BC studies.
Regarding this, Tian et al. developed an ultrasensitive reusable
cytosensor based on magnetic GCE (MGCE) modified with rGO/
MoS2 composite enabling increased surface area, rapid electron
transfer, and satisfactory biocompatibility. MGCE is advantageous
for the separation of CTCs labeled with magnetic materials due to
the presence of a powerful magnetic field. The cytosensor was
fabricated by the immobilization of immunomagnetic beads
Fig. 15. Sandwich assays generated by combining aptamer and antibodies. a) binary aptamer sandwich-type; b) antibody-aptamer sandwich-type; c) aptamer-antibody sandwich-
type.
29
Materials Today Chemistry 26 (2022) 101129
(Fe3O4-NPs conjugated with aptamer/maleimide-polyethylenei-
mine) onto the modified electrode surface and followed by the
introduction of the sample. The rGO/MoS2 composite and immu-
nomagnetic beads enhanced the signal via a synergistic catalysis
effect. Moreover, both were utilized as EC signal indicators and
enhancers without supplementary redox mediators. Further, the
developed cytosensor could be revitalized by simply withdrawing
the magnet. The developed cytosensor was inexpensive, versatile,
efficient, reproducible, and sensitive with an LR of 15e45 cells/mL
and a LOD of 6 cells/mL [167]. Another EC cytosensor was devel-
oped for the rapid detection of MCF-7 using GCE modified with
rGO/AuNPs composites as a support material and CuO nanozyme as
a signal amplifier. rGO/AuNPs provide enhanced surface area for
rapid electron transport, biocompatibility, and stability whereas
CuO nanozyme was responsible for the current signal magnifica-
tion enabling ultrasensitive detection of MCF-7. The developed
cytosensor detected MCF-7 through the surface recognition
P. Lakhera, V. Chaudhary, A. Jha et al.
between specific MUC-1 (over-expressed on the MCF-7 cell mem-
branes) and MUC1 aptamer. Under the optimized conditions, the
developed cytosensor was reproducible and selective with an LR
from 50e7  103 cells/mL and a LOD of 27 cells/mL. Moreover, the
cytosensor can easily detect the MCF-7 from the serum sample due
to the peculiar combination of MUC1 and MUC1 aptamer [168].
Another, GCE-based label-free and sandwich-type EC biosensor
were developed for the sensitive detection of MCF-7 cells through
DPV. The biosensor was developed by the surface modification of
GCE with a composite of 3D Gr and Au nanocages/MWCNT-NH2
functionalized with a primary antibody. Further, the developed
immunosensor was dipped in different concentrations of MCF-7
solutions and subsequently, ssDNA-secondary antibody solutions
were dropped. Due to the NM-based signal amplification, and DNA-
labeled antibodies, the proposed biosensor was stable, specific, and
sensitive for MCF-7 with an LR from 100-1,000,000 cells/mL and a
LOD of 80 cells/mL [169].
Biofouling and control of selectivity is a challenge for the con-
struction of reusable electrode surfaces, particularly in complex
media. Liu et al. proposed an EC aptasensor with an antifouling
interface for the quantification of cancer cells in complex biological
media. The antifouling interface was constructed by covalent
attachment of branched zwitterionic peptides on the PANI-
modified GCE. The developed interface exhibited a reduction in
non-specific cell and protein adsorption. The developed aptasensor
with MUC1 aptamer was able to detect MUC1-positive MCF-7 BC
cells with magnified selectivity and sensitivity with an LR from 50-
1,000,000 cells/mL and a LOD of 20 cells/mL. Moreover, the assay
performance of the cytosensor was the same without any fouling
due to the antifouling peptide [170]. Another aptasensor based on
GCE modified with a nanohybrid of TiO2 nanotube-rGO linked with
MUC1 aptamer was developed for the sensitive detection of MCF-
7 cells. The modified electrode with hybrid provides enhanced
surface area enabling more sites for MUC1 immobilization. The
developed aptasensor was sensitive for MCF-7 with an LR from 103-
107 cells/mL and a LOD of 40 cells/mL [171]. Recently, a label-free EC
aptasensor for MCF-7 detection based on GCE modified with an
rGO-chitosan-Au NPs composite was developed. The aptasensor
was developed by immobilization of AS1411 aptamer onto the
modified electrode surface and followed by incubation in an MCF-
7 cell line sample. After incubation, the cell lines interact with the
aptamer resulting in cell capture on the biosensor. The developed
aptasensor exhibited enhanced signal and magnified detection
limit due to the additive effects of composite enabling enhanced
surface area and electrical conductivity. The developed aptasensor
was reproducible, stable, sensitive, and selective for MCF-7 with an
LR from 10-1,000,000 cells/mL and a LOD of 4 cells/mL [172]. In
another recent study, MCF-7 was detected by a highly sensitive
biosensor based on the synergistic effect of self-assembled aptamer
inserted Arch-cruciform DNA on vanadium disulfide (VS2)/Au NPs
modified GCE. In the absence of cancer cells, a cruciform DNA
tagged with three terminal biotins was bound to arch DNA's top,
and further attached with streptavidin labeled HRP to catalyze the
HQ-H2O2 reaction on the modified GCE surface. Furthermore, the
cruciform DNA was released in the presence of MCF-7 cells and
decreased the concentration of immobilized HRP enabling enzyme-
mediated electrocatalysis inhibition (Fig. 16). The EC response of
the developed biosensor was negatively correlated with the con-
centration of cancer cells, with an LR from 10 to 100000 cells/mL,
and a LOD of 5 cells/mL. The developed biosensor may be used for
the diagnosis of CTC in real sample analysis due to the 2-D materials
and enzyme-based dual signal amplification [173].
The MDA-MB-231 cell lines are commonly used to diagnose
late-stage BC and are an acceptable model for triple-negative BC
due to the lack of HER2 [211]. Tang et al. developed a label-free EIS
30
Materials Today Chemistry 26 (2022) 101129
cytosensor for specific detection of MDA-MB-231 through the re-
action between the mannosyl GCE (mannose-C2NH2, mannose-
C6NH2, and galactose-C2NH2) and the overexpressed mannose re-
ceptors on the MDA-MB-231 cell surface. The fluorescent micro-
scopy revealed that the modified electrode was biocompatible and
specific to target cells. It was observed that the GCE modified with
mannose-C2NH2 exhibited magnified sensitivity in contrast to
mannose-C6NH2 whereas, galactose-C2NH2 modified electrode was
not able to detect the target cells. The developed cytosensor was
sensitive and specific for MDA-MB-231 with an LR from 10 to
100000 cells/mL and a LOD of 10 cells/mL [174]. Overexpression of
CD44 is associated with various cancer cell surfaces and plays a
pivotal role in cancer development through metastasis, invasion,
adhesion, and angiogenesis. Hyaluronic acid (HA) is a key ligand for
CD44 with high affinity. Recently, Zhou et al. developed a label-free
EIS cytosensor for the detection of MDA-MB-231 cells and CD44
overexpression by using GCE modified with HA-BSA-modified
AuNPs. The modified electrode exhibited enhanced capture of
CD44-overexpressing cancer cells (MDA-MB-231) due to specific
molecular recognition of HA. Under the optimal experimental
conditions, the developed cytosensor was sensitive and specific for
MDA-MB-231 with an LR from 2.0  102e 3.0  105 cells/mL and a
LOD of 128 cells/mL [175].
5.5. Detection of multiple BC biomarkers
Detection of a single biomarker is not reliable for early diag-
nosis, and prognosis of BC due to different molecular events
involved in BC development, intersubject variations in the
expression of biomarkers, and scarcity of specific biomarkers.
Therefore, multiplex determination of specific biomarkers can lead
to early, reliable, and personalized diagnosis of BC without false
positives [22]. EC biosensors are a promising platform for the
multiplex analysis of BC biomarkers at the different or same mo-
lecular level in contrast to conventional techniques. Various SPE/
GCE-based EC biosensors have been developed for the simulta-
neous determination of multiple BC biomarkers.
CA 15-3 and HER2-ECD are HER2-ECD are independent bio-
markers but the combination of these two plays a key role in the
diagnosis of high-risk BC patients. Therefore, Marques et al. devel-
oped a voltammetric sandwich-type immunosensor for the simul-
taneous determination of CA 15-3 and HER2-ECD based on SPdCE
modified with Au NPs. Further, the modified electrode was coated
with captured antibodies (anti-CA 15-3, and anti-HER2-ECD), and
the free surface was blocked by casein. After incubation with target
biomarkers solution and labeled detection antibodies, antigen-
antibody reactions were monitored by LSV. The developed immu-
nosensor was non-invasive and specific for CA 15-3, and HER2-ECD
[176]. In another study, a dual immunosensor was developed for the
simultaneous determination of Tumor Necrosis Factor-alpha (TNF),
and Receptor Activator of Nuclear Factor-kB Ligand (RANKL) by us-
ing SPdCEs, and neutravidin-functionalized magnetic microbeads
(Neu-MBs). The proposed biosensor was sandwich-type using bio-
tinylated specific capture antibodies, detector antibodies, and HRP-
labeled secondary antibodies, functionalized onto Neu-MBs. EC
detection was carried out by amperometric transduction at SPdCEs
using the H2O2/HQ system. The proposed immunosensor was
ultrasensitively and offered a detection limit of 3.0 pg/mL and
2.6 pg/mL for TNF and RANKL respectively [177].
Guo et al. reported a sandwich-type EC immunosensor for the
detection of MUC1 and MCF-7 cells based on Ag nanoclusters
mediated signal amplification. The immunosensor was developed
by immobilization of anti-MUC1 on a GCE followed by placing the
MUC1 sample. Further Ag nanoclusters were added for signal
amplification via deposition of metallic silver on Ag nanoclusters,
P. Lakhera, V. Chaudhary, A. Jha et al.
Fig. 16. Development and principle of cytosensor for MCF-7 using self-assembled aptamer inserted Arch-cruciform DNA on vanadium disulfide (VS2)/gold nanoparticles (Au NPs)
modified glassy carbon electrode. Adaptee from Ref. [173], MDPI, 2021.
and signals were monitored by SWV. The developed immunosensor
was sensitive and selective for MUC1 and MCF-7 with a detection
limit of 0.5 nM, and 50 cells/mL, respectively [178]. Another
sandwich-type immunosensor was developed for the detection of
MUC1 and MCF-7 cells using Ag NP labels and a poly(glutamic
acid)/MWCNT nanocomposite. The immunosensor was developed
by the immobilization of MUC1 binding aptamer on the surface of
GCE modified with poly (glutamic acid)/MWCNT nanocomposite,
and followed by the immobilization of another aptamer labeled
with Ag NPs for recognition of MCF-7 cells enabling magnified
selectivity and signal amplification. Under the optimal experi-
mental conditions, the developed immunosensor was reproducible,
stable, selective, and sensitive for MUC1 and MCF-7 with a LOD of
3 ng/mL, and 25 cells/mL, respectively [179].
Recently, a sandwich-type EC aptasensor was fabricated for the
simultaneous detection of CA 15-3 and CEA using AuNPs/3-D Gr
hydrogel nanocomposite modified GCE as a biosensing substrate,
and CA 15e3 and CEA aptamers attached to AuNPs/redox probe/Gr
nanocomposite as a biosensing probe. In this study, ferrocene and
hemin were used as redox probes for CA 15-3 and CEA respectively
for dual detection. CA 15e3 and CEA aptamers were immobilized
on the surface of the modified electrode. It was observed that the
surface modification was responsible for the augmented surface
area resulting in more immobilization of aptamer on its surface.
Additionally, the nanocomposites increased the sensitivity of the
aptasensor due to its high conductivity. Moreover, the aptamers
used as capture and reporter probes made this aptasensor more
specific in contrast to other BRE. The developed duplex aptasensor
31
Materials Today Chemistry 26 (2022) 101129
was reliable, sensitive, and specific for CA 15-3, and CEA with a
detection limit of 1.3  102 U/mL, and 11.2 pg/mL, respectively
[180]. Very recently, a label-free EC biosensor was developed for the
simultaneous dual detection of CA 15-3 and miRNA 21. The
biosensor was based on poly(3-aminobenzylamine)/two-dimen-
sional (2D) MoSe2/GO-NC modified dual SPGE, functionalized
independently with 2,3-diaminophenazine-Au NPs and toluidine
blue-Au NPs. Both of the redox probe-Au NPs were employed as
signaling molecules and aided the immobilization of anti-CA 15-3
antibodies and capture DNA-21 probes, respectively. The modified
electrodes exhibited a magnified immobilization of capture probe
and antibodies due to a high surface-to-volume ratio and superior
conductivity leading to efficient dual detection. The developed
biosensor was selective, and sensitive with a LOD of 1.2 fM and 0.14
U/mL for miRNA-21, and CA 15-3 respectively [181].
6. Challenges and future perspectives
EC biosensors based on SPEs and GCEs are a promising inex-
pensive alternative for the non-invasive diagnosis of circulating BC
biomarkers at different molecular levels due to their simplicity,
accuracy, portability, intrinsic miniaturization, compatibility with
semiconductor technologies, and standard microfabrication.
Despite various research, these biosensors face certain limitations
like complex protocols, multiple steps, and the preparation of
various reagents required for EC detection. Moreover, the discussed
biosensors are only prototyped and assessed under laboratory
conditions and far from the real world. None of them have been
P. Lakhera, V. Chaudhary, A. Jha et al.
used for a large number of real patient samples to evaluate their
reliability and validity [22]. Furthermore, these devices are not
commercially available due to insufficient clinical validation studies
regarding the intended motive of describing the procedure, preci-
sion, and selectivity. Additionally, a lack of practical solutions to
address the basic analytical issues related to non-specific binding,
toxicity, reproducibility, and stability [212]. The lack of communi-
cation between the researchers and physicians is another challenge
to the successful development of these devices. Storage and pro-
cessing of samples is also another challenge due to their perish-
ability. Therefore, a simple rapid diagnostic process is required
which includes in situ detection or automatic sample processing.
Moreover, a comprehensive validation via the large numbers of real
patient samples analysis and the comparison against other avail-
able competitive approaches remains to be addressed. For
commercialization, the developed devices should be reproducible,
stable, and robust through the optimization of different variables
by using the design of experiments.
In the future, advancements in multiplex analysis can magnify
the sensitivity and specificity of these biosensors by using a single
sample. Microfluidics-based lab-on-chip devices and recent ap-
proaches in nanotechnology open novel directions for designing
multiplexed, miniaturized, and entirely automated biosensors for
cancer diagnosis. These portable, high throughput and inexpensive
approaches impart low LOD and magnified sensitivity along with
simultaneous analysis of different biomarkers on a single chip.
Further, automation in sensor technology to involve various bio-
markers patterns software can enhance the potential of these de-
vices for newly discovered biomarkers for POC testing. Additionally,
advances in bioinformatics, medicine, materials science, nano-
technology, and synthetic biology may improve the reliability and
practical application of these devices through personalization.
Biofouling and control of selectivity is another challenge for the
construction of reusable electrode surfaces, particularly in complex
media. Biofouling is the colonization of undesirable biological
materials, and bacteria onto the electrode surfaces leading to
hampering the selectivity of EC sensors resulting in false-positive or
false-negative results. Recently, various strategies are used to
develop an antifouling surface using biocompatible materials (PEG,
zwitterions, biomimetic materials, synthetic peptides, etc.) but face
reduced EC sensitivity due to the poor electrical conductivity of the
polymer backbone [213e215]. Moreover, the properties of different
BRE also reduce the antifouling efficiency under different circum-
stances [213]. These limitations can be overcome by using con-
ducting polymers with other antifouling materials. A better
understanding of the fouling of electrodes at any phase can lead to
research for targeted antifouling agents. Thus investigating the
phenomena in their interface chemistry on the electrode and with
BRE can result in long storage life and better performance in the
assay. Additionally, a multifunctional probe comprising both
recognizing and antifouling effects may be another approach to
resolve such limitations.
7. Conclusion
SPEs and GCEs are ideal for analytical purposes due to their
individually unique properties and can be easily integrated with
other approaches to fit a specific application. EC biosensors when
integrated with SPEs and GCEs, provide a promising inexpensive
alternative for the non-invasive diagnosis of circulating BC bio-
markers. This comprehensive review focuses on the recent devel-
opment and fabrication of the EC bioscaffolds based on modified
SPEs and GCEs for the early diagnosis of diverse BC biomarkers at
different molecular levels like genomic, transcriptomic, and pro-
teomics. Further, CTC and multiplex determination of BC
32
Materials Today Chemistry 26 (2022) 101129
biomarkers are also carried out by using both electrodes. Consid-
ering the magnified sensitivity, most of the reported biosensors
have been made by surface modification of SPEs/GCEs with various
NMs, enzymes, and metals due to their unique properties. The
broad variability of the different approaches shows the prospect of
using various bioreceptors, bioassays, and signal amplification
strategies in connection with the electrode substrates and EC
techniques. Moreover, these methodologies deal in a wide range
from a simple label-free test involving analyte-induced changes in
various properties of the complex formed to very complicated
multistep but attractive methodologies enabling better recognition
of analytes. However, further research is still required to address
the different challenges like reliability, validity, sensitivity, and
selectivity in the determination of multianalyte. More research
needs to be carried out for the commercialization, and multiplex
analysis of various biomarkers via the fabrication of multiarray
biosensors with minimum error. Additionally, a multidisciplinary
collaboration should be taken between the various research/aca-
demic institutions and hospitals to improve the efficacy of devel-
oped devices with ample real patient samples. Recent
advancements concerning SPEs and GCEs-based EC biosensor
fabrication for early diagnosis of BC have the potential for simple
and cost-effective treatment for society. Therefore, this review will
impart relevant background for the design and fabrication of
innovative POC devices for diverse BC biomarker monitoring which
would open the way for augmenting cancer diagnosis and
therapeutics.
Author contributions
Conceptualization, writingdoriginal draft preparation Preeti
Kush, Praveen Lakhera, Vikas Chaudhary, and Amit Jha; wri-
tingdreview and editing, Parveen Kumar and Ranjit Singh.
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 article.
Data availability
Review article
Acknowledgment
Praveen Lakhera thanks to Indian Council of Medical Research,
India for the senior research fellowship award from grant number
5/3/8/38/ITR-F/2020-ITR for project titled “Design and develop-
ment of colorimetric technique-based point of care device for the
detection of Glycated Haemoglobin”. Preeti Kush and Ranjit Singh
thank to Shobhit University, Gangoh, Saharanpur, Uttar Pradesh
247341 Saharanpur, U.P.
References
[1] M. Hasanzadeh, N. Shadjou, M. de la Guardia, Early stage screening of breast
cancer using electrochemical biomarker detection, TrAC, Trends Anal. Chem.
91 (2017) 67e76.
[2] J. Ferlay, M. Ervik, F. Lam, M. Colombet, L. Mery, M. Pin ~ eros, A. Znaor,
I. Soerjomataram, F. Bray, Global Cancer Observatory: Cancer Today, inter-
national agency for research on cancer, Cancer Today, Lyon, France, 2018.
[3] S. Şahin, M.O. Caglayan, Z. Üstündag , Recent advances in aptamer-based
sensors for breast cancer diagnosis: special cases for nanomaterial-based
VEGF, HER2, and MUC1 aptasensors, Microchim. Acta 187 (10) (2020) 549.
[4] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019, CA A Cancer J. Clin. 69
(1) (2019) 7e34.
P. Lakhera, V. Chaudhary, A. Jha et al.
[5] V. Gajdosova, L. Lorencova, P. Kasak, J. Tkac, Electrochemical nanobiosensors
for detection of breast cancer biomarkers, Sensors (Basel) 20 (14) (2020)
4022.
[6] R. Ahirwar, N. Khan, S. Kumar, Aptamer-based sensing of breast cancer
biomarkers: a comprehensive review of analytical figures of merit, Expert
Rev. Mol. Diagn. 21 (7) (2021) 703e721.
[7] F. Vajhadin, S. Ahadian, J. Travas-Sejdic, J. Lee, M. Mazloum-Ardakani,
J. Salvador, G.E. Aninwene 2nd, P. Bandaru, W. Sun, A. Khademhossieni,
Electrochemical cytosensors for detection of breast cancer cells, Biosens.
Bioelectron. 151 (2020), 111984.
[8] C. Nún ~ ez, Blood-based protein biomarkers in breast cancer, Clinica chimica
acta, Int. J. Clinical. Chem. 490 (2019) 113e127.
[9] L. Wang, Early diagnosis of breast cancer, Sensors (Basel) 17 (7) (2017).
[10] S. Mittal, H. Kaur, N. Gautam, A.K. Mantha, Biosensors for breast cancer
diagnosis: a review of bioreceptors, biotransducers and signal amplification
strategies, Biosens. Bioelectron. 88 (2017) 217e231.
[11] I.M. Mostafa, Y. Tian, S. Anjum, S. Hanif, M. Hosseini, B. Lou, G. Xu,
Comprehensive review on the electrochemical biosensors of different breast
cancer biomarkers, Sensor. Actuator. B Chem. 365 (2022), 131944.
[12] D.C. Ferreira, M.R. Batistuti, B. Bachour, M. Mulato, Aptasensor based on
screen-printed electrode for breast cancer detection in undiluted human
serum, Bioelectrochemistry 137 (2021), 107586.
[13] L. Li, D. Feng, J. Zhao, Z. Guo, Y. Zhang, Simultaneous fluoroimmunoassay of
two tumor markers based on CdTe quantum dots and gold nanocluster
coated-silica nanospheres as labels, RSC Adv. 5 (128) (2015) 105992e105998.
[14] V. Beral, D. Bull, R. Doll, T. Key, Breast cancer and hormone replacement
therapy: collaborative reanalysis of data from 51 epidemiological studies of
52 705, Lancet 350 (1997) 9084.
[15] E. Asav, M.K. Sezgintürk, A novel impedimetric disposable immunosensor for
rapid detection of a potential cancer biomarker, Int. J. Biol. Macromol. 66
(2014) 273e280.
[16] D.R. Hansberry, A. John, E. John, N. Agarwal, S.F. Gonzales, S.R. Baker,
A critical review of the readability of online patient education resources from
RadiologyInfo, Org. Am. J. Roentgenol. 202 (3) (2014) 566e575.
[17] M. Mazloum- Ardakani, Z. Tavakolian- Ardakani, N. Sahraei,
S.M. Moshtaghioun, Fabrication of an ultrasensitive and selective electro-
chemical aptasensor to detect carcinoembryonic antigen by using a new
nanocomposite, Biosens. Bioelectron. 129 (2019) 1e6.
[18] H.A. Alhadrami, Biosensors: Classifications, medical applications, and future
prospective, Biotechnol. Appl. Biochem. 65 (3) (2018) 497e508.
[19] S. Carvajal, S.N. Fera, A.L. Jones, T.A. Baldo, I.M. Mosa, J.F. Rusling, C.E. Krause,
Disposable inkjet-printed electrochemical platform for detection of clinically
relevant HER-2 breast cancer biomarker, Biosens. Bioelectron. 104 (2018)
158e162.
[20] M. Li, D.-W. Li, G. Xiu, Y.-T. Long, Applications of screen-printed electrodes in
current environmental analysis, Curr. Opin. Electrochem. 3 (1) (2017)
137e143.
[21] M.H. Naveen, N.G. Gurudatt, Y.-B. Shim, Applications of conducting polymer
composites to electrochemical sensors: a review, Appl. Mater. Today 9
(2017) 419e433.
[22] S. Campuzano, M. Pedrero, J.M. Pingarro n, Non-invasive breast cancer
diagnosis through electrochemical biosensing at different molecular levels,
Sensors (Basel) 17 (9) (2017).
[23] P. Kush, P. Kumar, R. Singh, A. Kaushik, Aspects of high-performance and bio-
acceptable magnetic nanoparticles for biomedical application, Asian J.
Pharm. Sci. 16 (6) (2021) 704e737.
[24] T. Florence, Anodic stripping voltammetry with a glassy carbon electrode
mercury-plated in situ, J. Electroanal. Chem. Interfacial Electrochem. 27 (2)
(1970) 273e281.
[25] S. Azhari, P. Sathishkumar, R. Ahamad, F. Ahmad, A.R. Mohd Yusoff, Fabri-
cation of a composite modified glassy carbon electrode: a highly selective,
sensitive and rapid electrochemical sensor for silver ion detection in river
water samples, Anal. Methods 8 (28) (2016) 5712e5721.
[26] M. Li, Y.-T. Li, D.-W. Li, Y.-T. Long, Recent developments and applications of
screen-printed electrodes in environmental assaysda review, Anal. Chim.
Acta 734 (2012) 31e44.
[27] I.H. Cho, D.H. Kim, S. Park, Electrochemical biosensors: perspective on
functional nanomaterials for on-site analysis, Biomater. Res. 24 (2020) 6.
[28] B. Wang, U. Akiba, J.-i. Anzai, Recent progress in nanomaterial-based elec-
trochemical biosensors for cancer biomarkers: a review, Molecules 22 (7)
(2017) 1048.
[29] I.E. Tothill, Biosensors for Cancer Markers Diagnosis, Seminars in Cell &
Developmental Biology, Elsevier, 2009, pp. 55e62.
[30] R. Stern, Hyaluronidases in cancer biology, Hyaluronan Cancer Bio. (2008)
207e220. Elsevier.
[31] M. Liu, Z. Wang, T. Tan, Z. Chen, X. Mou, X. Yu, Y. Deng, G. Lu, N. He, An
aptamer-based probe for molecular subtyping of breast cancer, Theranostics
8 (20) (2018) 5772.
[32] P.F. Rostamabadi, E. Heydari-Bafrooei, Impedimetric aptasensing of the
breast cancer biomarker HER2 using a glassy carbon electrode modified with
gold nanoparticles in a composite consisting of electrochemically reduced
graphene oxide and single-walled carbon nanotubes, Microchim. Acta 186
(8) (2019) 495.
[33] S. Singh, A. Deep, G. Mohanta, V.K. Meena, Developments in the electro-
chemical bionanosensors for the predictive diagnosis of prostate and breast
33
Materials Today Chemistry 26 (2022) 101129
cancer, in: P. Chandra, Y.N. Tan, S.P. Singh (Eds.), Next Generation Point-of-
care Biomedical Sensors Technologies for Cancer Diagnosis, Springer
Singapore, Singapore, 2017, pp. 253e278.
[34] I. Diaconu, C. Cristea, V. Ha ^rceaga , G. Marrazza, I. Berindan-Neagoe,
R. S
andulescu, Electrochemical immunosensors in breast and ovarian cancer,
Clinica chimica acta, Int. J. Clinical. Chem. 425 (2013) 128e138.
[35] S. Campuzano, P. Ya n
~ ez-Seden~ o, J.M. Pingarro
 n, Electrochemical genosensing
of circulating biomarkers, Sensors (Basel) 17 (4) (2017).
[36] F. Arduini, L. Micheli, D. Moscone, G. Palleschi, S. Piermarini, F. Ricci, G. Volpe,
Electrochemical biosensors based on nanomodified screen-printed elec-
trodes: recent applications in clinical analysis, TrAC, Trends Anal. Chem. 79
(2016) 114e126.
[37] A. Sinha, Dhanjai, S.M. Mugo, H. Zhao, J. Chen, R. Jain, Chapter 5 - electro-
chemical immunosensors for rapid detection of breast cancer biomarkers, in:
Inamuddin, R. Khan, A. Mohammad, A.M. Asiri (Eds.), Advanced Biosensors
for Health Care Applications, Elsevier, 2019, pp. 147e169.
[38] E.C. Rama, A. Costa-García, Screen-printed Electrochemical Immunosensors
for the Detection of Cancer and Cardiovascular Biomarkers, Electroanalys. 28
(8) (2016) 1700e1715.
[39] F.M. Alkabban, T. Ferguson, Breast Cancer, StatPearls, StatPearls Publishing
Copyright © 2021, StatPearls Publishing LLC., Treasure Island (FL), 2021.
[40] M. Begum, S. Karim, A. Malik, R. Khurshid, M. Asif, A. Salim, S.A. Nagra,
A. Zaheer, Z. Iqbal, A.M. Abuzenadah, M.H. Alqahtani, M. Rasool, CA 15-3
(Mucin-1) and physiological characteristics of breast cancer from Lahore,
Pakistan, Asian Pacific Journal of Cancer Prevention : Asian Pac. J. Cancer
Prev. APJCP 13 (10) (2012) 5257e5261.
[41] E.A. Rakha, T.C. Putti, D.M. Abd El-Rehim, C. Paish, A.R. Green, D.G. Powe,
A.H. Lee, J.F. Robertson, I.O. Ellis, Morphological and immunophenotypic
analysis of breast carcinomas with basal and myoepithelial differentiation,
J. Pathol. 208 (4) (2006) 495e506.
[42] A. Bombonati, D.C. Sgroi, The molecular pathology of breast cancer pro-
gression, J. Pathol. 223 (2) (2011) 307e317.
[43] C.E. DeSantis, J. Ma, A. Goding Sauer, L.A. Newman, A. Jemal, Breast cancer
statistics, 2017, racial disparity in mortality by state, CA A Cancer J. Clin. 67
(6) (2017) 439e448.
[44] N.J. Ronkainen, S.L. Okon, Nanomaterial-based electrochemical immuno-
sensors for clinically significant biomarkers, Materials 7 (6) (2014)
4669e4709.
[45] A. Rasooly, J. Jacobson, Development of biosensors for cancer clinical testing,
Biosens. Bioelectron. 21 (10) (2006) 1851e1858.
[46] M. Perfe zou, A. Turner, A. Merkoçi, Cancer detection using nanoparticle-
based sensors, Chem. Soc. Rev. 41 (7) (2012) 2606e2622.
[47] C.L. Sawyers, The cancer biomarker problem, Nature 452 (7187) (2008)
548e552.
[48] P. Ranjan, A. Parihar, S. Jain, N. Kumar, C. Dhand, S. Murali, D. Mishra,
S.K. Sanghi, J.P. Chaurasia, A.K. Srivastava, R. Khan, Biosensor-based diag-
nostic approaches for various cellular biomarkers of breast cancer: a
comprehensive review, Anal. Biochem. 610 (2020), 113996.
[49] D. Sadighbayan, K. Sadighbayan, M.R. Tohid-kia, A.Y. Khosroushahi,
M. Hasanzadeh, Development of electrochemical biosensors for tumor
marker determination towards cancer diagnosis: recent progress, TrAC,
Trends Anal. Chem. 118 (2019) 73e88.
[50] F. Cui, Z. Zhou, H.S. Zhou, Reviewdmeasurement and analysis of cancer
biomarkers based on electrochemical biosensors, J. Electrochem. Soc. 167 (3)
(2020), 037525.
[51] F. Mollarasouli, S. Kurbanoglu, S.A. Ozkan, The role of electrochemical
immunosensors in clinical analysis, Biosensors (Basel) 9 (3) (2019).
[52] M. Sharifi, M.R. Avadi, F. Attar, F. Dashtestani, H. Ghorchian, S.M. Rezayat,
A.A. Saboury, M. Falahati, Cancer diagnosis using nanomaterials based
electrochemical nanobiosensors, Biosens. Bioelectron. 126 (2019) 773e784.
[53] M. Hassler, 3 - other commonly used biomedical coatings: pyrolytic carbon
coatings, in: M. Driver (Ed.), Coatings for Biomedical Applications, Woodhead
Publishing, 2012, pp. 75e105.
[54] N.B. Mincu, V. Lazar, D. Stan, C.M. Mihailescu, R. Iosub, A.L. Mateescu, Screen-
printed electrodes (SPE) for in vitro diagnostic purpose, Diagnostics (Basel,
Switzerland) 10 (8) (2020).
[55] E. Martínez-Perin ~
an, C. Gutierrez-Sa nchez, T. García-Mendiola, E. Lorenzo,
Electrochemiluminescence biosensors using screen-printed electrodes, Bio-
sens. (Basel) 10 (9) (2020) 118.
[56] Z. Taleat, A. Khoshroo, M. Mazloum-Ardakani, Screen-printed electrodes for
biosensing: a review (2008e2013), Microchim. Acta 181 (9e10) (2014)
865e891.
[57] R.A. Couto, J.L. Lima, M.B. Quinaz, Recent developments, characteristics and
potential applications of screen-printed electrodes in pharmaceutical and
biological analysis, Talanta 146 (2016) 801e814.
[58] E.C. Rama, A. Costa-García, Screen-printed electrochemical immunosensors
for the detection of cancer and cardiovascular biomarkers, Electroanalys. 28
(8) (2016) 1700e1715.
[59] L. Valdevit, J. Bauer, Chapter 13.1 - fabrication of 3D micro-/nanoarchitected
materials, in: T. Baldacchini (Ed.), Three-Dimensional Microfabrication Using
Two-Photon Polymerization, second ed., William Andrew Publishing, 2020,
pp. 541e576.
[60] L. Redivo, M. Stredanský, E. De Angelis, L. Navarini, M. Resmini,  
L. Svorc, Bare
carbon electrodes as simple and efficient sensors for the quantification of
caffeine in commercial beverages, R. Soc. Open Sci. 5 (5) (2018), 172146.
P. Lakhera, V. Chaudhary, A. Jha et al.
[61] S. Sharma, Glassy carbon: a promising material for micro- and nano-
manufacturing, Materials (Basel, Switzerland) 11 (10) (2018).
[62] Y. Yi, G. Weinberg, M. Prenzel, M. Greiner, S. Heumann, S. Becker, R. Schlo €gl,
Electrochemical corrosion of a glassy carbon electrode, Catal. Today 295
(2017) 32e40.
[63] Activation of Glassy Carbon Electrodes, 2020.
[64] C.X. Du, L. Han, S.L. Dong, L.H. Li, Y. Wei, A novel procedure for fabricating
flexible screen-printed electrodes with improved electrochemical perfor-
mance, IOP Conf. Ser. Mater. Sci. Eng. 137 (2016), 012060.
[65] P. Kumar, A. Deep, S.C. Sharma, L.M. Bharadwaj, Bioconjugation of InGaP
quantum dots for molecular sensing, Anal. Biochem. 421 (1) (2012)
285e290.
[66] A.L. Sharma, P. Kumar, A. Deep, Highly sensitive glucose sensing with multi-
walled carbon nanotubes e polyaniline composite, Polym.-Plast. Technol.
Eng. 51 (13) (2012) 1382e1387.
[67] M. Kukkar, A. Sharma, P. Kumar, K.H. Kim, A. Deep, Application of MoS2
modified screen-printed electrodes for highly sensitive detection of bovine
serum albumin, Anal. Chim. Acta 939 (2016) 101e107.
[68] A. Leniart, M. Brycht, B. Burnat, S. Skrzypek, An application of a glassy carbon
electrode and a glassy carbon electrode modified with multi-walled carbon
nanotubes in electroanalytical determination of oxycarboxin, Ionics 24 (7)
(2018) 2111e2121.
[69] M.R. Hasan, M.S. Ahommed, M. Daizy, M.S. Bacchu, M.R. Ali, M.R. Al-Mamun,
M.A. Saad Aly, M.Z.H. Khan, S.I. Hossain, Recent development in electro-
chemical biosensors for cancer biomarkers detection, Biosens. Bioelectron. X
8 (2021), 100075.
[70] W. Putzbach, N.J. Ronkainen, Immobilization techniques in the fabrication of
nanomaterial-based electrochemical biosensors: a review, Sensors 13 (4)
(2013) 4811e4840.
[71] F. Ricci, G. Adornetto, G. Palleschi, A review of experimental aspects of
electrochemical immunosensors, Electrochim. Acta 84 (2012) 74e83.
[72] G. Chang, H. Shu, K. Ji, M. Oyama, X. Liu, Y. He, Gold nanoparticles directly
modified glassy carbon electrode for non-enzymatic detection of glucose,
Appl. Surf. Sci. 288 (2014) 524e529.
[73] Z. Li, J. Zhang, Y. Zhou, S. Shuang, C. Dong, M.M.F. Choi, Electrodeposition of
palladium nanoparticles on fullerene modified glassy carbon electrode for
methane sensing, Electrochim. Acta 76 (2012) 288e291.
[74] L. Dhanapala, C.E. Krause, A.L. Jones, J.F. Rusling, Printed electrodes in
microfluidic arrays for cancer biomarker protein detection, Biosensors
(Basel) 10 (9) (2020).
[75] F. Wei, P.B. Lillehoj, C.M. Ho, DNA diagnostics: nanotechnology-enhanced
electrochemical detection of nucleic acids, Pediatr. Res. 67 (5) (2010)
458e468.
[76] S.Z. Mousavisani, J.-B. Raoof, A.P.F. Turner, R. Ojani, W.C. Mak, Label-free DNA
sensor based on diazonium immobilisation for detection of DNA damage in
breast cancer 1 gene, Sensor. Actuator. B Chem. 264 (2018) 59e66.
[77] T. García-Mendiola, S. Requena-Sanz, E. Martínez-Perin ~an, I. Bravo,
F. Pariente, E. Lorenzo, Influence of carbon nanodots on DNA-Thionine
interaction. Application to breast cancer diagnosis, Electrochim. Acta 353
(2020), 136522.
[78] D. Feng, J. Su, G. He, Y. Xu, C. Wang, M. Zheng, Q. Qian, X. Mi, Electrochemical
DNA sensor for sensitive BRCA1 detection based on DNA tetrahedral-
structured probe and poly-adenine mediated gold nanoparticles, Bio-
sensors (Basel) 10 (7) (2020).
[79] P.A. Rasheed, N. Sandhyarani, A highly sensitive DNA sensor for attomolar
detection of the BRCA1 gene: signal amplification with gold nanoparticle
clusters, Analyst 140 (8) (2015) 2713e2718.
[80] W. Wang, X. Fan, S. Xu, J.J. Davis, X. Luo, Low fouling label-free DNA sensor
based on polyethylene glycols decorated with gold nanoparticles for the
detection of breast cancer biomarkers, Biosens. Bioelectron. 71 (2015)
51e56.
[81] N. Hui, X. Sun, S. Niu, X. Luo, PEGylated polyaniline nanofibers: antifouling
and conducting biomaterial for electrochemical DNA sensing, ACS Appl.
Mater. Interfaces 9 (3) (2017) 2914e2923.
[82] G. Wang, R. Han, X. Su, Y. Li, G. Xu, X. Luo, Zwitterionic peptide anchored to
conducting polymer PEDOT for the development of antifouling and ultra-
sensitive electrochemical DNA sensor, Biosens. Bioelectron. 92 (2017)
396e401.
[83] S. Shahrokhian, R. Salimian, Ultrasensitive detection of cancer biomarkers
using conducting polymer/electrochemically reduced graphene oxide-based
biosensor: application toward BRCA1 sensing, Sensor. Actuator. B Chem. 266
(2018) 160e169.
[84] M. Hasanzadeh, M. Feyziazar, E. Solhi, A. Mokhtarzadeh, J. Soleymani,
N. Shadjou, A. Jouyban, S. Mahboob, Ultrasensitive immunoassay of breast
cancer type 1 susceptibility protein (BRCA1) using poly (dopamine-beta
cyclodextrine-Cetyl trimethylammonium bromide) doped with silver
nanoparticles: a new platform in early stage diagnosis of breast cancer and
efficient management, Microchem. J. 145 (2019) 778e783.
[85] Y.-M. Xia, M.-Y. Li, C.-L. Chen, M. Xia, W. Zhang, W.-W. Gao, Employing label-
free electrochemical biosensor based on 3D-reduced graphene oxide and
polyaniline nanofibers for ultrasensitive detection of breast cancer BRCA1
biomarker, Electroanalysis 32 (9) (2020) 2045e2055.
[86] H. Ehzari, M. Safari, M. Samimi, Signal amplification of novel sandwich-type
genosensor via catalytic redox-recycling on platform MWCNTs/Fe(3)O(4)
@TMU-21 for BRCA1 gene detection, Talanta 234 (2021), 122698.
34
Materials Today Chemistry 26 (2022) 101129
[87] R.M. Torrente-Rodríguez, S. Campuzano, E. Lo pez-Herna ndez, V.R. Montiel,
R. Barderas, R. Granados, J.M. Sa nchez-Puelles, J.M. Pingarron, Simultaneous
detection of two breast cancer-related miRNAs in tumor tissues using p19-
based disposable amperometric magnetobiosensing platforms, Biosens.
Bioelectron. 66 (2015) 385e391.
[88] A. Erdem, E. Eksin, G. Congur, Indicator-free electrochemical biosensor for
microRNA detection based on carbon nanofibers modified screen printed
electrodes, J. Electroanal. Chem. 755 (2015) 167e173.
[89] A.R. Cardoso, F.T.C. Moreira, R. Fernandes, M.G.F. Sales, Novel and simple
electrochemical biosensor monitoring attomolar levels of miRNA-155 in
breast cancer, Biosens. Bioelectron. 80 (2016) 621e630.
[90] H. Mohammadi, A. Amine, Spectrophotometric and electrochemical deter-
mination of MicroRNA-155 using sandwich hybridization magnetic beads,
Anal. Lett. 51 (3) (2018) 411e423.
[91] E. Povedano, V. Ruiz-Valdepen ~ as Montiel, M. Gamella, V. Serafín, M. Pedrero,
L. Moranova, M. Bartosik, J.J. Montoya, P. Y ~ ez-Seden
an ~ o, S. Campuzano,
J.M. Pingarro n, A novel zinc finger protein-based amperometric
biosensor for miRNA determination, Anal. Bioanal. Chem. 412 (21) (2020)
5031e5041.
[92] M. Zouari, S. Campuzano, J.M. Pingarro  n, N. Raouafi, Femtomolar direct
voltammetric determination of circulating miRNAs in sera of cancer patients
using an enzymeless biosensor, Anal. Chim. Acta 1104 (2020) 188e198.
[93] C. Pothipor, N. Aroonyadet, S. Bamrungsap, J. Jakmunee, K. Ounnunkad,
A highly sensitive electrochemical microRNA-21 biosensor based on inter-
calating methylene blue signal amplification and a highly dispersed gold
nanoparticles/graphene/polypyrrole composite, Analyst 146 (8) (2021)
2679e2688.
[94] C. Pothipor, J. Jakmunee, S. Bamrungsap, K. Ounnunkad, An electrochemical
biosensor for simultaneous detection of breast cancer clinically related
microRNAs based on a gold nanoparticles/graphene quantum dots/graphene
oxide film, Analyst 146 (12) (2021) 4000e4009.
[95] H.A. Rafiee-Pour, M. Behpour, M. Keshavarz, A novel label-free electro-
chemical miRNA biosensor using methylene blue as redox indicator: appli-
cation to breast cancer biomarker miRNA-21, Biosens. Bioelectron. 77 (2016)
202e207.
[96] A. Ebrahimi, I. Nikokar, M. Zokaei, E. Bozorgzadeh, Design, development and
evaluation of microRNA-199a-5p detecting electrochemical nanobiosensor
with diagnostic application in Triple Negative Breast Cancer, Talanta 189
(2018) 592e598.
[97] D. Wang, J. Wang, A sensitive and label-free electrochemical microRNA
biosensor based on Polyamidoamine Dendrimer functionalized Polypyrrole
nanowires hybrid, Microchim. Acta 188 (5) (2021) 173.
[98] M. Azimzadeh, M. Rahaie, N. Nasirizadeh, K. Ashtari, H. Naderi-Manesh, An
electrochemical nanobiosensor for plasma miRNA-155, based on graphene
oxide and gold nanorod, for early detection of breast cancer, Biosens. Bio-
electron. 77 (2016) 99e106.
[99] K. Zhang, N. Zhang, L. Zhang, H. Wang, H. Shi, Q. Liu, Label-free impedimetric
sensing platform for microRNA-21 based on ZrO2-reduced graphene oxide
nanohybrids coupled with catalytic hairpin assembly amplification, RSC Adv.
8 (29) (2018) 16146e16151.
[100] J. Zhang, D.Z. Wu, S.X. Cai, M. Chen, Y.K. Xia, F. Wu, J.H. Chen, An
immobilization-free electrochemical impedance biosensor based on duplex-
specific nuclease assisted target recycling for amplified detection of micro-
RNA, Biosens. Bioelectron. 75 (2016) 452e457.
[101] X. Ma, H. Xu, K. Qian, M. Kandawa-Schulz, W. Miao, Y. Wang, Electrochemical
detection of microRNAs based on AuNPs/CNNS nanocomposite with Duplex-
specific nuclease assisted target recycling to improve the sensitivity, Talanta
208 (2020), 120441.
[102] S. Sharma, J. Zapatero-Rodríguez, R. Saxena, R. O'Kennedy, S. Srivastava,
Ultrasensitive direct impedimetric immunosensor for detection of serum
HER2, Biosens. Bioelectron. 106 (2018) 78e85.
[103] E. Chocholova, T. Bertok, L. Lorencova, A. Holazova, P. Farkas, A. Vikartovska,
V. Bella, D. Velicova, P. Kasak, A.A. Eckstein, J. Mosnacek, D. Hasko, J. Tkac,
Advanced antifouling zwitterionic layer based impedimetric HER2 bio-
sensing in human serum: glycoprofiling as a novel approach for breast
cancer diagnostics, Sensor. Actuator. B Chem. 272 (2018) 626e633.
[104] Y.W. Hartati, L.K. Letelay, S. Gaffar, S. Wyantuti, H.H. Bahti, Cerium
oxide-monoclonal antibody bioconjugate for electrochemical immuno-
sensing of HER2 as a breast cancer biomarker, Sens. Bio-Sens. Res. 27
(2020), 100316.
[105] S.D. Tallapragada, K. Layek, R. Mukherjee, K.K. Mistry, M. Ghosh, Develop-
ment of screen-printed electrode based immunosensor for the detection of
HER2 antigen in human serum samples, Bioelectrochemistry 118 (2017)
25e30.
[106] Z. Lah, S.A.A. Ahmad, M.S. Zaini, M.A. Kamarudin, An electrochemical sand-
wich immunosensor for the detection of HER2 using antibody-conjugated
PbS quantum dot as a label, J. Pharmaceut. Biomed. Anal. 174 (2019)
608e617.
[107] M. Freitas, H.P.A. Nouws, E. Keating, C. Delerue-Matos, High-performance
electrochemical immunomagnetic assay for breast cancer analysis, Sensor.
Actuator. B Chem. 308 (2020), 127667.
[108] M. Freitas, H.P.A. Nouws, E. Keating, V.C. Fernandes, C. Delerue-Matos,
Immunomagnetic bead-based bioassay for the voltammetric analysis of the
breast cancer biomarker HER2-ECD and tumour cells using quantum dots as
detection labels, Mikrochim. Acta 187 (3) (2020) 184.
P. Lakhera, V. Chaudhary, A. Jha et al.
[109] M. Freitas, M.M. Neves, H.P. Nouws, C. Delerue-Matos, Quantum dots as
nanolabels for breast cancer biomarker HER2-ECD analysis in human serum,
Talanta 208 (2020), 120430.
[110] M. Shamsipur, M. Emami, L. Farzin, R. Saber, A sandwich-type electro-
chemical immunosensor based on in situ silver deposition for determination
of serum level of HER2 in breast cancer patients, Biosens. Bioelectron. 103
(2018) 54e61.
[111] H. Ehzari, M. Samimi, M. Safari, M.B. Gholivand, Label-free electrochemical
immunosensor for sensitive HER2 biomarker detection using the core-shell
magnetic metal-organic frameworks, J. Electroanal. Chem. 877 (2020),
114722.
[112] S. Centane, T. Nyokong, The antibody assisted detection of HER2 on a cobalt
porphyrin binuclear framework and gold functionalized graphene quantum
dots modified electrode, J. Electroanal. Chem. 880 (2021), 114908.
[113] H. Nasrollahpour, A. Naseri, M.-R. Rashidi, B. Khalilzadeh, Application of
green synthesized WO3-poly glutamic acid nanobiocomposite for early stage
biosensing of breast cancer using electrochemical approach, Sci. Rep. 11 (1)
(2021), 23994.
[114] K. Chan, H. Lim, N. Shams, S. Jayabal, A. Pandikumar, N. Huang, Fabrication of
graphene/gold-modified screen-printed electrode for detection of carci-
noembryonic antigen, Mater. Sci. Eng. C 58 (2016) 666e674.
[115] L. Zhao, C. Li, H. Qi, Q. Gao, C. Zhang, Electrochemical lectin-based biosensor
array for detection and discrimination of carcinoembryonic antigen using
dual amplification of gold nanoparticles and horseradish peroxidase, Sensor.
Actuator. B Chem. 235 (2016) 575e582.
[116] S. Lee, H. Lim, I. Ibrahim, A. Jamil, A. Pandikumar, N. Huang, Horseradish
peroxidase-labeled silver/reduced graphene oxide thin film-modified
screen-printed electrode for detection of carcinoembryonic antigen, Bio-
sens. Bioelectron. 89 (2017) 673e680.
[117] L. Cao, H. Xiao, C. Fang, F. Zhao, Z. Chen, Electrochemical immunosensor
based on binary nanoparticles decorated rGO-TEPA as magnetic capture and
Au@PtNPs as probe for CEA detection, Mikrochim. Acta 187 (10) (2020) 584.
[118] N. Li, Y. Wang, W. Cao, Y. Zhang, T. Yan, B. Du, Q. Wei, An ultrasensitive
electrochemical immunosensor for CEA using MWCNT-NH 2 supported PdPt
nanocages as labels for signal amplification, J. Mater. Chem. B 3 (9) (2015)
2006e2011.
[119] J. Han, L. Jiang, F. Li, P. Wang, Q. Liu, Y. Dong, Y. Li, Q. Wei, Ultrasensitive non-
enzymatic immunosensor for carcino-embryonic antigen based on palla-
dium hybrid vanadium pentoxide/multiwalled carbon nanotubes, Biosens.
Bioelectron. 77 (2016) 1104e1111.
[120] L. Zheng, X. Niu, J. Zhao, T. Liu, Y. Liu, Y. Yang, A label-free immunosensor for
CEA based on Pd-Ir bimetallic nanoparticles, J. Nanosci. Nanotechnol. 16 (6)
(2016) 5984e5990.
[121] Z. Tang, Z. Ma, Ultrasensitive amperometric immunoassay for carcinoem-
bryonic antigens by using a glassy carbon electrode coated with a
polydopamine-Pb (II) redox system and a chitosan-gold nanocomposite,
Microchim. Acta 184 (4) (2017) 1135e1142.
[122] Y. Luo, Y. Wang, H. Yan, Y. Wu, C. Zhu, D. Du, Y. Lin, SWCNTs@GQDs com-
posites as nanocarriers for enzyme-free dual-signal amplification electro-
chemical immunoassay of cancer biomarker, Anal. Chim. Acta 1042 (2018)
44e51.
[123] Y. Wang, G. Zhao, Y. Zhang, X. Pang, W. Cao, B. Du, Q. Wei, Sandwich-type
electrochemical immunosensor for CEA detection based on Ag/MoS2@Fe3O4
and an analogous ELISA method with total internal reflection microscopy,
Sensor. Actuator. B Chem. 266 (2018) 561e569.
[124] Y. Yang, M. Jiang, K. Cao, M. Wu, C. Zhao, H. Li, C. Hong, An electrochemical
immunosensor for CEA detection based on Au-Ag/rGO@PDA nanocomposites
as integrated double signal amplification strategy, Microchem. J. 151 (2019),
104223.
[125] S.A. Nakhjavani, H. Afsharan, B. Khalilzadeh, M.H. Ghahremani, S. Carrara,
Y. Omidi, Gold and silver bio/nano-hybrids-based electrochemical immu-
nosensor for ultrasensitive detection of carcinoembryonic antigen, Biosens.
Bioelectron. 141 (2019), 111439.
[126] Y. Lai, H. Huang, Z. Xia, S. Li, Y. Deng, X. Liu, A sandwich-type electrochemical
immunosensor using polythionine/AuNPs nanocomposites as label for ul-
trasensitive detection of carcinoembryonic antigen, Mater. Exp. 9 (5) (2019)
444e450.
[127] K. Liu, H. Deng, Y. Wang, S. Cheng, X. Xiong, C. Li, A sandwich-type photo-
electrochemical immunosensor based on ReS2 nanosheets for high-
performance determination of carcinoembryonic antigen, Sensor. Actuator.
B Chem. 320 (2020), 128341.
[128] A. Jing, Q. Xu, W. Feng, G. Liang, An electrochemical immunosensor for
sensitive detection of the tumor marker carcinoembryonic antigen (CEA)
based on three-dimensional porous nanoplatinum/graphene, Micro-
machines 11 (7) (2020).
[129] M. Jozghorbani, M. Fathi, S.H. Kazemi, N. Alinejadian, Determination of
carcinoembryonic antigen as a tumor marker using a novel graphene-based
label-free electrochemical immunosensor, Anal. Biochem. 613 (2021),
114017.
[130] S. Rauf, G.K. Mishra, J. Azhar, R.K. Mishra, K.Y. Goud, M.A.H. Nawaz, J.L. Marty,
A. Hayat, Carboxylic group riched graphene oxide based disposable elec-
trochemical immunosensor for cancer biomarker detection, Anal. Biochem.
545 (2018) 13e19.
[131] N.A. Alarfaj, M.F. El-Tohamy, H. Oraby, New label-free ultrasensitive elec-
trochemical immunosensor-based Au/MoS2/rGO nanocomposites for CA 27-
35
Materials Today Chemistry 26 (2022) 101129
29 breast cancer antigen detection, New J. Chem. 42 (13) (2018)
11046e11053.
[132] V.-A. Nguyen, H.L. Nguyen, D.T. Nguyen, Q.P. Do, L.D. Tran, Electro-
synthesized poly(1,5-diaminonaphthalene)/polypyrrole nanowires bilayer
as an immunosensor platform for breast cancer biomarker CA 15-3, Curr.
Appl. Phys. 17 (11) (2017) 1422e1429.
[133] R.S. Gomes, F.T.C. Moreira, R. Fernandes, M.G.F. Sales, Sensing CA 15-3 in
point-of-care by electropolymerizing O-phenylenediamine (oPDA) on Au-
screen printed electrodes, PLoS One 13 (5) (2018), e0196656.
[134] J. Amani, A. Khoshroo, M. Rahimi-Nasrabadi, Electrochemical immunosensor
for the breast cancer marker CA 15-3 based on the catalytic activity of a CuS/
reduced graphene oxide nanocomposite towards the electrooxidation of
catechol, Mikrochim. Acta 185 (1) (2017) 79.
[135] A. Khoshroo, M. Mazloum-Ardakani, M. Forat-Yazdi, Enhanced performance
of label-free electrochemical immunosensor for carbohydrate antigen 15-3
based on catalytic activity of cobalt sulfide/graphene nanocomposite, Sens.
Actuator. B Chem. 255 (2018) 580e587.
[136] T.S. Martins, J.L. Bott-Neto, O.N. Oliveira, S.A.S. Machado, A sandwich-type
electrochemical immunosensor based on Au-rGO composite for CA15-3 tu-
mor marker detection, Microchim. Acta 189 (1) (2021) 38.
[137] T.S.C.R. Rebelo, J.A. Ribeiro, M.G.F. Sales, C.M. Pereira, Electrochemical
immunosensor for detection of CA 15-3 biomarker in point-of-care, Sens.
Bio-Sens. Res. 33 (2021), 100445.
[138] L. Liu, J. Gao, L. Guo, J. Xu, Nitrogen-doped graphene modified glassy carbon
electrode for electrochemical determination of breast cancer marker car-
bohydrate antigen 15-3, Int. J. Electrochem. Sci. 12 (2017) 8280e8287.
[139] Y. Liu, X. Weng, K.-K. Wang, Y. Xue, A.-J. Wang, L. Wu, J.-J. Feng, A novel
enzyme-free sandwich-like electrochemical immunosensor for the detection
of carbohydrate antigen 15-3 based on hierarchical AuPd nanochain net-
works, Sensor. Actuator. B Chem. 247 (2017) 349e356.
[140] M. Hasanzadeh, E. Solhi, M. Jafari, A. Mokhtarzadeh, J. Soleymani, A. Jouyban,
S. Mahboob, Ultrasensitive immunoassay of tumor protein CA 15.3 in MCF-7
breast cancer cell lysates and unprocessed human plasma using gold
nanoparticles doped on the structure of mesoporous silica, Int. J. Biol. Mac-
romol. 120 (Pt B) (2018) 2493e2508.
[141] H. Jia, Q. Tian, J. Xu, L. Lu, X. Ma, Y. Yu, Aerogels prepared from polymeric b-
cyclodextrin and graphene aerogels as a novel host-guest system for
immobilization of antibodies: a voltammetric immunosensor for the tumor
marker CA 15-3, Mikrochim. Acta 185 (11) (2018) 517.
[142] M. Hasanzadeh, S. Tagi, E. Solhi, A. Mokhtarzadeh, N. Shadjou, A. Eftekhari,
S. Mahboob, An innovative immunosensor for ultrasensitive detection of
breast cancer specific carbohydrate (CA 15-3) in unprocessed human plasma
and MCF-7 breast cancer cell lysates using gold nanospear electrochemically
assembled onto thiolated graphene quantum dots, Int. J. Biol. Macromol. 114
(2018) 1008e1017.
[143] A. Saadati, S. Hassanpour, M. Hasanzadeh, N. Shadjou, A. Hassanzadeh,
Immunosensing of breast cancer tumor protein CA 15-3 (carbohydrate an-
tigen 15.3) using a novel nano-bioink: a new platform for screening of
proteins in human biofluids by pen-on-paper technology, Int. J. Biol. Mac-
romol. 132 (2019) 748e758.
[144] C. Zhang, D. Zhang, Z. Ma, H. Han, Cascade catalysis-initiated radical poly-
merization amplified impedimetric immunosensor for ultrasensitive detec-
tion of carbohydrate antigen 15-3, Biosens. Bioelectron. 137 (2019) 1e7.
[145] U. Eletxigerra, J. Martinez-Perdiguero, S. Merino, R. Barderas, V. Ruiz-
Valdepen ~ as Montiel, R. Villalonga, J.M. Pingarron, S. Campuzano, Estrogen
receptor a determination in serum, cell lysates and breast cancer cells using
an amperometric magnetoimmunosensing platform, Sens. Bio-Sens. Res. 7
(2016) 71e76.
[146] U. Eletxigerra, J. Martinez-Perdiguero, S. Merino, R. Barderas, V. Ruiz-
Valdepen ~ as Montiel, R. Villalonga, J.M. Pingarro  n, S. Campuzano, Electro-
chemical magnetoimmunosensor for progesterone receptor determination.
Application to the simultaneous detection of estrogen and progesterone
breast-cancer related receptors in raw cell lysates, Electroanalysis 28 (8)
(2016) 1787e1794.
[147] G. Bezerra, C. Co rdula, D. Campos, G. Nascimento, N. Oliveira, M.A. Seabra,
V. Visani, S. Lucas, I. Lopes, J. Santos, F. Xavier Jr., M.A. Borba, D. Martins,
J. Lima-Filho, Electrochemical aptasensor for the detection of HER2 in human
serum to assist in the diagnosis of early stage breast cancer, Anal. Bioanal.
Chem. 411 (25) (2019) 6667e6676.
[148] T. Harahsheh, Y.F. Makableh, I. Rawashdeh, M. Al-Fandi, Enhanced apta-
sensor performance for targeted HER2 breast cancer detection by using
screen-printed electrodes modified with Au nanoparticles, Biomed. Micro-
devices 23 (4) (2021) 46.
[149] A. Tabasi, A. Noorbakhsh, E. Sharifi, Reduced graphene oxide-chitosan-aptamer
interface as new platform for ultrasensitive detection of human epidermal
growth factor receptor 2, Biosens. Bioelectron. 95 (2017) 117e123.
[150] B. He, Differential pulse voltammetric assay for the carcinoembryonic anti-
gen using a glassy carbon electrode modified with layered molybdenum
selenide, graphene, and gold nanoparticles, Microchim. Acta 184 (1) (2017)
229e235.
[151] Z. Si, B. Xie, Z. Chen, C. Tang, T. Li, M. Yang, Electrochemical aptasensor for
the cancer biomarker CEA based on aptamer induced current due to for-
mation of molybdophosphate, Microchim. Acta 184 (9) (2017) 3215e3221.
[152] Z. Shekari, H.R. Zare, A. Falahati, Developing an impedimetric aptasensor for
selective labelefree detection of CEA as a cancer biomarker based on gold
P. Lakhera, V. Chaudhary, A. Jha et al.
nanoparticles loaded in functionalized mesoporous silica films,
J. Electrochem. Soc. 164 (13) (2017) B739eB745.
[153] W. Jiang, L. Liu, L. Zhang, Q. Guo, Y. Cui, M. Yang, Sensitive immunosensing of
the carcinoembryonic antigen utilizing aptamer-based in-situ formation of a
redox-active heteropolyacid and rolling circle amplification, Microchim. Acta
184 (12) (2017) 4757e4763.
[154] X. Zhou, S. Guo, J. Gao, J. Zhao, S. Xue, W. Xu, Glucose oxidase-initiated
cascade catalysis for sensitive impedimetric aptasensor based on metal-
organic frameworks functionalized with Pt nanoparticles and hemin/G-
quadruplex as mimicking peroxidases, Biosens. Bioelectron. 98 (2017) 83e90.
[155] M.A. Nawaz, S. Rauf, G. Catanante, M.H. Nawaz, G. Nunes, J.L. Marty, A. Hayat,
One step assembly of thin films of carbon nanotubes on screen printed
interface for electrochemical aptasensing of breast cancer biomarker, Sen-
sors 16 (10) (2016).
[156] S. Yang, F. Zhang, Q. Liang, Z. Wang, A three-dimensional graphene-based
ratiometric signal amplification aptasensor for MUC1 detection, Biosens.
Bioelectron. 120 (2018) 85e92.
[157] H. Wang, J. Sun, L. Lu, X. Yang, J. Xia, F. Zhang, Z. Wang, Competitive elec-
trochemical aptasensor based on a cDNA-ferrocene/MXene probe for
detection of breast cancer marker Mucin1, Anal. Chim. Acta 1094 (2020)
18e25.
[158] R. Ahirwar, A. Dalal, J.G. Sharma, B.K. Yadav, P. Nahar, A. Kumar, S. Kumar, An
aptasensor for rapid and sensitive detection of estrogen receptor alpha in
human breast cancer, Biotechnol. Bioeng. 116 (1) (2019) 227e233.
[159] H.A. Samie, M. Arvand, Label-free electrochemical aptasensor for proges-
terone detection in biological fluids, Bioelectrochemistry 133 (2020),
107489.
[160] Y. Park, M.S. Hong, W.H. Lee, J.G. Kim, K. Kim, Highly sensitive electro-
chemical aptasensor for detecting the VEGF(165) tumor marker with PANI/
CNT nanocomposites, Biosens. (Basel) 11 (4) (2021).
[161] M. Shamsipur, L. Farzin, M. Amouzadeh Tabrizi, F. Molaabasi, Highly sensi-
tive label free electrochemical detection of VGEF165 tumor marker based on
“signal off” and “signal on” strategies using an anti-VEGF165 aptamer
immobilized BSA-gold nanoclusters/ionic liquid/glassy carbon electrode,
Biosens. Bioelectron. 74 (2015) 369e375.
[162] X.-M. Fu, Z.-J. Liu, S.-X. Cai, Y.-P. Zhao, D.-Z. Wu, C.-Y. Li, J.-H. Chen, Elec-
trochemical aptasensor for the detection of vascular endothelial growth
factor (VEGF) based on DNA-templated Ag/Pt bimetallic nanoclusters, Chin.
Chem. Lett. 27 (6) (2016) 920e926.
[163] A. Bagheri Hashkavayi, B.S. Cha, S.H. Hwang, J. Kim, K.S. Park, Highly sensitive
electrochemical detection of circulating EpCAM-positive tumor cells using a
dual signal amplification strategy, Sensor. Actuator. B Chem. 343 (2021),
130087.
[164] R. Zhang, Q. You, M. Cheng, M. Ge, Q. Mei, L. Yang, W.-F. Dong, Z. Chang,
Multifunctional gold nano-cytosensor with quick capture, electrochemical
detection, and non-invasive release of circulating tumor cells for early cancer
treatment, Front. Bioeng. Biotechnol. 9 (2021), 783661-783661.
[165] M. Amouzadeh Tabrizi, M. Shamsipur, R. Saber, S. Sarkar, N. Zolfaghari, An
ultrasensitive sandwich-type electrochemical immunosensor for the deter-
mination of SKBR-3 breast cancer cell using rGO-TPA/FeHCFnano labeled
Anti-HCT as a signal tag, Sensor. Actuator. B Chem. 243 (2017) 823e830.
[166] Z. Rahimzadeh, S.M. Naghib, E. Askari, F. Molaabasi, A. Sadr, Y. Zare,
M. Afsharpad, K.Y. Rhee, A rapid nanobiosensing platform based on
herceptin-conjugated graphene for ultrasensitive detection of circulating
tumor cells in early breast cancer, Nanotechnol. Rev. 10 (1) (2021) 744e753.
[167] L. Tian, J. Qi, K. Qian, O. Oderinde, Y. Cai, C. Yao, W. Song, Y. Wang, An ul-
trasensitive electrochemical cytosensor based on the magnetic field assisted
binanozymes synergistic catalysis of Fe3O4 nanozyme and reduced gra-
phene oxide/molybdenum disulfide nanozyme, Sensor. Actuator. B Chem.
260 (2018) 676e684.
[168] L. Tian, J. Qi, K. Qian, O. Oderinde, Q. Liu, C. Yao, W. Song, Y. Wang, Copper (II)
oxide nanozyme based electrochemical cytosensor for high sensitive
detection of circulating tumor cells in breast cancer, J. Electroanal. Chem. 812
(2018) 1e9.
[169] Y. Yang, Y. Fu, H. Su, L. Mao, M. Chen, Sensitive detection of MCF-7 human
breast cancer cells by using a novel DNA-labeled sandwich electrochemical
biosensor, Biosens. Bioelectron. 122 (2018) 175e182.
[170] N. Liu, J. Song, Y. Lu, J.J. Davis, F. Gao, X. Luo, Electrochemical aptasensor for
ultralow fouling cancer cell quantification in complex biological media based
on designed branched peptides, Anal. Chem. 91 (13) (2019) 8334e8340.
[171] M. Safavipour, M. Kharaziha, E. Amjadi, F. Karimzadeh, A. Allafchian, TiO2
nanotubes/reduced GO nanoparticles for sensitive detection of breast cancer
cells and photothermal performance, Talanta 208 (2020), 120369.
[172] F. Shafiei, R.S. Saberi, M.A. Mehrgardi, A label-free electrochemical apta-
sensor for breast cancer cell detection based on a reduced graphene oxide-
chitosan-gold nanoparticle composite, Bioelectrochemistry 140 (2021),
107807.
[173] J. Quan, Y. Wang, J. Zhang, K. Huang, X. Wang, H. Jiang, Aptamer embedded
arch-cruciform DNA assemblies on 2-D VS(2) scaffolds for sensitive detection
of breast cancer cells, Biosensors (Basel) 11 (10) (2021).
[174] Y.H. Tang, H.C. Lin, C.L. Lai, P.Y. Chen, C.H. Lai, Mannosyl electrochemical
impedance cytosensor for label-free MDA-MB-231 cancer cell detection,
Biosens. Bioelectron. 116 (2018) 100e107.
36
Materials Today Chemistry 26 (2022) 101129
[175] Y. Zhou, Y. Wan, M. Yu, X. Yuan, C. Zhang, Hyaluronic acid-based label-free
electrochemical impedance analysis for cancer cell quantification and CD44
expression, Microchem. J. 160 (2021), 105622.
[176] R.C.B. Marques, E. Costa-Rama, S. Viswanathan, H.P.A. Nouws, A. Costa-
García, C. Delerue-Matos, M.B. Gonz alez-García, Voltammetric immuno-
sensor for the simultaneous analysis of the breast cancer biomarkers CA 15-3
and HER2-ECD, Sensor. Actuator. B Chem. 255 (2018) 918e925.
[177] A. Valverde, V. Serafín, J. Garoz, A. Montero-Calle, A. Gonza lez-Corte
s,
M. Arenas, J. Camps, R. Barderas, P. Y ~ ez-Seden
an ~ o, S. Campuzano,
J.M. Pingarro n, Electrochemical immunoplatform to improve the reliability
of breast cancer diagnosis through the simultaneous determination of
RANKL and TNF in serum, Sensor. Actuator. B Chem. 314 (2020), 128096.
[178] Q. Guo, X. Li, C. Shen, S. Zhang, H. Qi, T. Li, M. Yang, Electrochemical
immunoassay for the protein biomarker mucin 1 and for MCF-7 cancer cells
based on signal enhancement by silver nanoclusters, Microchim. Acta 182
(7) (2015) 1483e1489.
[179] S. Yazdanparast, A. Benvidi, M. Banaei, H. Nikukar, M.D. Tezerjani,
M. Azimzadeh, Dual-aptamer based electrochemical sandwich biosensor for
MCF-7 human breast cancer cells using silver nanoparticle labels and a pol-
y(glutamic acid)/MWNT nanocomposite, Microchim. Acta 185 (9) (2018) 405.
[180] Z. Shekari, H.R. Zare, A. Falahati, Dual assaying of breast cancer biomarkers
by using a sandwichetype electrochemical aptasensor based on a gold
nanoparticlese3D graphene hydrogel nanocomposite and redox probes
labeled aptamers, Sensor. Actuator. B Chem. 332 (2021), 129515.
[181] C. Pothipor, S. Bamrungsap, J. Jakmunee, K. Ounnunkad, A gold nanoparticle-
dye/poly(3-aminobenzylamine)/two dimensional MoSe2/graphene oxide
electrode towards label-free electrochemical biosensor for simultaneous
dual-mode detection of cancer antigen 15-3 and microRNA-21, Collo. Surf. B
Biointer. 210 (2022), 112260.
[182] M. Sharifi, A. Hasan, F. Attar, A. Taghizadeh, M. Falahati, Development of
point-of-care nanobiosensors for breast cancers diagnosis, Talanta 217
(2020), 121091.
[183] P. Balraj, A. Khoo, L. Volpi, J. Tan, S. Nair, H. Abdullah, Mutation Analysis of
the BRCA1 Gene in Malaysian Breast Cancer Patients, 2002.
[184] L.A. Salas, S.N. Lundgren, E.P. Browne, E.C. Punska, D.L. Anderton,
M.R. Karagas, K.F. Arcaro, B.C. Christensen, Prediagnostic breast milk DNA
methylation alterations in women who develop breast cancer, Hum. Mol.
Genet. 29 (4) (2020) 662e673.
[185] N.J. Ronkainen, S.L. Okon, Nanomaterial-based electrochemical immuno-
sensors for clinically significant biomarkers, Mater. (Basel, Switzerland) 7 (6)
(2014) 4669e4709.
[186] M. El Aamri, G. Yammouri, H. Mohammadi, A. Amine, H. Korri-Youssoufi,
Electrochemical biosensors for detection of MicroRNA as a cancer biomarker:
pros and cons, Biosens. (Basel) 10 (11) (2020).
[187] M. Negrini, M. Ferracin, S. Sabbioni, C.M. Croce, MicroRNAs in human cancer:
from research to therapy, J. Cell Sci. 120 (Pt 11) (2007) 1833e1840.
[188] X. Tang, G. Tang, S. Ozcan, Role of microRNAs in diabetes, Biochim. Biophys.
Acta 1779 (11) (2008) 697e701.
[189] P. Miao, B. Wang, Z. Yu, J. Zhao, Y. Tang, Ultrasensitive electrochemical
detection of microRNA with star trigon structure and endonuclease medi-
ated signal amplification, Biosens. Bioelectron. 63 (2015) 365e370.
[190] S. Li, X. Yang, J. Yang, J. Zhen, D. Zhang, Serum microRNA-21 as a potential
diagnostic biomarker for breast cancer: a systematic review and meta-
analysis, Clin. Exp. Med. 16 (1) (2016) 29e35.
[191] M. Zubair, S. Wang, N. Ali, Advanced Approaches to Breast Cancer Classifi-
cation and Diagnosis, 2021, p. 11.
[192] F.S. Felix, L. Angnes, Electrochemical immunosensors - a powerful tool for
analytical applications, Biosens. Bioelectron. 102 (2018) 470e478.
[193] S.A. Lim, M.U. Ahmed, CHAPTER 1 introduction to immunosensors, immu-
nosensors, Royal Soc. Chem. (2019) 1e20.
[194] M.J. Duffy, D. Evoy, E.W. McDermott, CA 15-3: uses and limitation as a
biomarker for breast cancer, Clin. Chim. Acta 411 (23e24) (2010)
1869e1874.
[195] C. Tse, A.-S. Gauchez, W. Jacot, P.-J. Lamy, HER2 shedding and serum HER2
extracellular domain: biology and clinical utility in breast cancer, Cancer
Treat Rev. 38 (2) (2012) 133e142.
[196] V.V. Levenson, Biomarkers for early detection of breast cancer: what, when,
and where? Biochim. Biophys. Acta 1770 (6) (2007) 847e856.
[197] D.W. Kufe, Mucins in cancer: function, prognosis and therapy, Nat. Rev.
Cancer 9 (12) (2009) 874e885.
[198] B. Bohunicky, S.A. Mousa, Biosensors: the new wave in cancer diagnosis,
Nanotechnol. Sci. Appl. 4 (2011) 1.
[199] J. Wu, Z. Fu, F. Yan, H. Ju, Biomedical and clinical applications of immuno-
assays and immunosensors for tumor markers, TrAC, Trends Anal. Chem. 26
(7) (2007) 679e688.
[200] M.-H. Schlageter, J. Larghero, B. Cassinat, M.-E. Toubert, C. Borschneck, J.-
D. Rain, Serum carcinoembryonic antigen, cancer antigen 125, cancer antigen
15-3, squamous cell carcinoma, and tumor-associated trypsin inhibitor con-
centrations during healthy pregnancy, Clin. Chem. 44 (9) (1998) 1995e1998.
[201] A. Ravelli, J.M. Reuben, F. Lanza, S. Anfossi, M.R. Cappelletti, L. Zanotti,
A. Gobbi, C. Senti, P. Brambilla, M. Milani, Breast cancer circulating bio-
markers: advantages, drawbacks, and new insights, Tumor Biol. 36 (9)
(2015) 6653e6665.
P. Lakhera, V. Chaudhary, A. Jha et al.
[202] J. Li, S. Li, C.F. Yang, Electrochemical biosensors for cancer biomarker
detection, Electroanalysis 24 (12) (2012) 2213e2229.
[203] T. Meyer, G.J. Rustin, Role of tumour markers in monitoring epithelial
ovarian cancer, Br. J. Cancer 82 (9) (2000) 1535e1538.
[204] P. De Cremoux, C. Tran-Perennou, B. Brockdorff, E. Boudou, H. Magdel,
A. Lykkesfeldt, Validation of real-time RT-PCR for analysis of human breast
cancer cell lines resistant or sensitive to treatment with antiestrogens,
Endocr. Relat. Cancer 10 (3) (2003) 409e418.
[205] E.M. Cormier, M.F. Wolf, V.C. Jordan, Decrease in estradiol-stimulated pro-
gesterone receptor production in MCF-7 cells by epidermal growth factor
and possible clinical implication for paracrine-regulated breast cancer
growth, Cancer Res. 49 (3) (1989) 576e580.
[206] B. Bozal-Palabiyik, M. Lettieri, B. Uslu, G. Marrazza, Electrochemical detection
of vascular endothelial growth factor by molecularly imprinted polymer,
Electroanalysis 31 (8) (2019) 1458e1464.
[207] M. Shahdordizadeh, R. Yazdian-Robati, M. Ramezani, K. Abnous,
S.M. Taghdisi, Aptamer application in targeted delivery systems for diagnosis
and treatment of breast cancer, J. Mater. Chem. B 4 (48) (2016) 7766e7778.
[208] D. Ou, D. Sun, X. Lin, Z. Liang, Y. Zhong, Z. Chen, A dual-aptamer-based biosensor
for specific detection of breast cancer biomarker HER2 via flower-like nano-
zymes and DNA nanostructures, J. Mater. Chem. B 7 (23) (2019) 3661e3669.
37
Materials Today Chemistry 26 (2022) 101129
[209] Z. Li, M.A. Mohamed, A.M. Vinu Mohan, Z. Zhu, V. Sharma, G.K. Mishra,
R.K. Mishra, Application of electrochemical aptasensors toward clinical di-
agnostics, Food Env. Monitor.: Rev. Sens. (Basel) 19 (24) (2019).
[210] R. Abd-Ellatief, M.R. Abd-Ellatief, Electrochemical aptasensors: current
status and future perspectives, Diagnostics (Basel, Switzerland) 11 (1)
(2021).
[211] J. Welsh, Chapter 40 - animal models for studying prevention and treatment
of breast cancer, in: P.M. Conn (Ed.), Animal Models for the Study of Human
Disease, Academic Press, Boston, 2013, pp. 997e1018.
[212] S. Menon, M.R. Mathew, S. Sam, K. Keerthi, K.G. Kumar, Recent advances and
challenges in electrochemical biosensors for emerging and re-emerging in-
fectious diseases, J. Electroanal. Chem. 878 (2020), 114596.
[213] P.H. Lin, B.R. Li, Antifouling strategies in advanced electrochemical sensors
and biosensors, Analyst 145 (4) (2020) 1110e1120.
[214] M.J. Russo, M. Han, P.E. Desroches, C.S. Manasa, J. Dennaoui, A.F. Quigley,
R.M.I. Kapsa, S.E. Moulton, R.M. Guijt, G.W. Greene, S.M. Silva, Antifouling
strategies for electrochemical biosensing: mechanisms and performance
toward point of care based diagnostic applications, ACS Sens. 6 (4) (2021)
1482e1507.
[215] J. Xu, H. Lee, Anti-biofouling strategies for long-term continuous use of
implantable biosensors, Chemosens. 8 (3) (2020).