12 (2022) 100259

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Biosensors and Bioelectronics: X

journal homepage: www.journals.elsevier.com/biosensors-and-bioelectronics-x

Peptide-based optical biosensors: A promising approach for early-stage

cancer detection
Gurpreet K. Soni , Saima , Priya Manhas , Rohit K. Sharma *
Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University, Sector-14, Chandigarh, 160014, India

A R T I C L E I N F O A B S T R A C T

Keywords: Cancer is the leading cause of 10 million deaths worldwide in 2020, which is exponentially increasing per year.
Optical cancer detection The early detection and effective treatment of cancer can save millions of lives worldwide. Cancer diagnostic
Peptide nucleic acid methods are scarce due to their high cost, laborious instrumentation, high maintenance, time-consuming, and
Targeting peptide
complex nature. Furthermore, current cancer detection methods are also inconvenient and necessitate the use of
Antifouling peptide
Self-assembled peptide
technical experts. However, peptide-based optical biosensors are fast, and compact with a low detection limit and
Cell-penetrating peptide high selectivity, hence having a lot of potential for diagnosing different types of cancer. The present work aims to
provide an overview of various types of peptide-based optical biosensors (OBS) for the early-stage detection of
cancer. Here, the peptides with high specificity, biocompatibility, small size, and numerous conjugation possi­
bilities incorporated with nanomaterials, are reported for the fabrication of optical biosensors to diagnose cancer.
Peptide-based optical biosensors are known to overcome the limitations of the conventional method by providing
high sensitivity, and portability, with rapid and real-time detection of all types of cancer cells. Moreover, in
comparison to conventional methods, cancer can be detected in a quite few million tumor tissues using optical
biosensors. This review mainly focused on the optical biosensors such as colorimetric, fluorescence, surface
plasmon resonance (SPR), chemiluminescence (CL), and photoelectrochemical derived from the different types of
peptides, including their working principle and conjugation with peptides. We have also discussed the advan­
tages and disadvantages of current peptide-based optical biosensors for early-stage cancer detection, as well as
future prospects.

1. Introduction mammography, sonography, polymerase chain reaction, and

Cancer is the second most lethal disease causes the death of 10
million people worldwide in 2020 (Sung et al., 2021). The death rate
will be expected to reach 12 million by the year 2030 (World Health
Organization, Fact sheet, 2022). However, it has also been reported that
30–50% of human lives can be saved by the early-stage detection,
diagnosis, and treatment of cancer (Office for National Statistics UK,
2016). The early-stage detection mainly focuses on the detection of
cancer/premalignant at the initial phase, which could increase the
survival rate or morbidity reduction. The effectiveness of early diagnosis
can be seen in the ratio of late detection of cervical cancer versus early
detection reported to be 19.8/3.1 per 100,000 (Crosby et al., 2022). The
non-recognition ability of the human body toward cancer and less
concentration of biomarkers is a great hurdle in its early detection. The
available standard methods for cancer detection include magnetic
resonance imaging (MRI), molecular breast imaging, tissue biopsy,
enzyme-linked immunosorbent assays (Kaur et al., 2022). These tech­
niques lack behind due to the laborious instruments, inexperienced staff,
high cost, complex setup, painful treatment, high maintenance, high
sample volume (blood, fluids), and long-time procedural duration
(Mittal et al., 2017). The limitations of these techniques open a new
opportunity for researchers to develop new methods for cancer detec­
tion at the initial stage with high sensitivity, low cost, fast response, and
user-friendly nature.
Biosensors are integrated receptor-transducer devices, which include
the receptor in conjugation with a transducer (physical/chemical) and
detector. The receptor part embraces the biological recognition elements
such as peptides, nucleic acids, antibodies, enzymes, protein, whole
cells, and tissue slices to interact with the analyte (Askim and Suslick,
2017). Depending on the concentration of the analyte (biomarker),
transducer produces a signal which is quantified by the detector. Bio­
sensors can be classified into various categories based on the signal

* Corresponding author.
E-mail address: rohitksg@pu.ac.in (R.K. Sharma).

https://doi.org/10.1016/j.biosx.2022.100259
Received 15 May 2022; Received in revised form 8 September 2022; Accepted 22 September 2022
Available online 6 October 2022
2590-1370/© 2022 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
G.K. Soni et al.
transducer - optical, optoelectronic, electrochemical, thermometric,
magnetic, and piezoelectric (Farré and Barceló, 2007). The utilization of
optical transducers and biological recognition elements in one probe
originate the new class of biosensors called optical biosensors. Optical
biosensors are a non-invasive replacement for available biosensing
techniques for cancer detection. They offer remarkable advantages over
electrochemical and other biosensors such as high sensitivity and
selectivity, multiple component detection, lower signal-to-noise ratio,
lower immunity to electromagnetic interference, ease in fabrication,
smaller size, direct label free and real time detection (Sołoducho and
Cabaj, 2015). Moreover, signal transduction is independent of the target
in them and can easily adaptable to the environment. These sensors
provide relevant results in the case of single molecular determination
and distant sensing. Also, the incorporation of multidisciplinary fields
including nano/micro technology, chemistry, molecular biology, mi­
croelectronics, and biotechnology results in the fabrication of optical
biosensor which provides a reliable device for healthcare (Chen et al.,
2019). Optical biosensors for cancer basically detect the biomarkers
such as micro ribonucleic acid (miRNA), circulating tumor cells (CTC),
exosomes, proteins, and deoxyribonucleic acid (DNA), which can be
collected from plasma, blood, serum, saliva, and urine (Badr et al., 2014;
Underwood et al., 2020).
Over the last decade, optical sensors for cancer detection mainly
comprise the recognition method, based on antibodies and antigens
(Holford et al., 2012). Although antibodies possess high specificity but
high production costs, non-availability of synthetic production methods,
limited methods for modification, and narrow self-life, restrict their
applications in cancer detection (Urbanek et al., 2011) Therefore, arti­
ficial molecules such as aptamer and peptide based optical sensors
dominating the cancer market in recent years (J. J. Zhang et al., 2017,
Roberts and Gandhi, 2022). Aptamers are oligonucleotides with a single
strand (ssDNA/RNA) that can preferentially bind to a particular target
via the formation of defined architectures. Whereas, peptides are short
polymers in which amino acids are bonded together via an amide bond.
Depending upon the nature, number, and sequence of amino acids
peptides show distinctive properties such as high specificity and selec­
tivity, cell penetration, low toxicity, easy synthesis, and surface modi­
fication. These synthetic materials (aptamer and peptide) efficiently
replaced antibodies by taking the advantage of additional features such
as high stability, high specificity, low-cost easy synthesis, and modifi­
cation (Liu et al., 2018). Targeting peptides and aptamers with biolog­
ical recognition ability can target the tumor cells and tissues through
outstanding ability with respect to target biomarkers. Peptide nucleic
acids (PNAs) are another class of peptides that mimic the DNA/RNA by
replacing the deoxyribose/ribose phosphate backbone with pseudo
peptide-N-(2-aminoethyl)-glycine (Corkill and Rapley, 2008). The
non-existence of deoxyribose/ribose sugar in PNAs makes them highly
stable toward enzymatic degradation in a biological environment. The
replacement of phosphate backbone with N-(2-aminoethyl)-glycine
build stable PNA/DNA and PNA/RNA hybridized pairs, as compared to
DNA/RNA due to the reduction of electrostatic interaction (IgIoi, 1998).
PNA’s unique properties make it superior to aptamers/nucleic acids in
cancer detection. Moreover, peptide based sensors have several advan­
tages over their larger analogues such as enzymes and proteins. Enzy­
me/protein based sensors suffer from limited lifetime, high cost,
complex synthesis methods, activity dependence over pH, inhibitors,
ionic strength, and temperature which are smoothly overcome by pep­
tide based sensors (Economou et al., 2017; Shahdeo et al., 2022). Besides
this, the problem of low response and low sensitivity with whole cell
biosensors can also be solved by this new class of biosensors (Chen et al.,
2017). Additionally, peptides exhibit special features like antifouling
property, self-assembly, and cell penetration depending upon the
sequence and nature of amino acids (Wang et al., 2021). The ability of
peptides to exhibit numerous properties and advantages over already
reported methods motivates the authors to gather peptide based optical
biosensors in this review article, particularly for early cancer detection.
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Biosensors and Bioelectronics: X 12 (2022) 100259
The current review article embraces the fabrication and application
of peptide based optical biosensors for early-stage cancer diagnosis,
especially in the last decade. The optical biosensing of cancer bio­
markers has been divided into five parts depending upon the nature and
application of peptides - (i) peptide nucleic acid (PNA) based detection
(ii) targeting peptide based detection (iii) antifouling peptide based
detection (iv) self-assembled peptide based detection (iv) cell pene­
trating peptide (CPP) based detection (Fig. 1a). The sensing mechanism
of various optical biosensors such as fluorescence, colorimetric, surface
plasmonic resonance, chemiluminescence, and photo electrochemical is
methodized with the usage of a particular peptide. Close attention has
been given to the recently developed user-friendly biosensors for cancer
detection. At last, we summarized the loopholes and future direction to
develop optical biosensors in the field of cancer.
2. Peptide based optical biosensors for early-stage cancer
detection
Nowadays, peptides are becoming nearly an ideal candidate for
fabricating selective and sensitive optical biosensors due to their various
advantages over nucleic acids, antibodies, and enzymes. A peptide with
antifouling, self-assembly, cell penetrating, and target recognition
properties conjugate with an optical probe for the fabrication of peptide
based optical sensors. The conjugation of peptides can be carried out
before and after the synthesis of optical probe (nanoparticles (NPs),
quantum dots (QDs), and carbon dots (CDs)) (Valetti et al., 2014). The
single-step peptide conjugation prior to the formation of the optical
probe shows advantages over other processes in terms of high quantum
yield, maintenance of peptide activity, and uniform particle size. The
variety of techniques used for peptide immobilization includes electro­
static binding, physical adsorption, covalent binding, and specific
recognition (Rajchakit and Sarojini, 2017). Peptides with positively or
negatively charged amino acid side chains can adsorb electrostatically
on nanoparticles capped with anionic ligands (citrate, mercaptopro­
pionic acid, lipoic acid) or cationic ligands (cysteamine) respectively
(Katz and Willner, 2014). Peptides with phenylalanine can also adsorb
on the surface of nanoparticles via self assembly through the formation
of a specific architecture. The most common approach for peptide
conjugation to the metallic nanoparticles involves the covalent bonding
via the formation of Au–S bond where the cysteine containing peptides
can directly synthesize the gold nanoparticles (AuNPs)/silver nano­
particles (AgNPs) via the formation of a metal thiolate bond (Jazayeri
et al., 2019). The second covalent approach is based on click chemistry
where copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) can be
used for peptide conjugation (Lutz and Zarafshani, 2008). Another
alternative way involves the utilization of suitable bifunctional linkers
with additional features such as control over the peptide orientation of
the nanoparticle surface. The bifunctional linkers (polyethylene glycol
(PEG), ethyl(dimethylaminopropyl)carbodiimide / N-hydrox­
ysuccinimide (EDC/NHS)) have a thiol group at one side, and carboxylic
acid group on another side (Casciaro et al., 2017). Here, the thiol group
helps in the formation of Au–S where the –COOH group is used in the
coupling with the –NH2 group of peptides. Among all covalent conju­
gation maintain the properties of peptides along with toxicity minimi­
zation of nanoparticles which makes it advantageous over other
conjugation methods.
Optical biosensor is an analytical device incorporating a receptor
(peptides, antibodies, nucleic acids, antibodies, etc.) optical transducer,
and detector (Damborský et al., 2016). The transducer part comprises
optical probes such as AuNPs, AgNPs, QDs, CDs, etc. which are further
integrated with the receptor. The basic sensing mechanism of optical
biosensors is based on the alteration of the characteristic signal of the
optical probe in the presence of an analyte (Seitz, 1989). The in­
teractions of receptor moieties with analyte initiate the various bio­
recognition events such as hybridization, protein/peptide binding,
metabolic reaction, etc. which produces the physical or chemical change
G.K. Soni et al.
Fig. 1. (a) Peptide based optical biosensors for early-stage cancer detection. (b) Schematic representation for the working principle of optical sensors.
in the system. These changes are then converted to optical/electronic
signals by the transducer and detected by the detector. The optical
biosensors can be categorized on the basis of various optical principles
such as SPR, absorbance, fluorescence, chemiluminescence, photo­
luminescence, refractive index, surface-enhanced Raman spectroscopy
(SERS), scattering, reflectance, diffraction, etc. covering the UV–visible,
infrared (IR) and near-infrared (NIR) region of electromagnetic spectra
(Fig. 1b) (Jerónimo et al., 2007; Law et al., 2020).
This review article mainly focuses on the SPR, colorimetric, fluori­
metric, chemiluminescence, and photoelectrochemical (PE) based op­
tical biosensors. Optical sensors derived from the excitation of surface
plasmons are assigned as SPR sensors (Homola, 2008). SPR phenomena
have been widely employed in the fabrication of biosensors and che­
mosensors. The collective/coherent oscillation of free conduction elec­
trons in metal nanoparticles referred to as SPR is categorized into
localized surface plasmon resonance (LSPR) and propagating surface
plasmon resonance (PSPR) (Chen and Ming 2012). The LSPR phenom­
ena is observed in metallic nanoparticles whereas, PSPR is mainly seen
in metallic films and optical fibers. The enhancement of electromagnetic
field by both phenomena is leading to the application of SPR in refrac­
tive index measurement (Zeng et al., 2005), fluorescence enhancement
(Sokolov et al., 1998), absorbance (Su et al., 2007), SERS (Chen and
Burstein, 1980), etc. Among all these, fluorescence has become the
utmost used optical technique for the detection of cancer due to its
excellent sensitivity as compared to other optical techniques. Fluores­
cence occurs when molecules excite by incident light followed by the
emission of photons from a single excited state to the ground state (Shin
et al., 2021). Various parameters of fluorescence such as fluorescence
intensity, fluorescence decay, high quantum yield, anisotropy, excita­
tion, and emission spectra provide great help in data analysis (Li et al.,
2019). Contradictory to this, colorimetric is an absorption-based oldest
analytical method used for the naked-eye detection of various analytes.
This straightforward technique can simplify the data analysis by eval­
uating the UV–Vis peak with high absorbance and spectral analysis at
200–800 nm (Liu et al., 2020). Divergent to all above mentioned tech­
niques, electrochemiluminescence (ECL) is a prominent chem­
iluminescence technique based on the production of ECL luminophore at
the surface of the electrode under applied voltage (Babamiria et al.,
2019). The produced luminophore undergoes an electron transfer
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Biosensors and Bioelectronics: X 12 (2022) 100259
process to create an excited state for luminescent signals (Ma et al.,
2020). On the other hand, photoelectrochemical (PEC) is opposite to
ECL which utilizes the light for the excitation of electrons and photo­
current to observe signals. The photoelectrochemical sensors work on
the change in photocurrent/potential caused by the interactions be­
tween photoelectrochemical material and target moieties (Shu, and
Tang, 2020). Both methods are equally sensitive for the detection of
various analytes by reducing the background noise. Based on all these
methods, peptide based optical biosensors have been discussed
depending on the nature and function of the peptide.
2.1. Peptide nucleic acid based optical sensors
Peptide nucleic acids are modified DNA/RNA analogues in which
negatively charged phosphate backbone is replaced by neutral N-2-
aminoethylglycine repeating units, introduced in 1990 by biochemist
Peter Nielsen and organic chemist Ole Buchardt (Singh et al., 2010;
Saadati et al., 2019). Considering the superior properties of PNA such as
high chemical stability, easy chemical modification, resistance towards
enzyme degradation, stable hybrid complex formation (DNA/PNA,
RNA/PNA, PNA/DNA/PNA) with high melting point and binding en­
ergy, high specificity for nucleic acids, stand invasion and extraordinary
ionic strength, it is widely used for the development of sensors, chips,
and arrays (Shi et al., 2015). The immobilization of PNA on optical
converters opens a new path for sensitive and selective determination of
various cancers (Gupta et al., 2017). A colorimetric sensor based on
PNA-gold nanoparticles (PNA-AuNPs) has been developed to detect two
mutations in exon 11 of c-Kit (Lee et al., 2011). The reported method
provides the selective and sensitive (lower limit of detection (LOD) 500
fM) detection of specific mutations by avoiding the uncontrolled ag­
gregation of PNA-AuNPs caused by electrostatic interactions. Corre­
spondingly, naked eye detection of human c-Myc mRNA in colon tumor
was performed by controlled aggregation of AgNPs by PNA/PNA-RNA
triplex with a sensitivity of 50 nM (Li et al., 2016). In response to
c-Myc mRNA, the AgNPs-PNA platform showed color change based on a
single base mismatch and provides visual detection. In another report,
seven mutations in codon 12 and 13 of the Kisten rat sarcoma viral
oncogene homologue (KRAS) gene were validated with the utilization of
AuNPs and PNAs for colorectal cancer (CRC) (Zhao and Lin, 2017). The
G.K. Soni et al. Biosensors and Bioelectronics: X 12 (2022) 100259

rapid, label free colorimetric method was based on the aggregation of
AuNPs in the presence of PNA which was further restricted by hybrid
complex formation between PNA and target DNA. A distinctive
approach illustrated the modification of gold nanorods (AuNRs) by PNA,
complementary to the target DNA for colorimetric sensing of point
mutations in the KRAS gene due to pancreatic ductal adenocarcinoma
(Tadimety et al., 2019). The study successfully differentiated between
the wild type and mutant sequence of KRAS with a sensitivity of 10
ng/mL in the serum sample. In another report, homopurine PNA was
efficiently bonded to homopyrimidine DNA and PNA-DNA/DNA triplex
which can be further conjugated with cyanine dye (DiSC2) to provide
color variation (Xu et al., 2020). This system helped in designing of
colorimetric sensing probe for micro-RNA for cancer detection. The
advancement in PNA-based colorimetric sensors for cervical cancer
detection was reported by the incorporation of the smartphone (Naor­
ungroj et al., 2021). The reported method provided the quantification of
Human papillomavirus (HPV) DNA by examining the color change in
AuNPs induced via pyrrolidinyl peptide nucleic acid (acpcPNA).
Another excellent class of optical biosensors belongs to fluorescence
in which fluorescently modified PNAs are widely employed in the bio­
sensing and bioimaging of cancer cells. A disposable fluorescent
biosensor was reported for early detection of lung cancer based on PNA
and poly(3-alkoxy-4-methylthiophene) as a fluorescent probe (Yildiz
et al., 2013). The positively charged fluorescent probe was fabricated on
poly(vinylidene fluoride) thin paper, which further corroborated with
PNA and target miRNA. Based on the formation of a hybrid complex
between reporter-miRNA/PNA and reporter-miRNA, two types of sig­
nals are produced which led to quantitative and qualitative detection. In

Fig. 2. (a) Schematic representation for tumor cell selective fluorescence detection based on ATP aptamer and PNA/peptide/PNA copolymer. (b) The comparison of
protease activatable aptamer probe (PA-SP) with conventional probe (C-SP) shows the high cell selectivity. Reprint with permission from (Xiang et al., 2021). ©2021
John Wiley & Sons, Inc.

4
G.K. Soni et al.
another report, point mutations in the KRAS oncogene was detected
utilizing the two types of molecular beacons (i) thiazole orange PNA
molecular beacon (TO-PNA-MB), (ii) phosphothiolated DNA molecular
beacon (PS-DNA-MB) (Kam et al., 2012). From these two systems,
TO-PNA-MB effectively differentiated KRAS and SNP in vitro and in vivo
to provide the sensitive and selective detection of KRAS mutations.
Similarly, the fluorescence probe from 15/16-mer PNA efficiently
distinguished the mutant target and wild type HER2 based on the single
base mismatch principle (Metaferia et al., 2013). During the synthesis,
PNAs can be easily modified with dyes, for example, the modification of
three different PNAs with three different fluorescent dyes carboxy
fluorescein (FAM)-PNA21, 6-carboxy-X-rhodamine (ROX)-PNA125b
and cyanine 5(Cy5)-PNA96 was reported (Ryoo et al., 2013). PNAs were
complementary to miRNA (miR-21, miR-125b, and miR-96), providing
quantitative monitoring based on the quenching of dyes with nanosized
graphene oxide (NGO). A different type of approach was adopted for the
fluorescence detection of KRAS2 mRNA in lung cancer cells (Sonar et al.,
2014). Fluorescence of single-stranded PNA-IGF1 tetrapeptide-thiol or­
ange complex escalated upon interaction with KRAS2 RNA. Corre­
spondingly, sensing of colorectal cancer was reported based on the
fluorescence enhancement of thiazole orange-peptide nucleic acid mo­
lecular beacons (TO-PNA-MB) (Kam et al., 2014). Thiazole Orange and
other cyanine dyes absorbed light near 500 nm, are highly sensitive to
the local environment, and lead to a remarkable increase in signal/noise
ratio upon binding with RNA/DNA. These types of spectral properties of
cyanine dyes are not appropriate for in vivo applications due to cell
auto-fluorescence. To overcome this limitation, the use of Bis Quinoline
(BisQ) and PNA conjugate to detect single point mutations in cancer
cells was reported (Kolevzon et al., 2016). Similarly, a red-emitting
BisQ-PNA probe was utilized for on-site fluorescent detection of
mRNA in human cancer tissues (Hashoul et al., 2019). A completely
distinctive approach was demonstrated for tumor overexpressed prote­
ase based on PNA/peptide/PNA copolymer (Fig. 2.) (Xiang et al., 2021).
The structure switching activity of adenosine triphosphate (ATP)
aptamer was locked by PNA/peptide/PNA copolymer and selective
cleavage of peptide by protease reduced the hybridization of PNA/ap­
tamer initiating the tumor cell selective molecular detection.
The single stranded PNAs show strong interactions with various
types of nanomaterials and act as a base for several sensing platforms.
The covalently linked PNA with fluorescent nanodiamonds (NDs) using
EDC/NHS chemistry for the detection of lung cancer was reported,
where the intrinsic luminescent properties of NDs-PNA complex
quenched upon interaction with target DNA (Gaillard et al., 2014). The
quenching of fluorescent label PNA with nanosheets and molecular
organic frame works (MOF) is the basic model for the detection of
several analytes. In this context, fluorescent dye labeled PNAs illustrated
for miRNA sensing using the nano metal-organic framework (NMOF,
UiO-66) and carbon nitride nanosheets (Wu et al., 2015; Liao et al.,
2015). A method for the conjugation of PNA on silicon oxide surface was
reported based on the click and thio-yne chemistry for early diagnosis of
cancer (Veerbeek et al., 2018). Thiol-PNA and azido-PNA were incor­
porated covalently on silicon surface via 1, 8-nonadyne as branching
moiety. Depending upon the target DNA quenching of fluorescent dyes
took place, providing selective and sensitive detection. Based on the
conjugation of thiolated PNA with magnetic beads (MBs) multistep
fluorescent sensing strategies had been reported for lung cancer (Wang
et al., 2020; D. Zhang et al., 2020). The initiator for atom transfer radical
polymerization (ATRP) was fabricated on PNA/DNA hybrid carbox­
y/sulfonate-Zr4+-phosphate chemistry. Further, the fluorescent signal of
deposit fluorescein-o-acrylate (FA) is accelerated by countless polymer
chains. Similarly, a dual-recognition fluorescent probe for circulating
tumor DNA (ctDNA) was reported, where the PNA-MBs complex selec­
tively distinguished the mutated ctDNA from wild type DNAs with a
detection limit of 0.3161 pM (Chen et al., 2020).
PNA directed templated reactions offer a fascinating approach to the
imaging and detection of DNA/RNA. The photocatalyzed cleavage of the
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Biosensors and Bioelectronics: X 12 (2022) 100259
linker in the presence of Ru2+-catalyst to expose rhodamine for miRNA
detection was reported (Sadhu and Winssinger, 2013). PNA act as a
template between the Ru2+-catalyst and enclosed rhodamine in the
presence of target miRNA. Contradictory, fluorescent biosensor reported
for prostate cancer based on the Michael addition reaction between two
PNA probes (Metcalf et al., 2016). A convenient method was developed
for EGFR mutated cancer cells based on fluorescence resonance energy
transfer (FRET) phenomena regulated by fluorescein
isothiocyanate-PNA (FITC-PNA) and dabcyl-DNA (Shigeto et al., 2019).
The fluorescent signal of FITC-PNA was quenched by dabcyl-DNA,
which was further regained in the presence of target mRNA due to the
formation of the FITC-PNA/mRNA hybrid. Correspondingly, PNA based
FRET was reported between the gold nanoparticles and carbon dots for
the HeLa cell line (Gao et al., 2020). PNA induced the aggregation of
AuNPs and restrict the FRET between AuNPs and CDs. When PNA/DNA
complex formed AuNPs remained free in the solution leading to the
quenching of CDs by FRET. A bioluminescence resonance energy
transfer (BRET) phenomenon was employed for smartphone based
quantification of three miRNA sequences (miR-21, -31, -141) (Chang
et al., 2020). The work utilized the PNA for selective DNA detection
which further conjugated with luciferase to form H-Luc-PNA conjugate.
The displacement of the PNA duplex was carried out by target RNA to
accomplish logic-gate operation (AND operation). An interesting probe
fabrication was carried out by coating the covalent complex (PNA-­
photo-cleavage linker and alginate hydrogel) on a microneedle array
(Sulaiman et al., 2019) (Fig. 3a and b). Treatment of the fabricated
microneedle with target DNA sequence was responsible for the forma­
tion of the PNA-DNA complex followed by the fluorescence quenching
and target detection (Fig. 3c and d). A distinctive approach was
employed based on PNA clamping-assisted fluorescence melting curve
analysis for the detection of non-small cell lung cancer (J.Y. Han et al.,
2016; Kim et al., 2017). This method effectively provided the prediction
of treatment response and mutation diagnosis for lung cancer in the
plasma sample.
A surface plasmon resonance phenomenon was used for the bio­
sensing of ctDNA (Bellassai et al., 2021). Sensor fabrication was carried
out by PNA complementary to target DNA, oligopeptide CEEEEE,
AuNPs@KRAS, and poly-L-lysine based antifouling layer. Target DNA
captured by the PNA probe initiated the changes in the SPR signal. The
radiolabeled PNAs showed high potential for non-invasive molecular
imaging of cancer cells. 64Cu-PNA-carboxyfluorescein/DOTA probe was
successfully fabricated for non-invasive PET imaging of miR-146b-5p
and miR-146a for lung and thyroid cancer detection (Croci et al., 2019).
2.2. Targeting peptide-based optical sensors
Over the past few years, significant interest has emerged in the
development of targeting peptide-based sensors for the early identifi­
cation and measurement of cancer (Karimzadeh et al., 2018). Antitumor
monoclonal antibodies have been widely studied as targeting agents for
cancer detection (Martinelli et al., 2009). However, the use of antibodies
as a targeting biosensor is associated with some limitations. The anti­
tumor monoclonal antibodies have a large size and are non-specifically
taken up by the liver and reticuloendothelial system (Aina et al., 2002).
On contrary, the targeting peptide-based detectors have benefits over
antibodies as they are relatively small-sized, less immunogenic, easy to
synthesize, exhibit high specificity, and excellent flexibility concerning
their conjugation potentials and hence are extensively used for
biosensor construction (Wang et al., 2022). The targeting peptide-based
optical biosensors generally involve electrochemiluminescence, fluo­
rescence resonance energy transfer, and optical analytical methods. The
ECL method is highly sensitive, facile, and generally involves the ben­
efits of both electrochemical and chemiluminescence methods, owing to
which, it has been widely explored for peptide-based biosensing in the
early detection of cancer (Qi and Zhang, 2020). In this context, recently,
a peptide-based electrochemiluminescence biosensor with graphitic
G.K. Soni et al.
phase carbon nitride nanosheet loaded with luminol capped gold
nanoparticles (Lum-AuNPs@g-C3N4) was reported for the early diag­
nosis of ovarian malignancy through highly sensitive detection of
phosphatidylserine positive (PS (+)) exosomes (Fig. 3e and f) (Liu et al.,
2021).
Similarly, a unique electrogenerated chemiluminescence peptide-
based biosensor (ECL-PB) has been reported for the specific determi­
nation of prostate-specific antigen (PSA) tumor marker for the early
detection and examination of recurrence of prostate cancer (Qi et al.,
2014). The peptide with sequence (CHSSKLQK) had been employed as a
molecular recognition element based on its target-induced cleavage
within Nafion films incorporated with gold nanoparticles and (Ru(bpy)3
2+
) as ECL emitting species. The reported method identified the PSA
with a 0.8 pg/mL detection limit. In another report, an ultra-sensitive
and selective electrochemiluminescence assay was developed for the
highly efficient detection of cyclin A2 via ECL graphene-up conversion
hybrid (Wu et al., 2014). Cyclin A2 is an effective indicator for
early-stage cancer and the developed ECL biosensor can be potentially
used for the screening of chemotherapeutic drugs and for early-stage
evaluation of treatments of cancers. Similarly, a selective and sensitive
biosensor based on ECL was reported for simultaneous detection of two
matrix metalloproteinases (MMPs) i.e., MMP-2 and MMP-7, associated
with the growth of cancer (Gao et al., 2017). This multiplexed biosensor
was designed based on the co-immobilization of the Ir1 and Ru1 labeled
peptides on a gold electrode using self-assembly. The authors reported
that the sensor allowed the detection of MMP-2 and MMP-7 with
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Biosensors and Bioelectronics: X 12 (2022) 100259
Fig. 3. (a) Schematic representation for the detection
of circulating nucleic acids in skin interstitial fluid
with the help of hydrogel-coated microneedle plat­
form, microneedle array coated with PNA, and algi­
nate hydrogel for target DNA. (b) Scanning electron
micrograph (SEM) of microneedle without and with
coating. (c) Schematic illustration of working princi­
ple of microneedle based array. (d) Swelling kinetics
of hydrogel coated microneedles. Reprinted with
permission from (Sulaiman et al., 2019). Copyright
(2019) American Chemical Society. (e) Pathway
illustration for the peptide-Lum-AuNPs@g-C3N4
nanoprobe preparation. (f) Schematic representation
for the construction of ECL biosensor for phosphati­
dylserine (PS)-positive exosomes. Reproduced with
permission of Elsevier with (Liu et al., 2021). Creative
Commons Attribution License (5293591498830).
detection limits of 5 ng/mL and 10 pg/mL, respectively.
Fluorescence resonance energy transfer is another important
analytical technique for cancer detection, utilizing fluorogenic peptide-
based biosensors. FRET is observed when the emission spectrum of a
donor (fluorophore) is overlapped by the absorption spectrum of an
acceptor (quencher). In FRET the donor should be at a suitable distance
from the acceptor in order to accommodate the distance-dependent
fluorescence resonance energy transfer (Zadran et al., 2012). Recently,
a 5-carboxy-fluorescein (FAM)-labeled peptide containing Arg6 assem­
bled with the negatively charged carbon nanoparticles (CNPs) was used
for convenient and highly sensitive detection of trypsin based on FRET
phenomenon (Hou et al., 2020). The activation of trypsin is known to
exceed Pancreatic secretory trypsin inhibitor (PSTI) control and acti­
vated other proteases for self-digestion, which can cause cell damage
and may lead to pancreatic cancer. The reported method was simple and
highly selective due to the specific cleavage of the six-arginine based
synthetic peptide by trypsin and efficient to determine the trypsin ac­
tivity, with the detection limit of about 0.7 μM. Similarly, a simple FRET
sensing platform based on peptide-functionalized upconversion@silica
nanoparticle (UCNP@SiO2@Cy5-pep) had been developed for the in
vitro and in vivo detection of caspase-9 activity which is significant in
diseases diagnosis and for the development of anti-cancer drugs (Nitin
et al., 2009). In the fluorophore-conjugated peptide detectors,
peptide-based nanomaterials have been also explored owing to their
high affinity and specificity. In this context, HEKMs micelles were re­
ported, which consist of a peptide as a recognition element, a matrix
G.K. Soni et al. Biosensors and Bioelectronics: X 12 (2022) 100259

metalloproteinase-2 (MMP-2)-regulated FRET probe for NIR fluores­
cence imaging, a gadolinium probe (Gd) for MRI and the last element
includes the mitochondrial damage segment KLA and the chemotherapy
drug doxorubicin (DOX) for synergetic antitumor treatment (Wang
et al., 2019). Apart from FRET, metal-enhanced fluorescence (MEF) is
one of the evolving detection methods as here the emission intensity can
be considerably enhanced by positioning the fluorescent molecules near
the metal nanostructure. Based on the MEF, a proteolytic enzyme
nanosensor was reported which comprised of AuNPs connected to both
single-strand DNA (ssDNA) and peptide (DEVD) (Fig. 4) (Choi et al.,
2020). The reported nanosensor was recognized and cleaved by
caspase-3, a proteolytic enzyme that is associated with apoptotic cells
and the diagnosis of cancers (Fig. 4a and b). Based on the immunoflu­
orescence assay the CSP3 (QQLPSS STSTYP) peptide was identified as an
effective targeting probe that can specifically and sensitively bound to
SiHa cells and serves as a potential candidate for diagnostic applications
of cervical cancer (Li et al., 2017). Similarly, a peptide-templated gold
nanoparticle as a nanosensor was reported for rapid detection of mul­
tiple post-translational modification (PTM) enzymes (J. Zhang et al.,
2020). For the early detection of pancreatic cancer, a peptide-22 con­
jugated to the fluorescent dye cyanine 7 (peptide-22-Cy7) was reported
for targeted imaging based on the high targeting toward the low-density
lipoprotein receptor (LDLR) which is usually overexpressed and
distributed over pancreatic cancer cells as biological target (Han et al.,
2019). An AuNPs based biosensor was reported for the determination of
chymase activity in kidney tissue (Chang et al., 2018). In this study, an
AuNPs-based fluorescence peptide probe was constructed containing
FITCAcp-DRVYIHPFHLDDDDDC peptide comprising an enzyme (chy­
mase) substrate (DRVYIHPFHL), a spacer (DDDDD), and cysteine (C)
was conjugated to 16 nm-AuNPs. The authors observed that the reported
AuNPs-peptide probe required only 15 min for the detection of chymase
activity. In another report, cationized polyacrylamide (CPAA) and a
synthetic pentadecapeptide, EPPT1 (YCAREPPTRTFAYWG) were stud­
ied for the detection of biomarkers, needed for the prevention and
efficient treatment of colorectal cancer (Bloch et al., 2012). It has been
observed that fluorescence colonoscopy connected with a fluorescent
probe specific for a polyp biomarker can improve the early detection of
cancer. A tumor-targeting, NIR fluorescent peptide (LS301, sequence
cypate-cyclo (cGRDSPC)K) was investigated for early detection and
endoscopic resection of polyps in a genetically engineered rat model of
colorectal cancer (Jones et al., 2018). Similarly, a water-soluble probe
consisting of a cyclic peptide (GE-137 peptide) that binds to human
tyrosine kinase c-Met, conjugated to the fluorescent cyanine dye was
investigated for the early detection of cancer (Burggraaf et al., 2015).
The authors reported that the intravenous administration of GE-137
cause-specific accumulation in c-Met expressed tumors. Similarly,
folate and FITC conjugated EC17 peptide was clinically studied for the
specific intraoperative detection of folate receptor-α targeting ovarian
cancer using fluorescence imaging (Tummers et al., 2015). As a fluo­
rescent contrast agent, the potential of epidermal growth factor (EGF)
labeled with Alexa Fluor® 647 was evaluated to image epidermal
growth factor receptor (EGFR) expression in oral neoplasia using
high-resolution fluorescence imaging (Nitin et al., 2009). The report
illustrated that the peptide-dye conjugate was efficiently and easily
entered in less than 30 min through the oral epithelium with a pene­
tration depth of more than 500 μm. Similarly, an epidermal growth
factor conjugated with Cy5.5 dye was used as a specific NIR contrast
agent to specifically bind the EGFR-positive tumors in mice (Ke et al.,
2003). Correspondingly, the peptide sequence SNFYMPL was used as a
fluorescent contrast agent that enables specific binding to dysplasia in
Barrett’s esophagus (Li et al., 2010). The authors reported that the
fluorescence intensity was increased with the progression of tissue from
normal to benign intestinal metaplasia to dysplasia. Also, hepsin, a
prostrate biomarker was detected using heparin targeting peptides via
phage display techniques (Kelly et al., 2008). The targeted peptides were
conjugated with fluorescent nanoparticles in order to improve the
pharmacokinetics which displayed an efficient biomarker-based detec­
tion of prostate cancer. Likewise, the peptide AADNAKTKSFPV (AAD)
had been identified using phage display selection to guide tissue biopsy
and for the early detection of gastric cancer (GC) (Zhang et al., 2012).
Moreover, the reported peptide enables the distinction of the neoplastic
from normal gastric mucosa. Based on the near-infrared fluorescence
optical imaging Cy5.5-conjugated mono-, di-, and tetrameric RGD pep­
tides were assessed for tumor-targeting efficacy and to investigate the
effect of multimerization on integrin avidity (Cheng et al., 2005). The
authors reported that all the dye conjugated peptides are appropriate for
integrin expression imaging and the tetramer showed better imaging
characteristics than monomer and dimer. Recently, the FITC-labeled
peptide (NH2-Lys-Gly-Pro-Arg-Ser-Leu-SerGly-Lys-FITC) conjugated to
graphene oxide (GO) by non-covalent or covalent linkages were

Fig. 4. Schematic diagram for the detection of proteolytic enzyme based on MEF-caspase-3 biosensor. The restricted metal enhancement was activated by caspase-3
by increasing the distance between AuNPs and FITC. (a) Fluorescent spectrum of bifunctional AuNPs without ssDNA and (b) with ssDNA after proteolytic reaction by
caspase-3. Reprinted with permission from (Choi et al., 2020). Copyright (2020) American Chemical Society. (c) Description of the modification of the FO-SPR sensor
along with the schematic illustration for the detection of PDGF-BB. (d) Schematic representation for home-made FO-SPR biosensing detection system. Reproduced
with permission of Elsevier (Qian et al., 2019). with Creative Commons Attribution License (5293550401354).

7
G.K. Soni et al.
reported as nanoprobes for MMP9 detection (Lin et al., 2018). In another
study, tetraphenylethylene (TPE) based peptide micelles have been
studied for the monitoring of cellular apoptosis (Qin et al., 2021). In this
study, a self-assemble amphiphilic polymer was constructed by linking a
Poly(histidine) chain polypeptide (H9) with
amino-carboxylpolyethylene glycol (NH2-PEG8-COOH) on one side and
TPE was attached to H9 using DEVD peptide as the core. The anticancer
drug doxorubicin was loaded and based on the aggregation-induced
emission (AIE) phenomenon of TPE, the loading and release of DOX
were processed.
2.3. Antifouling peptide based optical biosensors
The selective and sensitive performance of any optical sensor in bio
fluids (saliva, blood, serum plasma, urine, and cell lysate), is highly
affected by nonspecific adsorption (fouling) and background contami­
nation or biosensor malfunction (Banerjee et al., 2011). To resolve this
issue numerous antifouling materials had been fabricated on the sensor
surface. Among all, synthetic antifouling peptides suppress the available
antifouling materials due to their high biocompatibility, easy synthesis,
flexible and tunable structures. Antifouling peptides comprise electric
neutrality and strong hydrophilic properties which makes them superior
resistant materials toward protein adsorption (Ye et al., 2015). Modu­
lation of these properties can be easily carried out by varying the
sequence, number, and type of amino acid residues in a particular
peptide. The immobilization of antifouling peptides onto the surface of
the optical sensor includes the electrografting, self-assembly, or poly­
merization methods from which self-assembly is the easiest way (Jiang
et al., 2020). An ECL ratiometric sensor was reported for DNA methyl­
transferase (MTase) detection and to study the antifouling properties of
the novel peptide DKDKDKDPPPPC (Li et al., 2019). The characteriza­
tion of the peptide with Differential Pulse Voltammetry (DPV) and
fluorescein diacetate (FDA) confirmed the antifouling nature of the
peptide. The detection principle of the ECL sensor initiates a dual ECL
signal based on the HCR amplification method. High concentrations of
Dam MTase decreased the Au@luminol intensity and increased the ECL
intensity of PTC-NH2 which allowed the selective detection of DNA
MTase. Similarly, another ECL sensor was fabricated by immobilization
of DKDKDKDPPPPC peptide on AuNPs via thiol bond formation (Hao
et al., 2020). Further, CdS QDs coupled to the peptide by EDC/NHS
chemistry were reported to study the electrochemiluminescence reso­
nance energy transfer (ERET) between QDs and Cy5 dye. The ECL based
sensor successfully reported the detection of carcinoembryonic antigen
(CEA) based on ratiometric ECL signal getting from Au-luminol (positive
potential) and QDs (negative potential). Apart from the immobilization
of antifouling peptides on the sensor surface, these peptides were also
used for the synthesis of antifouling magnetic beads to detect miRNA 21
in biological media (Hao et al., 2021). The dendritic peptide and hairpin
DNAzyme are anchored on MBs to acquire an antifouling surface and
from the two signal amplification methods, the first one comprised of
the DNAzyme on MBs which caused cycle amplification of nucleic acids.
The product of the first amplification (DNA) activated the hybrid chain
reaction (HCR) on the GCE/TiO2 NNs/PANI electrode to generate the
second amplification with respect to the ECL signal. The proposed sensor
can detect miRNA with an LOD of 0.13 fM, and the antifouling MBs can
serve as a separating tool.
An antifouling peptide was utilized for the development of a home-
made fiber optic surface plasmon resonance (FO-SPR) sensor for a
platelet-derived growth factor (PDGF-BB) (Qian et al., 2019). The
FO-SPR sensor assembly is constructed by immobilization of antifouling
peptide and PDGF-BB specific aptamer on gold-coated optical fibers
(Fig. 4c and d). The interaction of PDGF-BB with aptamer resulted in
refractive index alteration and detection of a target with a LOD of 0.35
pM. A newly developed SPR based sensor for ctDNA had been reported
which consists of a dual-functional surface layer, PNA probe, and AuNPs
(Bellassai et al., 2021). The dual-functional layer was introduced to the
8
Biosensors and Bioelectronics: X 12 (2022) 100259
sensor surface using cationic poly-L-Lysine (PLL) with anionic peptide
CEEEEE to prevent nonspecific adsorption, whereas PNA was immobi­
lized for targeting ctDNA. Owing to the antifouling property of
dual-layer and PNA KRAS p.G12D-mutated DNA can be detected with
LOD 2.5 aM in human plasma. Apart from these, multifunctional pep­
tides consisting of a number of domains along with antifouling proper­
ties, have been utilized in early cancer detection. A multifunctional
peptide SS-IMVTESSDYSSY-KK-FHYQRDTPKSYN has been utilized for
the fabrication of a direct-real-time SPR sensor for PD-L1 exosomes (Mao
et al., 2021). The peptide comprised of four different regions depending
upon the sequence and type of amino acids- SS (antifouling region),
IMVTESSD (assembly region), YSSY (binding region), and
FHYQRDTPKSYN (recognizing region). The Ser residues with hydro­
philic properties prevented the non-specific adsorption of biomolecules
via hydration. The multifunctional peptide enhanced the interactions
among graphene and target molecules and ensure the selective detection
of PD-L1 exosomes. In another report, RVRRFFF peptide with anti­
fouling (FFF) and recognition (RVRR) properties were employed for
fluorimetric detection of MDA-MB-231 cells (Li et al., 2019). Nitro­
benzoxadiazole (NBD) modified peptide (RVRRFFF-NBD)
self-assembled via π–π interactions and resulted in quenching of fluo­
rescence. Here FFF group provided the antifouling property as well as
helped in the self-assembly of the peptide. In the presence of
MDA-MB-231 cells self-assembled structure was disrupted due to the
specific binding of the peptide with the target and resulting in the regain
of fluorescence.
These antifouling peptides are a magnificent candidate for the
fabrication of PEC sensors, for example, an optical PEC based sensor was
reported for human chorionic gonadotropin (hCG) using EKEKE­
KEPPPPC (antifouling) and PPLRINRHILTR (recognition) peptide in one
probe electrode (Gu et al., 2021). The recognition and antifouling pep­
tide were attached to CBO/Au photocathode via Au–NH2 and Au–S
chemistry respectively. The steric hindrance caused by the peptide-hCG
complex reduces the photocurrent and leads to the selective detection of
the target in complex media. In another report, PEC cytosensor for HeLa
cells based on zwitterionic peptide (EESKSESKSGGGGC), As1411
aptamer, and ITO/TiO2/ZnIn2S4 matrix have been reported (Fan et al.,
2019). The decrease in photocurrent response upon binding of HeLa
cells to AS1411 aptamer exhibited sensitive and selective detection with
LOD of 34 cells/mL. Similarly, the PEC sensor for neuron specific
enolase (NES) analysis was fabricated using EKEKEKEPPPPC peptide,
anti-NSE, and monocrystal perovskite magnesium dititanate (MgTi2O5)
as a basic matrix (Xu et al., 2022). Peptide immobilized on MgTi2O5
surface using polydopamine (PDA) provided the antifouling property as
well as enhanced the sensitivity of the sensor. Similarly, the PEC
immunosensor was illustrated in which zwitterionic peptide KAEA­
KAEAPPPPC was used to provide an antifouling interface to novel het­
erojunction photocathode Au/ZIS/CIS (Hu et al., 2022). The proposed
PEC immunosensor provided a new approach for cardiac troponin I
(cTnI) detection at the pM level.
2.4. Self-assembled peptide based optical biosensors
Self-assembly of bio-molecules such as peptides, proteins, lipids, and
DNA provides the efficient pathway for the engineering of ordered 1D,
2D, and 3D nano/micro structures (Singh et al., 2020). Depending upon
the specific recognition, these ordered structures are basically facilitated
by the non-covalent interactions including the π-π stacking interactions,
hydrophobic forces, hydrogen bonding, electrostatic interactions, and
van der Waals interactions (Brar et al., 2021). Recently self-assembly of
peptides has been reported to form ordered nanostructures such as
nanospheres, nanotapes, nanotubes, and nanofibrils (Gazit, 2007).
Inspired by these, molecular self-assembled peptides have been widely
used in the field of bio-sensing and biomedical science. By utilizing the
self-assembly property, a novel peptide nanofibrils based colorimetric
method has been developed for HepG2 detection using B
G.K. Soni et al.
(OH)2AEAEAELRARARL-OH (C. Chen et al., 2013). The peptide was
modified with phenylboronic acid (BA-P) to further conjugate with
catechol alizarin red dye (ARS) for colorimetric probe fabrication. The
interactions of ARS with BA-P resulted in the self-assembly of peptides
into nanofibrils and the color changed from wine-red to orange yellow.
After the interactions with target HepG2 cells, the self-assembly of
peptides get disordered, and the change in color can be seen by the
naked eyes. Apart from this, peptides can be self-assembled on the metal
surface via the formation of a covalent bond between the –SH group of
Cys and metal. In this context, cysteine terminated WLEAAYQRFLC
(WC10) peptide self-assembled on ECL matrix for human breast cancer
cell (MCF-7) detection (L. Zhang et al., 2017). The high affinity between
WC10 and MCF-7 along with enzyme catalysis-based signal amplifica­
tion and recognition property of lectin Con A provided the highly sen­
sitive detection with a LOD of 150 cell/mL.
Self-assembly of peptides is susceptible to molecular environment, a
small change due to various factors such as pH, solvent, temperature,
etc. disrupting the ordered structure. The growth of cancer cells is
facilitated by a slightly acidic environment (pH 6.7–7.1), which
9
Biosensors and Bioelectronics: X 12 (2022) 100259
motivates the researchers to design the pH-based biosensors for cancer
detection. In this regard, pH responsive nanoparticles were reported
based on the self-assembly of oligopeptide ([CH3CONH]-K(DEAP)SKSK
(DEAP)-[CONH2]) (Zhao et al., 2014). The peptide had been modified
with fluorescent dye and BHQ-1 for FRET based detection of the tumor.
The non-fluorescent supramolecular nanoparticles of self-assembled
peptide quickly changed to disassembled fluorescent peptide mole­
cules upon binding with the tumor at pH 6.7–7.1. This pH-based
detection is quite useful for easy and effective tumor diagnosis in clin­
ical. Similarly, pH triggered hydrophilic peptide (SKDEEWHKNNFPLSP)
and hydrophobic signal molecule tetraphenylethylene based fluorescent
detection of VEGFR2 was reported (Qian et al., 2018). The peptide was
modified with TPE to assemble non-fluorescent nanoparticles which
regain their fluorescence during the binding with VEGFR2 in acidic pH.
The peptides with self-assembling properties can be modified
conveniently with various fluorophores (TPE, TAMRA, NBD) for the
early diagnosis of cancer. A fluorescent light-up probe for caspase-3 was
illustrated based on the aggregation induced emission (AIE) by TPE-
GFFYK(DVEDEE-Ac) (Han et al., 2016). The –COOH terminal of DVED
Fig. 5. (a) The chemical structures of RVRRFFF (C-3)
and nitrobenzoxadiazole NBD. (b) Schematic repre­
sentation for peptide self-assembly based detection of
furin C-3. Reprinted with permission from (Li et al.,
2019). Copyright (2019) American Chemical Society.
(c) Schematic representation of RGDs and TAT con­
jugated NaYF4:Yb3+/Er3+ NPs for Human Epithelioid
Cervix Carcinoma detection. Reprinted with permis­
sion from (Kostiv et al., 2016). Copyright (2016)
American Chemical Society.
G.K. Soni et al.
segment had been cleaved in the presence of caspase-3 resulting in
fluorescence activation due to the absence of hydrophilic groups. Fol­
lowed by this, enzyme hydrolysis in the biological environment
enhanced the AIE of the TPE-GFFYK peptide via self-assembly. Similarly,
amphiphilic peptide RVRRFFF was modified with nitrobenzoxadiazole
(NBD) fluorophore to fabricate non-fluorescent micelles by
self-assembly (Li et al., 2019) (Fig. 5a). The RVRR domain in the peptide
was selectively bound to furin and turn-on the quenched fluorescence in
the hydrophobic environment (Fig. 5b). An interesting approach was
reported by fabricating the fluorescent sensor for breast cancer detection
using peptide ANSFLEELRHSSLERECIEEICDFEEAKEIFQN-AN33 and 2D
material molybdenum disulfide (MoS2) (Dou et al., 2019). The peptide
modified TAMRA (tetramethyl-rhodamine) fluorophore (TAM­
RA-AN33) loses its activity during the binding with MoS2 via a
self-assembled mechanism. The binding of protein C receptor (PROCR)
with TAMRA-AN33-MoS2 broke the ordered structure and enabled the
detection via activation of the fluorophore.
Peptides comprising the different domains with distinctive ap­
proaches can be synthesized and utilized for cancer detection without
modification, for example, SPR sensor was fabricated for PD-L1 exosome
based on SS-IMVTESSDYSSY-KK-FHYQRDTPKSYN and gold chip modi­
fied with graphene (Mao et al., 2021). Peptide consists of different do­
mains with a different function, where IMVTESSD act as a self-assembly
domain that participated in the formation of ordered and crystallo­
graphic nanostructures. The binding of Pd-L1 exosome disrupted the
ordered assembly resulting in the alteration of the refractive index value.
These changes provided the effective and convenient detection of the
Pd-L1 exosome for early stage cancer diagnosis. The reported self
assembled peptides based optical biosensors for early cancer detection
have been listed in Table 1.
2.5. Cell penetrating peptide based optical sensors
Cell penetrating peptides act as an astonishing agent for detecting
the targeted sites of diseases and delivering drugs. The first CPP was
discovered in 1988 via the discovery of the Human immunodeficiency
virus (HIV) trans activator of transcription protein (TAT) (Figueiredo
et al., 2014). In general, a CPP is a short peptide sequence that either has
35 or less than 35 amino acid residues. Cell penetrating peptides have
caught much attention due to their ambiguous characteristics such as
low cytotoxicity, an ability to enable receptor-independent transport
across cell membranes when linked to oligonucleotides or proteins, and
the ability to cross the cell plasma membrane during the process of
endocytosis. Thus, CPPs are classified into three categories-on the basis
of their physicochemical properties: cationic, hydrophobic, and
amphipathic. Cationic CPPs contain highly positive net charges that
originate from the basic short strands of arginine and lysine and are most
employed in diagnosis due to their preferable interactions with cell
surface (El-Sayed and Harashima, 2009). Based on these, CPPs played an
important role in modern biosensor development in various fields, such
as clinic diagnosis and earlier detection of diseases. A report has suc­
cessfully demonstrated the application of CPPs for colorimetric
screening and imaging of cancer cells (P. Chen et al., 2013). In this,
colorimetric detection of the matrix metalloproteinase matrilysin
Table 1
List of reported self-assembled peptide based optical biosensors for early cancer detection.
Sequence of Peptide Type of Cancer
B(OH)2AEAEAELRARARL-OH Liver Cancer
WLEAAYQRFLC (WC10) Breast cancer
[CH3CONH]-K(DEAP)SKSK(DEAP)-[CONH2] Skin Cancer
SKDEEWHKNNFPLSP Breast Cancer
TPE-GFFYK(DVEDEE-Ac Colon Cancer
RVRRFFF Breast Cancer
ANSFLEELRHSSLERECIEEICDFEEAKEIFQN-AN33 Breast cancer
SS-IMVTESSDYSSY-KK-FHYQRDTPKSYN Lung Cancer
10
Biosensors and Bioelectronics: X 12 (2022) 100259
(MMP-7) was carried out based on the CPP peptide (JR2EC) function­
alized gold nanoparticles (GNPs). GNPs were modified through the
cysteine in the middle of the JR2EC sequence (NAADLEKAIEA­
LEKHLEAKGPCDAAQLEKQLEQAFEAFERAG), via thiol-gold linkage.
MMP-7 can recognize and cleave the peptide which was further recog­
nized by GNPs aggregation. A LSPR shift and a color change from red to
blue can detect the MMP-7 in the salivary gland of cancer patients with a
LOD of 5 nM.
An interesting report was presented on chemiluminescence based on
the cell adhesive RGD and TAT peptide decorated lanthanide NPs
(NaYF4:Yb3+/Er3+) for Human Epithelioid Cervix Carcinoma detection
(Kostiv et al., 2016). The click chemistry had been applied for decorating
the NaYF4:Yb3+/Er3+ NPs with RGD and TAT peptides (Fig. 5c). Further,
the targeting affinity of utilized peptides facilitates further it to get
localized at the membrane of HeLa cells helping in the imaging of
Human Epithelioid Cervix Carcinoma and also in photothermal therapy.
In another report, a signal amplification strategy based on Ru(bpy)2+- 3
encapsulated silica nanoparticles for the electrochemiluminescent
cytosensing and sensitive quantification of drug-induced apoptotic
cancer cells was also carried out (Wu et al., 2012). Here, an
arginine-glycine-aspartic acid-serine (RGDS) tetrapeptide was func­
tionalized with multiwalled carbon nanotubes (MWCNTs). The formed
RGDS-MWCNTs composites were then immobilized on a graphene
nanotubes surface as an anchorage layer in order to effectively capture
the cells via the specific combination of RGD domains with integrin
receptors on the cell surface. Similarly, a simple and sensitive electro­
generated chemiluminescence biosensor was developed to monitor
matrix metalloproteinase 2 (MMP-2) by employing a CPP peptide
(CGPLGVRGK) as a molecular recognition substrate and thus help in the
detection of the malignancy (Dang et al., 2016). Bis(2, 2′ -bipyr­
idine)-4′ -methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-
bis(hexafluorophosphate) (Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 (Ru1)
was used as ECL-emitting species and covalently labeled onto the pep­
tide through lysine via acylation reaction to form Ru1-peptide. ECL
peptide-based biosensor was further fabricated by self-assembling the
ECL probe onto the surface of the gold electrode. The decreased ECL
intensity was linear in the concentration range of 1–500 ng/mL of
MMP-2. Moreover, the ECL biosensor is successfully applied to the
detection of MMP-2 secreted by living cells, such as HeLa cells.
In another example, a novel intracellular protease fluorescence
“turn-on” sensor based on the nanoconjugate of graphene oxides and
peptide substrates was developed and used for early stage malignancy
detection (Wang et al., 2011). The peptide was cleaved from the
GO-peptide conjugate after being transported into cells by intracellular
proteases which further results in a significant increase in fluorescence
emission because of the release of the fluorophore from GO. The fluo­
rescence peaks drastically enhanced with increasing caspase-3 concen­
tration in the range from 7.25 to 362 ng/mL and LOD was found to be
7.25 ng/mL. Further FRET phenomena has been used for MMPs detec­
tion based on the nona-arginine (R9), octa-glutamate (E8), labeled with
Cy5 and Cy7, respectively, and MMP2/9 cleavable linker (PLGCAG)
(Savariar et al., 2013). When MMPs in the tumor tissue cleave the linker,
FRET between Cy7 and Cy5 was interrupted producing red fluorescence
by Cy5 in the tumor tissue. Recently, a group has developed a
Transduced signal LOD Reference
Colorimetric 18.6 mM Chen et al. (2013)
ECL 150 cell/mL Zhang et al. (2017)
Fluorescence - Zhao et al., 2014)
Fluorescence - Qian et al. (2018)
Fluorescence 0.54 pM Han et al., 2016
Fluorescence 0.11 ng/μL Li et al. (2019)
Fluorescence 0.76 nM Dou et al., 2019
SPR 20 particles/mL Mao et al. (2021)
G.K. Soni et al.
cell-permeable biosensor to detect deubiquitinating enzyme (DUB) ac­
tivity in intact cancer cells (Safa et al., 2019). The reporter incorporated
the previously established RWRWR β-hairpin CPP with a short
DUB-binding peptide sequence (LRGG), and a fluorescent molecule
(AFC). They observed that the conjugation of the four amino acid
binding sequence diminished the net uptake of the peptide-based re­
porter compared to the uptake of the RWRWR CPP itself. RWRWR up­
take in HeLa cells was found to occur rapidly for 90 min to be sufficiently
internalized which clearly demonstrated that the efficiency of the CPP
should be considered when designing CPP-conjugated therapeutics or
biosensors. In a similar report, it has been observed that the conjugation
of green fluorescent protein (GFP) as a model cargo onto seven different
CPPs, such as penetratin, octa-arginine, TAT, Transportan, and Xentry
(Patel et al., 2019) has been successfully applied for cancer cell detec­
tion. The study demonstrated that uptake of the cargo was not only
dependent on the CPP but also on the cell line it was tested in, including
HeLa, HEK, and HepG2. A group of researchers used nanomaterial such
as quantum dots and developed a novel antibody-free assay to study
protein kinase (Shiosaki et al.2013). A cationic peptide was modified
with a fluorophore that interacts electrostatically with anionic QDs
causing FRET from the QDs to the fluorophore. The fluorescence lifetime
of the fluorescent probes which is also called FLIM can also be used for
monitoring biological processes. Based on this, researchers have re­
ported a cell-penetrating peptide biosensor for dynamic monitoring of
phosphorylation by Abl kinase (Damayanti et al., 2013). FLIM, which is
not confounded by photobleaching or cellular autofluorescence, was
employed to detect phosphorylation-dependent fluorophore lifetime
shifts (1–2 ns) in living cells. In order to accurately probe diseased sites,
many CPPs-mediated molecular probes such as activatable CPP (ACPP)
have been used for cancer diagnosis. Further, research has shown that
ACPP can be activated by matrix metalloproteinase over-expressed in
tumors and imaging can be carried out using fluorescent agents. In a
report, a research group designed and developed a novel intravenously
administered peptide-dye conjugate (AVB-620) for breast cancer diag­
nosis (Miampamba et al., 2017). Experimental results indicated that
AVB-620 can visualize the tumors under a fluorescence imaging camera
based on the FRET between Cy5 and Cy7. Moreover, AVB-620 was in the
clinical phase I study in patients with breast cancer (Unkart et al., 2017).
The result indicated that AVB-620 was safe, and well-tolerated at doses
for tumor-specific fluorescence detection which was obtained by
intra-operative imaging of surgical specimens after administration of
AVB-620. In addition, a similar type of report has been also documented
where they also designed and synthesized CPPs-mediated molecular
probes as imaging agents (Zhu et al., 2018). They firstly synthesized
three thermally activated delayed fluorescence (TADF) chemicals
(4CzIPN, NAI-DPAC, BTZ-DMAC) and TADF was loaded into amphi­
philic CPPs (F6G6(rR)3R2) which was capable of self-assembling into
nanoparticles in water to constructed TADF nanoparticles as imaging
agents.
With the emergence of nanotechnology, CPPs-mediated nanoplat­
forms as imaging agents for diagnosis have attracted widespread
attention (Onoshima et al., 2015). Because of their advantages of low
cytotoxicity, good biocompatible, high cell membrane permeability,
small size, large surface areas, abundant functional group on their sur­
face, and easily modify. One excellent example was based on
CPPs-mediated quantum dots as imaging agents for biological diagnosis
(P. Zhang et al., 2018; Yang et al., 2020). However, CPPs-mediated QDs
not only possess the advantages of QDs but also have high cell mem­
brane permeability. A near-infrared semiconducting polymer dots
coated with a CPP (R8-Pdots) for cell tracking in deep organs has been
designed (Z. Zhang et al., 2018) which presented low cytotoxicity and
high cell membrane permeability for MCF-7 cells. Meanwhile, a clear
and strong fluorescence signal was recorded in vitro and in vivo. The
results of cell tracking capability of Pdots in live mice in vivo indicated
that MCF-7 cells labeled with R8-Pdots were visualized in real-time,
which are powerful for whole-body fluorescence imaging in vivo. In
11
Biosensors and Bioelectronics: X 12 (2022) 100259
addition, mesoporous silica nanoparticles, superparamagnetic iron
oxide nanoparticles (SPIONs), and gold nanoparticles have also been
explored for imaging agents and diagnosis, on account of their low
cytotoxicity and easy functionalization (Grasso et al., 2019)]. Fluo­
rescently labeled, PEG-coated SPIONs were successfully bioconjugated
with gH625-derived peptide from the CPP family for breast cancer im­
aging ((Perillo et al., 2017). The uptake of the bioconjugated NPs into
cytosolic low polarity locations seems consistent with the
endosomal-lysosomal pathways of endocytosis, which appears to be the
major cellular uptake mechanism for most nanosystems. Thus, in
contrast to free gH625 crossing cell plasma membranes by direct
translocation, the CPP-capped PEGylated SPIONs undergo enhanced
endocytosis.
It has been noticed recently that some CPPs have been used for
cancer diagnosis using molecular imaging techniques like optical im­
aging, wherein their sensitivity depends on the efficient delivery of
contrast agents to the cytoplasm and/or nuclei of the target tissue.
ACPPs have many advantages over other optical and MR-based probes.
First, their amenability to dual labeling for MR and fluorescence pro­
vides an ideal correlation between tomographic imaging that can detect
tumors anywhere in the body with higher resolution. This added infor­
mation may be particularly useful in the staging of certain cancers, in
which tumor infiltration of a capsule plays a critical role in prognosis.
Thirdly, the increase in molecular size further reduces the high back­
ground uptake, previously seen for unadorned ACPP’s and allows for
additional tumor accumulation based on enhanced permeability and
retention although still remaining small enough to penetrate into solid
tumors. For all of these reasons, ACPPs may be useful for clinical pur­
poses in the early stage of cancer. Thus in a report, the cell-penetrating
peptide-ACPPD (EEEEEEEEPLGCAGRRRRRRRRRC) has been applied
for the successful delivery of quantum dots to reveal targeted tumor
imaging and thus help in increasing the chances of penetration of the
nanomaterial through the cell for highly specific cell imaging of the
cancer cells (Olson et al., 2010). Another such theranostic application
based on the use of CPP-coated silica NPs were described (Yu et al.,
2016; De La Torre et al., 2015). Where the mesoporous silica NPs were
coated with CPPs and a fluorogenic helps in the detection of the HeLa
cell line.
In another report, a plasmonic gold nanostar platform was designed
to increase the Raman signal via the surface-enhanced resonance Raman
scattering (SERRS) effect (Fales et al., 2013). SERS imaging and
photodynamic therapy using this construct were demonstrated on
BT-549 breast cancer cells. The TAT peptide allowed for effective Raman
imaging and photosensitization with the nanoparticle after the 1h in­
cubation period. In the absence of the TAT peptide, nanoparticle accu­
mulation in the cells was not sufficient to be observed by Raman imaging
or to produce any photosensitization effect after this short incubation
period. This report shows the first application of combined SERS im­
aging and photosensitization from a theranostic nanoparticle construct.
3. Future perspective
The present review highlights the recent progress made in the optical
sensing of cancer using peptide-based biosensors. The peptides exhibit
stability against denaturation, specificity, and chemical flexibility.
These features of peptides can contribute to the future development of
novel biosensors for targeted cancer detection with an improved limit of
detection, sensitivity, and range of linearity. It is important to note that
although the reported peptide-based biosensors for optical sensing are
focused on specific biomarker-based cancer detection and showed great
potential for the early-stage diagnosis of cancer, still these methods are
associated with some limitations. Also, most of the reported detection
methods can detect one biomarker at a time which limited the simul­
taneous multiple detections of different cancers present in different parts
of the body. Multiple biomarkers-based detections by one biosensor are
highly required for successful cancers diagnosis. It is worth mentioning
G.K. Soni et al.
that the peptides incorporated with nanoparticles, nanocavities, 2D
materials, and fibers can be used for the development of novel optical
biosensors with the high specificity of different biomarkers of cancer.
However, the progression of the development of such novel and sensi­
tive biosensors will primarily depend on the advancement of new ma­
terials exhibiting unique shapes and structures. Since most of the
reported peptide-based biosensors show good analysis in solution or cell
lysate but due to their complicated components, the precise molecular
recognition is difficult in real samples of blood and interstitial fluid. In
this context, material-based synergetic recognition can be a more
effective approach. The increasing research for the development of
nanomaterials on peptide-based optical sensors demonstrated their
significance in early cancer detection and hence can be used to create
the biosensors for easy and successful clinical diagnostics. Also, peptide-
based biosensors can be used to fabricate the commercial portable bio­
chips. The new sensing principles and electrochemical signaling can be
used for the development of peptide-based biochips models. The
development of biochips of peptide-based biosensors will provide a new
prospect for the fast and convenient biological analysis of early-stage
detection of cancer. It is important to note that there is a certain gap
between fundamental research and clinical applications however, the
development of these biochips can move the peptide-based biosensing
approach from laboratories to the real world for easy and decentralized
cancer diagnosis.
4. Conclusion
Interdisciplinary research of biomolecular nanomaterials brings
great challenges and opportunities to push the development of effective
peptide-based optical biosensors with a special emphasis on cancer
detection. Currently, strategies for employing the concept of peptide-
based nanomaterials are being diversified toward the different roles in
the sensing process including the recognition element. Various aspects
of the biosensors, such as type of peptide nanomaterial, functionaliza­
tion tool, detection method, signal amplification, and the corresponding
sample preparation, selectivity, sensitivity, stability, etc., have also been
discussed in detail. In addition, the above-summarized examples of
peptide-based biosensors reported so far in the literature, can be eval­
uated with their advantages and limitations so as to stress their potential
for further development in this field. It is still clear that we have wit­
nessed explosive growth in achievements related to the utilization of
peptide-based optical biosensors and their type as targeted, cell pene­
trating, antifouling, self assembles, etc., with nanomaterial-based bio­
sensors. We further aimed for providing the reader with an introduction
to the peptide-based biosensor field in cancer detection. Although sig­
nificant advances in the development of peptide-based optical bio­
sensors have been made, this field is still in its infancy. The remaining
challenges for the subsequent development and application of peptide-
based optical biosensors as a promising solution should be addressed
optimally in future. For this purpose efforts are being ongoing for
delivering effective peptide based optical biosensors for early stage
cancer detection.
CRediT authorship contribution statement
Gurpreet K. Soni: Conceptualization, Writing – original draft,
Writing – review & editing. Saima: Writing – review & editing. Priya
Manhas: Writing – review & editing. Rohit K. Sharma: Supervision,
Conceptualization, Writing – review & editing, Overall, Supervision,
Research support.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
12
Biosensors and Bioelectronics: X 12 (2022) 100259
Data availability
No data was used for the research described in the article.
Acknowledgments
R.K.S. acknowledges the support of the Department of Science and
Technology (DST), India-Project No. DST/ TDT/AGRO-10/2019. G.K.S.
thanks University grant commission (UGC), India and P.M thanks
Council of Scientific and Industrial Research (CSIR), India for their
respective research fellowships.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biosx.2022.100259.
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