AMITY INSTITUTE OF BIOTECHNOLOGY

MATERIAL SCIENCE: ASSIGNMENT

PROJECT TITILE : 2D CARBON MATERIALS AS A

PLATFORM FOR BIOSENSING
COURSE : B.Tech (BT)
SEMESTER : 03
NAME OF THE STUDENT : PARISHA GARG
ENROLLMENT NO. : A0504118062
BATCH : 2018 -22
NAME OF THE FACULTY : DR. MONALISA PAHARI

SIGNATURE OF FACULTY
 ABSTRACT

The increasing demands of bioassay and biomedical applications have significantly promoted
the rational design and fabrication of a wide range of functional nanomaterials. Coupling
these advanced nanomaterials with biomolecule recognition events leads to novel sensing and
diagnostic platforms. Because of their unique structures and multi functionalities, two-
dimensional nanomaterials, such as graphene and graphene-like materials (e.g., graphitic
carbon nitride, transition metal dichalcogenides, boron nitride, and transition metal oxides),
have stimulated great interest in the field of optical biosensors and imaging because of their
innovative mechanical, physicochemical and optical properties. Depending on the different
applications, the graphene and graphene-like nanomaterials can be tailored to form either
fluorescent emitters or efficient fluorescence quenchers, making them powerful platforms for
fabricating a series of optical biosensors to sensitively detect various targets including ions,
small biomolecules, DNA/RNA and proteins. This review highlights the recent progress in
biosensors based on 2D carbon nanomaterials and their imaging applications. Finally, the
opportunities and some critical challenges in this field are also addressed.

Keywords: graphene, optical biosensors, graphene-like materials, field-effect transistor,

biosensor devices.
 INTRODUCTION

A biosensor is an analytical device which can detect a biomolecule-related element with an

appropriate transducer to generate a measurable signal from the sample. The biosensor
system illustrated in Figure 1 shows a typical platform, consisting of a bioreceptor interfaced
with a transducer. The bioreceptor has to be capable of recognizing the biomolecule element,
for instance, enzymes, antibodies, DNA, RNA, and cells. In an incorporation, the biological
signal that can be detected in various quantities by the receptor is transduced by physical,
chemical, optical, thermal or electrochemical actions into observable information and
analysed quantitatively. The first generation of biosensor devices was introduced by Clark
and Lyons [9] to monitor chemical components in the blood of a surgical patient and to
record quantitative biomolecule contents in blood. Since then, the applications of biosensors
in the biomedical field and global healthcare have become indispensable for human life
improvements in order to spatially analyse diseases through a patient, to detect and to
diagnose biomolecules, to integrate with drug delivery and food safety. In many efforts of the
investigations, major requirements of biosensors are that the receptor has to not only be
highly selective and specific to the biomolecular element, but also that the transducer needs to
be ultrasensitive and with sufficient reproducibility for reliable real-time measurements. For
more precision, the presence of chemical binding or biological specificity to an analyte in a
labelled technique is used to ensure that only the labelled biological activities give a strong
signal. However, this technique requires a labelling process in preparation and involves the
fluorescent dyes, chemiluminescent molecules, photoluminescent nanoparticles and quantum
dots. Alternatively, the label-free technique uses molecular, physical, mechanical, electrical,
optical properties and charge interaction to monitor binding activities. The label-free methods
can provide real-time tracking in biomolecular events and give more direct information about
the target biomolecules without interference effects from the labelling procedure. Currently,
label-free biosensors are essential for personalized genomics, cancer diagnostics, and drug
development where the sensitivity is one of the key requirements that needs to be engineered
for state-of-the-art biosensors.
The emergence of graphene, a two-dimensional (2D) nanomaterial, has played a tremendous
role in the electronic and sensor communities. Graphene is defined as “a single-atom-thick
sheet of hexagonally arranged, sp2-bonded carbon atoms occurring within a carbon material
structure” [3]. The nano-thickness graphene film with 100 μm of lateral size is observed as
carbon planes connected together by van der Waals forces acting over a distance of about
0.335 nm. The properties of graphene are those of a semi-metal and are stable under ambient
circumstances. This contradicts the general belief that a 2D material could not exist and be
thermodynamically stable. The charge transport and electronic properties of graphene are due
to its unique electronic band structure. In particular, among existing nanomaterials graphene
has a large surface area (2630 m2/g) being available for direct interaction in a wide range of
biomolecules [10]. Graphene can be engineered with structural defects using low-cost
fabrication methods due to the migration of heteroatoms, oxidation, and reduction by
chemical modification. The uses of graphene-based materials for biosensing involve two
points of view. One is based on charge-biomolecule interactions at π-π domains, electrostatic
forces and charge exchange leading to electrical variations in the pristine graphene. The other
uses are the effect of defects, disorder, the chemical functionalization to immobilize the
molecular receptors onto the surface of graphene oxide (GO), reduced graphene oxide (RGO)
and graphene-based quantum dots (GQDs). Recently, several excellent reviews have focused
on the interactions of graphene, GO and RGO-based biosensors with their biomolecular
targets. In this review, we focus on the graphene-based material properties due to their
fabrication process, surface chemistry and the photoluminescent graphene so called GQDs.
These graphene properties are utilized and integrated in biosensors for biological and medical
applications.
Graphene and graphene-based materials
the induction of the conjugated πdomains and the defect-derived photoluminescence
emissions—are used to account for the generation of photoluminescence in graphene
nanomaterials. Graphene oxide (GO) is generally produced from graphite using strong acids
and an oxidant treatment, and the GO is subsequently transformed to reduced GO (rGO) [9].
Because of the chemical synthesis, some oxygen-containing functional groups and structural
defects are inevitably involved on the surface of the GO and the derived rGO. Because the
oxygen containing functional groups on the GO confine the π electrons within the sp2-carbon
nanodomains, GO can fluoresce in a wide range of wavelengths from the near-infrared
(NIR)to ultraviolet. In most cases, these two mechanisms occur simultaneously to produce
the photoluminescence emission. In addition to graphene, other 2D graphene-like nanosheets
have also exhibited strong photoluminescence, such as g-C3N4 and MoS2. For example, g-
C3N4, which is synthesized via heat treatment of nitrogen-rich precursors, is composed of a
high degree of condensation of the tris-triazine unit, which produces strong
photoluminescence. Strong photoluminescent emission is alsoobservedforMoS2 nano sheets
due to their direct gap electronic structure. The advantage of their unique structures and
compositions gives these 2D graphene and graphene-like nanomaterials great potential for
various optical applications based on inherent persistent luminescence. In addition to the
inherent persistent luminescence, these fascinating 2D nanomaterials have received
significant interest as nano quenchers in fluorescent biosensors based on Förster (or
fluorescence) resonance energy transfer (FRET) because of their high fluorescence quenching
capabilities, good biocompatibility and large surface areas. In this process, the
photoexcitation energy is transferred from a donor fluorophore to an acceptor molecule based
on the spectral overlap between the emission of the donor and the absorbance of the acceptor.
Although GO itself is fluorescent, it can also serve as an efficient nano quencher and is able
to quench fluorescence to a large extent. The quenching capability of GO far surpasses that of
conventional organic quenchers. It has been estimated that the quenching ability of pristine
graphene is as large as 103, and the quenching by GO is attainable even at a distance of
30nm.However, there attraction in the use of graphene/GO as a nano quencher results from
the fact that the FRET effect is independent of the emission spectra of the donor [9].
 DISCUSSION

1. Engineering of biosensors using 2D carbon-based

nanomaterials
Previously, the structures and outstanding properties of graphene-based materials using
different synthesis methods have been exploited in biosensing applications since graphene
is a semi-metal with ultra-high charge mobility giving excellent electronic properties,
having large surface area, being capable of being functionalized on its surface. There are
many possible approaches to engineer the receptor for targeting biomolecules. In the
biomedical field, pristine graphene is not only referred to as an oxide-free graphene
presenting π-π stacking, non-covalent interactions and high electrostatic force, but it also
offers an infinite surface at a molecular level. Therefore, graphene provides for a high
possibility of active sites for charge-biomolecular interactions due to the large specific
surface area leading to a sensing enhancement as well as supporting the desired
functionalization to target biomolecules to improve the selectivity. Figure 6 illustrates the
points of view of the possible interactions of the graphene-based material system. For
example, the pure graphene area as shown in the figure can provide a charged area to
absorb any charged molecules or metal ions as well as interactions at a vacancy defect.
The functionalized graphene area is able to directly detect the biomolecules by its own
oxide components due to the synthesis in which lots of epoxide, hydroxy land carboxyl
groups are formed on the edge and surface sites. In addition, the functionalized graphene
allows binding of heteroatoms, nanoparticles (NPS), quantum dots (QDs), DNA,
enzymes, proteins, antigens, antibodies, and other specific molecules [10].

Figure 2. Schematic illustration of the graphene-based materials that can be immobilised with biomolecules as the
receptor.
1.1 Engineering of Pristine Graphene: Biomolecules-Based Biosensors
In the graphene-based biosensors, graphene is able to enhance the sensitivity and
LOD as well as the performance of the biosensor device by improving the charge
or electron transfer between graphene and the biomolecules due to its
extraordinary properties. For example, as seen in Figure 3, a label-free and
portable apt a sensor utilizes pristine graphene as the electrode in a field-effect
transistor device. The GFET biosensor is used to detect the Pb2+ ions in
children’s blood, in which the blood matrix is very complicated. The mechanism
to distinguish Pb2+ ions from common ions in the blood, including Na+, K+,
Mg2+ and Ca2+ at lower 0.1 M/L, is intrinsically p-doping on the CVD graphene
and the surface engineering by G-quadruplex, Thrombin binding aptamer (TBA),
and 8–17 DNAzyme. ThelowestconcentrationofGFETaptasensorisat37.5ng/L,
which was approximately one thousandth of the safety limit (100 µg/L) for Pb2+
in blood. A GFET construction shows a similar Pb2+ detecting platform but using
G-quadruplex as the receptor. The mechanism is due to the electrostatic potential
change after the lead combines to the double layer of DNA/CVD graphene
electrodes leading to the shift in Dirac point in the band structure of graphene. For
this device, the LOD is only 163.7 ng/L for the first signal verification of
DNA/GFET. Carcinoembryonic antigen (CEA) is a protein that can be measured
in the blood of a cancer patient. Recently, a label-free immunosensor based on the
antibody-modified graphene FET was reported. The surface modification is
applied via a non-covalent functionalization and π-stacking using a pyrene and a
reactive succinimide ester group to interact with graphene. The GFET biosensor
shows the specific monitoring of the CEA protein in real-time with high
sensitivity of <100 pg/mL. In the precise quantitative measurement of DNA
concentrations as well as binding affinities and kinetics of DNA hybridization, an
array of six CVD graphene-based FETs was fabricated in a single multiplexed
sensor for DNA analysis [18]. The concentration of oligonucleotides can be
measured as low as 10pM in which the single-single-base mutations can be
analysed in real time.
 Figure 3. Schematic illustrations of Graphene-based biosensors: (a) Pb2+ in blood biosensor based on
GFET; [15](b) Pb2+ biosensor based on graphene/ DNA; [9](c) CEA protein biosensor based on graphene/
anti-CEA; [19](d) real-time binding kinetics and affinity of DNA hybridisation based on GFET; [18](e)
paper-based biosensor for human papilloma virus(HPV) detection; [11](f) a lipid-based modified graphene
electrochemical biosensor [10].

In the use of electrochemical properties of graphene material, a novel paper-based

biosensor for human papillomavirus (HPV) detection was reported. The graphene-
polyaniline (G-PANI) electrode is modified using an anthraquinone-labelled pyrrolidinyl
peptide nucleic acid (acpcPNA) probe (AQ-PNA) and printed by inkjet printing method.
In a presence of surface engineering of a negatively charged amino acid on graphene
electrode through the electrostatic attraction, a synthetic 14-base oligonucleotide target
with a sequence corresponding to human papillomavirus (HPV) type 16 DNA is
measured the electrochemical signal response of the AQ label to identify the primary
stages of cervical cancer. On the development of electrochemical technology, graphene
microelectrodes integrated with bilayer lipid membranes (BLMs) have shown promising
results in both static and stirred experiments [12]. Moreover, due to the support made of
lipid film, the biosensor achieves a good reproducibility, reusability, high selectivity,
rapid response times, long-shelf life, and high sensitivity. This enables a direct
potentiometric measurement. Nikolelis et al. have also reported the use of the graphene
microelectrodes in detecting toxicants, i.e., carbofuran in fruit, saxitoxin, cholera toxin
and for diagnosis of D-dimers, urea and cholesterol as seen in Figure 3f.
1.2. Engineering of Biomolecules-Functionalized Graphene Based
Biosensors
Biosensors based on the subtype of graphene materials or functionalized graphene
GO, RGO, and GQD are widely used in medicine, biomedical, and bioimaging
regimes. This is due to their extremely large surface area and ability to interact
with various types of molecules. In addition, the outstanding properties of
solubility, biocompatibility, and functionalization play an important role in
sensing mechanisms. Currently, as shown in Figure 8, the GO-based FET has
simply demonstrated glucose detection without an enzymatic glucose solution. In
this device, GO is used as the selective material to glucose while the sensitivity of
the sensor is enhanced down to 1 µM by adding CuNPS and AgNPs. Interestingly,
functionalized graphene oxide (GO) ink has been printed on a pentacene FET for
detecting artificial DNA and circulating tumour cells. Upon capturing the DNA by
its phosphate group, the negative charge attracts holes at the grain-boundary of the
pentacene layer and induces the collision or scattering in the region of the
pentacene layer. Therefore, the mobility of the FET changes extremely achieving
a high sensitivity of 0.1 pM and this could be improved for mass-scale production
of printed biosensors. In a platform for ultra-sensitive urea detection, the RGO
surface is decorated by the new construction of layer-by-layer assemblies of
polyethyleneimine (PEI) and urease. The RGO FET of urea detection can be
analysed as the change of pH in liquid gate, by which shifting the Dirac point at
the minimum voltage of <500 mV. The limit of urea detection is down to 1 µM
with very fast response and good long-term stability. In addition, the introduction
the Cu2+ improves the LOD down to 0.01 µM. In recent complex platforms, FET
biosensors using RGO combined with PtNPS and anti-BNP have been explored as
a brain natriuretic peptide (BNP) detector at the early stage level [8]. The BNP is a
recognized biomarker and it is very important in heart failure diagnosis and
prognosis. The RGO FET achieves the lowest of detection at 100 fM in a human
whole blood sample. Surface plasmon resonance (SPR) is a widely used technique
to investigate biochemical reactions in scientific research and medical diagnosis.
In particular, SPR provides label-free biosensing and real-time monitoring of
biomolecule interactions. However, for small molecules or at low concentrations
of the targets, the SPR signal is not sufficient to be analyzed. To improve the SPR
signal and biosensing performance, linking layers of GO have been introduced
into the SPR sensor system. The sensor chip consists of gold sensor chips using
PMMA as an intermediate membrane, monolayer of CVD graphene on top, and
the biotin-SA conjugate, respectively. The SA molecules allow the biosensor to
select the immobilized biomolecules containing biotin. The linking layers of GO
in the system provides a number of binding sites for biomolecules due to the large
surface area. However, a GO thickness of more than 10 nm strongly limited the
optical absorption leading to a sensitivity reduction. Recently, there has been a
demonstration showing the improvement and control of the plasmonic coupling
mechanism in GOSPR-based immune affinity biosensors by adding carboxyl
groups. The GO-COOH SPR chip can be improved four times over the SPR angle
shift and achieved the lowest antibody detection limit of 0.01 pg/mL. Other work
has reported the reduction of GO-based SPR fabricated by thermal reduction at
high temperature, the so-called RGO SPR, with a thickness of 8.1 nm. The
performance of the RGO SPR biosensor shows a response to rabbit
immunoglobulin G (rabbit IgG) with a LOD of 0.0625 µg/mL. Currently,
fluorescence biosensors based on the GQDs have gained much attention as an
alternative choice due to their ease of the synthesis, good stability, fast tissue
internalization, and biocompatibility. The fluorescence biosensor relies on the
energy transfer between the electron donor and acceptor which is powerful for
drug delivery and biomolecular interactions at the nanoscale. Fluorescence
resonance energy transfer (FRET) is a mechanism to describe the energy transfer
between two fluorescent molecules, where one is a donor being in an excited state
and ready to transfer to the other one or an acceptor via a non-radiative dipole-
dipole coupling [4].
Figure 4. Schematic illustration of functionalized graphene-based biosensors: (a) a glucose detection based
on GO FET; [6](b) DNA detection based on printing GO/ pentacene FET; [7](c) urea platform biosensor
based on Urease/PEI/RGO FET; [8](d) Heart failure detection based on Pt NPS/RGO FET; [13](e) Biotin-
SA/GO SPR chip; [13](f) BSA biosensor based on GO/COOH enhanced SPR; [1] (g) Rabbit IgG detection
based on RGO SPR; [5](h) FRET biosensor based on GQD/PEG aptamer/MoS2 [12]

This simple technique has been reported in a novel FRET based on GQD-PEG
aptamer/MoS2 for the detection of epithelial cell adhesion molecule (EpCAM),
which is a glycosylated membrane protein expressed on the surface of circulating
tumour cells (CTCs) [15]. In the mechanism, GQD is used as the FRET donor that
emits fluorescence at 466 nm under an excitation of 360 nm. MoS2 having a good
quenching ability is the acceptor. When the PEG is conjugated onto GQD, the
PEGylated/GQD exhibits a stronger fluorescence emission because of the
quantum confinement. Then the PEGylated/GQD is conjugated with the aptamer
via the van der Waals binding causing a proximity of GQD and MoS2 and
quenching GQD. When the EpCAM protein with a strong binding affinity is
introduced, the GQD could label on EpCAM aptamer and restores its
fluorescence. Therefore, the EpCAM target protein can be monitored by the
fluorescence emission.
2. FLUROSCENT BIOSENSORS USED FOR DETECTION-
OPTICAL BIOSENSORS

2.1 Fluorescent biosensors for detecting ions and small molecules

based on graphene and graphene-like materials
Since the pioneering research on the sensitive and specific determination of DNA
and proteins using the GO-based FRET strategy, a large number GO/ rGO-based
optical sensors have been designed with high sensitivities towards different
targets. GO-based FRET biosensors can also be employed for assay of metal ions.
The specific interactions between metal ions and DNA base pairs have increased
interest in establishing an innovative oligonucleotide-based metal ion detection
strategy. In particular, the specific interactions of Ag+ ions with cytosine–cytosine
(C– Ag+–C) and Hg2+ ions with thymine–thymine (T– Hg2+–T) have been
widely explored to fabricate a series of Ag+ and Hg2+ ion sensors. For example,
using a T-rich mercury-specific oligo nucleotide (MSO), Fanetal demonstrated a
GO- based FRET biosensorforthedetectionofHg2+ [5].The addition of Hg2+ leads
to the evolution of the stem-loop structure of the FAM-labelled MSO probe,
increasing the distance between the GO and the fluorophore, which is no longer
quenched by GO. The fluorescence intensity of the MSO provides a quantitative
readout for the Hg2+ concentration. This sensor exhibited excellent specificity
against various interfering metal ions and exhibited a detection limit of 30nM.
With a similar detection scheme, they also reported an Ag+ sensor using a silver-
specific oligonucleotide (SSO) probe that contains C-rich nucleic acids coupled
with GO (figure 1(A)). This sensor exhibited a high sensitivity with a low
detection limit of 5nM, which meets the requirement of the US Environmental
Protection Agency (EPA) for drinking water. Furthermore, the Ag+ possessed
satisfactory selectivity when other metal ions are added with a 10-fold increased
concentration. Other groups have also used this specific interaction and further
modified the sensor designs. Yang’s group replaced the traditional fluorophore
dye with graphene quantum dots (GQDs) to label oligo deoxy ribonucleotide, and
they developed a sensitive Hg2+ fluorescent sensor. The fluorescence detection of
Hg2+ based on DNA duplexes of poly(dT)and GO was also achieved by Li’ group
[20]. Furthermore, Sun et al demonstrated a sensitive turn on and label-free
fluorescent sensor for Hg2+ detection that employed [Ru (b p y)2(pip)]2+ as the
signal reporter and GO as the nano quencher. When the target was introduced, the
signal report was intercalated into the formed double-stranded DNA (dsDNA) via
the T–Hg2+–T base pairs, and the newly formed nanostructures were desorbed
from the GO surface, resulting in the recovery of the fluorescence of the[Ru(b p
y)2(pip)]2+. The coupling functional of enzymes with the GO based FRET
mechanism provides enormous opportunities to construct different types of
fluorescent sensors. As seen in figure5 (B), Fanetal investigated the interactions
between GO and an8–17 DNAzyme, and they fabricated a sensitive turn-on
fluorescent sensor for Pb2+ based on the fact that Pb2+ could specifically modulate
the interactions via cleavage of the 17S substrate. By taking advantage of the
preferable cleavage capacity of exonuclease III to dsDNA compared with single-
stranded DNA (ssDNA) and the excellent fluorescence quenching ability of GO,
Tang et al presented an Exo III-aided turn-on fluorescence mode for the sensitive
detection of Hg2+. Besides, to further improve the detection sensitivity, various
signal amplification routes combined with the GO-based FRET mechanism have
been introduced.

Figure5. A. Schematic illustration of the fluorescence sensor for Ag+ ions based on the target induced
conformational change of SSO and the interaction between the fluorogenic SSO probe and GO; [17] B.
Schematic for the Pb2+ modulated interactions between DNAzyme and GO. Inset is the fluorescence spectrum
of a mixture of DNAzyme and GO upon interaction with zero M and to mM of Pb 2+ in aTris -HCl buffer (50
mM, pH7.4) solution containing50 mM of NaCl; [16] C. Schematic of the proposed detection mechanism (for
clarity, pristine graphene issued to represent GO) [4]
2.2 Fluorescent biosensors for nucleic acid detection based on
graphene and graphene like materials
Because of the strong non covalent binding of GO with ssDNA, Lu et al for the
first time constructed a graphene platform to sensitively detect DNA. The strong
interaction between GO and the dye-labelled ssDNA induced the complete
fluorescence quenching of the dye. The introduction of a target DNA caused it
to hybridize with dye-labelled DNA, thereby altering the conformation of the dye-
labelled DNA and releasing the newly formed dsDNA from the surface of the GO.
Fluorescence restoration was observed and could be used as an off–on model to
sensitively detect the target DNA. As shown in figure 5(A), Fan’s group further
extended this GO-based sensing system to a multi-colour sensor for the
determination of multiple DNA targets in the same solution. As for the detailed
mechanism, molecular dynamics simulations were further employed to illustrate
the interaction between the ssDNA and the dsDNA with GO. They showed that
the ssDNA was stably adsorbed onto the surface of GO. This was attributed to the
π–π stacking interaction between the ring structures in the nucleobases and the
hexagonal cells of the graphene. In contrast, the dsDNA was desorbed from the
GO surface because the nucleobases were efficiently shielded within the densely
negatively charged phosphate backbone of the dsDNA. In another report, our
group claimed that the ssDNA constrained on the functionalized graphene can be
efficiently protected from DNAase cleavage, which can improve the specificity of
its response to the complementary DNA (figure 5(B)). To demonstrate the
versatility and further improve the sensitivity of this type of GO based FRET
DNA sensor, rational designs of signal probe have been widely reported. For
example, a molecular beacon (MB)provides new opportunities to detect DNA
targets with a high sensitivity and specificity due to their unique thermodynamics
and inherent structural constraint [14]. Incorporation of GO with a MB
significantly enhances the sensitivity of the FRET biosensor with a detection limit
of 0.1nM Moreover, this method can be used for detection of single-base
mismatched target DNA. It is worth noting that quantum dots (QDs) have also
been introduced into the GO-based FRET biosensor to form a GO/MB-QDs
sensing platform, which has displayed good selectivity and high sensitivity.
Additionally, Srivatsan et al developed GO-based FRET telomere sensor using a
fluorescent peptide nucleic acid probe. Conjugated polyelectrolytes were also
introduced into this GO-based FRET DNA sensor to enhance the detection
sensitivity.

Figure6. (A)Schematic demonstration of the GO-based multicolour DNA analysis [3] (B)Schematic
illustration of the constraint of DNA molecules on functionalized graphene and its effects. (I)The ssDNA can
be effectively constrained on the graphene surface via adsorption. (II)DNase I can digest free DNA but not
graphene bound DNA. (III)The constrained DNA shows improved specificity response to wards target
sequences that can distinguish the complementary and single-mismatch targets. [14]

The many studies on GO-based FRET biosensors have sparked new interest in
other graphene-like 2D layered nanomaterials. Similarly, the FRET mechanism
between the graphene-like nanomaterials and the dye-labelled ssDNA was also
utilized to fabricate a broad spectrum of DNA biosensors. Zhang et al reported
that a MoS2 nanosheet possessed a high fluorescence quenching ability, and they
successfully employed it as an ovel sensing platform to detect DNA and small
molecules [21]. The van der Waals force between the nucleobases and the basal
plane of the MoS2 triggers the fluorescence quenching of the dye.
The weak interaction between the dsDNA and the MoS2 nanosheet results in the
release of dsDNA and the recovery of the fluorescence signal. Thus, theMoS2
nanosheet exhibits great promise for the sensitive as say of DNA and small
molecules. Given the different types of TMDs, such as MoS2, TiS2 and TaS2, a
comparative study on their FRETDNA sensors was implemented. The results
indicated that the DNA sensor based on TaS2 nanosheets possessed the best
performance among the three TMD sensors [21]. Similarly, Ju et al systematically
studied the interaction between g-C3N4 nanosheets and DNA and proposed a
Universal FRET-based sensing strategy for the sensitive detection of DNA and
DNA-related targets, such as metal ions, small molecules and proteins (figure 6).
More importantly, a signal amplification method such as enzyme-mediated target
recycling can also be easily combined with this universal sensing platform to
further improve the detection sensitivity. By integrating this strategy with the
excellent fluorescence quenching ability of 2D graphene-like materials, several
research groups have also focused on the rational design of DNA fluorescent
sensing platforms with different signal amplification strategies. Jiang et al
proposed a highly sensitive and specific strategy for detection of microRNA using
WS2 nanosheets as the nano quenchers and duplex-specific nuclease (DSN)as the
signal amplification protocol. By taking advantage of this signal amplification
protocol, the researchers showed that this novel fluorescent sensor exhibited a
high sensitivity and specificity with a low detection limit of 300fM. With the
assistance of the hybridization chain reactions, a MoS2-basedamplified
fluorescence DNA sensor was also reported by the Li group [7].
 CONCLUSION

In this review, we discuss our points of view on the intrinsic properties of graphene and its
surface functionalization concerned with the transduction mechanisms in biomedical
applications. We also explain several well-known techniques used for the synthesis of
graphene-based materials and their properties. A variety of graphene-based materials have
been made consisting of pristine graphene and the functionalization of graphene oxide,
reduced graphene oxide and graphene quantum dot. The mechanisms are discussed with
respect to the most recent biosensing devices for drug delivery, biosensors, healthcare
sensors, bioimaging, and other novel techniques.

Due to their unique properties, graphene and a series of graphene-like 2D nanomaterials (e.g.,
graphitic carbon nitride, boron nitride, transition metal dichalcogenides and transition metal
oxides) have many applications in biosensors and nanomedicine. By taking advantage of their
unique planar structure, their intrinsic tunable optical properties, their high surface area and
their abundant chemical compositions and diverse biological effects, graphene and graphene-
like nanomaterials can be well designed to construct a wide range of optical sensing
platforms for the sensitive detection of various targets. In this review, we addressed the recent
progress in optical biosensors and imaging applications based on graphene and graphene-like
materials. Specifically, the graphene and graphene-like nanomaterials can be tailored to form
either a fluorescent emitter or an efficient fluorescence quencher, making them powerful
platforms for the fabrication of a series of optical biosensors to sensitively detect various
targets including ions, small biomolecules, DNA/RNA and proteins.

Graphene is one of the most well-known 2D materials. The major characteristic and
properties of graphene are outstanding, i.e., zero-bandgap semiconductor, linear-like at the
Dirac point, relativistic-like charge velocity, ultra-high charge mobility, transparency, large
surface area, non-toxicity, having proximity induced ability, high tensile strength and high
thermal conductivity, etc. However, to maintain those properties, graphene has to be perfectly
proper. Alternative techniques by chemical synthesis that serve for ambient circumstances are
also presented. In the synthetic procedures, the graphite is modified and functionalized by
various oxygen-containing groups. In addition, the functionalized graphene is always
contaminated by impurities, defects, and disorder. Hence, the structure of graphene would be
importantly changed, especially the electronic properties will be distorted. On the other hand,
the presence of the oxygen-containing groups and the ability to functionalize onto the
graphene-based structure are important in the electrochemistry of the biosensing applications
such as for labelling biomolecule recognition, for enhancing the sensing signal, for increasing
the number of active sites and active area, and for probing biomolecules in an imaging
application. A novel variety of the graphene-based biosensors is presented in the last section.
The engineering of biosensing platforms, the mechanisms and techniques are discussed.
Many new approaches are discussed in detail in this review. Since graphene material has
been very well established, other 2D materials have now been explored. This opens up a wide
range of possibilities and plays a crucial role in sensor and biosensor applications utilizing the
largest surface area. In the upcoming future, these 2D novel materials will be further
developed and tailored for specificity of bioreceptors. These materials can be employed and
integrated in different sensor and biosensor platforms giving an ultra-high sensitivity and may
provide a solution to some challenges, such as early stage cancer detection.
 FUTURE PERSPECTIVE

Although considerable attempts have been made, the development of functional graphene and
graphene-like nanomaterials and their exploration for biomedical applications still remains
grand challenges. First, simpler and more reliable synthetic approaches for the controllable
and reproducible preparation of grapheneandgraphene-like2Dnanomaterialsneedto be
established to precisely control the size, morphology, surface chemistry and composition. By
achieving this, the accurate tuning of their fluorescent behaviours is likely to be attained,
further optimizing their optical parameters. In particular, a better understanding of their
detailed emission mechanisms is critical and plays an important role in the design of high
performance 2Dnanomaterials. Additionally, the surface chemistry routes, including
modification and functionalization, are needed to further enhance their stabilities, optimize
their optical properties and minimize their toxicity. Besides, researchers should also focus on
the safety of graphene and graphene-like nanomaterials by studying their long-term toxicity,
cellular-uptake mechanisms and intracellular and in vivo metabolic pathways, which are
critical for applications in imaging, drug delivery and therapy. With the rapid development of
material fabrication approaches and characterization techniques, it is anticipated that major
advancements in graphene and graphene-like nanomaterials and their potential applications in
optical sensors and bioimaging will emerge in the future.
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