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Nanotechnology Reviews 2022; 11: 96–116
Review Article
Hassan A. Alhazmi, Waquar Ahsan*, Bharti Mangla, Shamama Javed, Mohd. Zaheen Hassan,
Mufarreh Asmari, Mohammed Al Bratty, and Asim Najmi
Graphene-based biosensors for disease
theranostics: Development, applications, and
recent advancements
https://doi.org/10.1515/ntrev-2022-0009 biosensors. Recent clinical trials and patents as well as
received September 8, 2021; accepted November 14, 2021 market trends and opportunities associated with graphene-
Abstract: Graphene, owing to its unique chemical struc- based biosensors are also summarized. The application of
ture and extraordinary chemical, electrical, thermal, optical, graphene-based biosensors in the detection of SARS-CoV-2
and mechanical properties, has opened up a new vista of causing COVID-19 is also reviewed.
applications, specifically as novel sensing platforms. The Keywords: graphene, biosensors, fabrication, functiona-
last decade has seen an extensive exploration of graphene lization, application, SARS-CoV-2, COVID-19, detection
and graphene-based materials either alone or modified with
nanoparticles and polymers for the fabrication of nanoscale
biosensors. These biosensors displayed excellent conduc-
tivity, high sensitivity, and selectivity, good accuracy, and 1 Introduction
precision, rapid detection with low detection limits as well as
long-term stability. The unmatched properties of graphene Diagnosis of diseases and their biomarkers requires accu-
and graphene-based materials have been applied for the rate and highly sensitive methods and to achieve it, a
detection of a number of chemical and biological molecules number of conventional and novel methods are available
successfully for the diagnosis of a variety of diseases, patho- [1,2]. Conventional methods include polymerase chain
gens, and biomarkers of the diseases. This review is aimed to reaction, lateral flow immunoassay, electrochemical methods,
cover the fabrication methods, functionalization techniques, DNA sequencing and microarrays and fluorescence micro-
and biomedical applications along with the recent advance- array, and enzyme-linked immunosorbent assay (ELISA)
ments in the field of development of graphene-based techniques [3,4]. These techniques however require highly
precise instruments, costly reagents, complicated sample
preparation steps, and tedious quantification methods in
order to achieve accurate and sensitive detection [5,6].
* Corresponding author: Waquar Ahsan, Department of In addition, these techniques have limitations when it
Pharmaceutical Chemistry, College of Pharmacy, Jazan University,
comes to detecting the disease in real-time. Novel methods
P.O. Box 114, Jazan, Saudi Arabia,
e-mail: wmohammad@jazanu.edu.sa, tel: +966-552144370
include the use of sensors that are comparatively inexpen-
Hassan A. Alhazmi: Department of Pharmaceutical Chemistry, sive, simple, and highly specific techniques for the detec-
College of Pharmacy, Jazan University, P.O. Box 114, Jazan, tion of target biomolecules. These sensors can be used in
Saudi Arabia; Substance Abuse and Toxicology Research Center, real-time to monitor and diagnose diseases and therefore
Jazan University, P.O. Box 114, Jazan, Saudi Arabia have broad clinical applications [7–10]. The added advan-
Bharti Mangla: Department of Pharmaceutics, School of
tages associated with sensors are their use in detecting the
Pharmaceutical Education and Research, Jamia Hamdard,
New Delhi 110062, India diseases at an early stage and requires minimal invasive
Shamama Javed: Department of Pharmaceutics, College of methods.
Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia These sensors are fabricated using nanomaterials
Mohd. Zaheen Hassan, Mufarreh Asmari: Department of that further improve their chemical and electrical proper-
Pharmaceutical Chemistry, College of Pharmacy, King Khalid
ties and therefore their sensitivity [10]. A number of
University, Abha, 61413, P.O. Box 9004, Saudi Arabia
Mohammed Al Bratty, Asim Najmi: Department of Pharmaceutical
nanomaterials are being used to fabricate these sensors,
Chemistry, College of Pharmacy, Jazan University, P.O. Box 114, and graphene and graphene-based nanomaterials have
Jazan, Saudi Arabia shown exceptional properties with enhanced signal
Open Access. © 2022 Hassan A. Alhazmi et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0
International License.
Recent applications of graphene-based biosensing 97
detection [11–13]. Graphene-based nanomaterials also Recent studies concerning graphene-based biosensors
support the incorporation of a number of biological mole- including the application in the COVID-19 testing along
cules such as proteins, enzymes, antibodies, DNA, and with the clinical trials and patents are also summarized.
cells owing to their excellent biocompatibility with these
molecules [13]. Sensors immobilized with biological mole-
cules are called biosensors and are used in the detection of
various molecules and cells [14,15]. 2 Development of graphene-based
Graphene, first discovered in 2004, is a carbon-based
nanomaterial arranged in form of a single layer of sp2
biosensors
carbon sheet having atoms arranged in a honeycomb lat-
tice [16,17]. It possesses a number of unique properties 2.1 Fabrication methods
including high intrinsic mobility, unequaled flexibility,
large surface area, and excellent electronic transporta- There exists an industrial revolution with respect to gra-
tion capabilities. The two-dimensional structure of gra- phene and its utilization into several products around us
phene allows its functionalization with a number of including drugs and their delivery, medical devices, as
linker molecules making it a highly sensitive and selec- well as many products and devices that improve the
tive candidate for biosensing applications [18–20]. Gra- quality of life. Generally, graphene is produced using
phene is available in a number of forms and each form two main strategies and each strategy includes different
has its own properties and applications. These forms fabrication methods. In a top-down strategy, graphene
include the few-layer graphene, multilayer graphene, derivatives can be obtained from any carbon source
graphene oxide (GO), reduced graphene oxide (rGO), such as graphite flakes or powder that is exfoliated
and graphene nanoplatelets (GNP), of which GO and mechanically or electrochemically and is subjected to
rGO are used for biosensing applications [21–27]. chemical oxidation–reduction reactions. For instance,
Apart from the biological applications, graphene-based scotch tape techniques, sonication in a liquid phase,
sensors are also used in nonmedical fields including nano- Hummers method, Brodie methods, etc., are the top-
electronics, optoelectronics, nanocomposites, supercapaci- down techniques used in the fabrication of graphene
tors, field-effect transistors (FET), solar cells, pH sensors as derivatives. In contrast, the bottom-up strategy depends
well as gas sensors [28–30]. Graphene offers ultrahigh on the synthesis of graphene layers from the carbon atom
loading capacity for the biomolecules and drugs along bases and involves numerous fabrication methods such
with strong adsorptive capabilities and high mechanical as chemical vapor deposition, epitaxial growth, thermal
strength, making it one of the most widely used nano- pyrolysis, etc. All these fabrication techniques have their
materials for the development of biosensors. Since every own advantages and disadvantages [33–37]. Fabrication
atom of graphene is present on the surface, the molecular is a critical step in the development of graphene-based
interaction and transportation of electrons from the biosensors as it can influence the nature of graphene.
adsorbed molecules through graphene are easy [31,32] Graphene and its derivatives are known to be excellent
and result in the ultrasensitive detection of biomolecules. supporting materials that have been exploited exten-
Graphene-based biosensors have opened up new vistas for sively in the development of biosensors. A typical bio-
the early diagnosis of a number of life-threatening condi- sensor consists of two layers of receptors and transducers
tions and severe diseases along with real-time health mon- that are attached to each other, as shown in Figure 1.
itoring. For instance, several graphene biosensors showed
a successful diagnosis of various cancer forms and cardio-
vascular diseases by detecting their biomarkers. Real-time
monitoring of disease would help change the quality of life
of the patients, and the graphene-based electrochemical
biosensors showed immense potential for implantable
devices that would detect the biomarkers in real-time
assessing the severity of the disease.
In this review, the development of graphene-based
biosensors with their biomedical applications is discussed.
The fabrication and immobilization methods used in the Figure 1: A typical biosensor device with graphene layer as a
development of graphene-based biosensors are reviewed. transducer and biomolecules as a receptor.
98 Hassan A. Alhazmi et al.
The upper layer is made of receptors that are com- this method, the ink solution is prepared and sprayed using
posed of biomolecules such as enzymes, proteins, DNA, various techniques including screen-printing, inkjet printing,
or antibodies with specific biorecognition to the targeted nozzle-jet printing, and laser scribing printing. Generally,
analytes. These receptor molecules that are attached to these printing techniques are similar to each other in principle
the transducer layer are very sensitive to physicochem- with small variations; for instance, screen printing is used for
ical changes upon biomolecular interactions and are cap- a thicker nanofilm and is commonly employed for industrial-
able to convert them into measurable signals [38–40]. scale fabrication due to its simplicity and utilization of pre-
Herein, graphene and its derivatives work as transducers fabricated mesh-covered frames to allow the transfer of the
for these signals that can be detected electrochemically, desired pattern to the electrode surface. Direct spay of ink
optically, or thermally. Therefore, graphene-based biosen- solution can be achieved using inkjet printing that exhibits
sors are fabricated according to the detection technique, and more controlled drop size and high-resolution pattern, while
electrochemical biosensors are more commonly employed in nozzle-jet printing is similar to inkjet printing with the addi-
comparison to optical biosensors [41,42]. tion of external pressure. Therefore, nozzle-jet printing is sui-
In the electrochemical biosensors, the designed sen- table for more viscous ink solutions. Meanwhile, laser scribing
sors usually consist of a three-electrode system: working printing is an advanced technique with high fabrication flex-
electrode, counter electrode also known as the auxiliary ibility for many substrates and supporting materials as well as
electrode, and the reference electrode. These electrodes small size sensors. However, printing using instrumental
are coated with graphene-based nanomaterials using fab- techniques require special handling skills and need prepara-
rication methodologies including coating, direct growth tion of ink solution critically.
methods, direct deposition methods, and printing-based Another method of fabrication of biosensors is the
methods. Direct coating is the most commonly employed direct growth of nanomaterials on the surface of elec-
technique for the fabrication of biosensors due to its sim- trodes. This method gained more attention recently due
plicity, cost-effectiveness, and it does not require any spe- to several controllable parameters during fabrication such
cific instrument [40]. Usually, a solution/gel containing as time, temperature, pH, pressure, and concentration.
graphene or graphene derivatives is used to coat the sur- This strategy depends on the recruitment of several experi-
face of electrodes using methods such as drop-casting, dip mental environments to catalyze the nanomaterial growth
coating, spin coating, and blade coating. Each coating on the electrode surface via numerous techniques such as
technique has its advantages and disadvantages, and thermal, hydrothermal decomposition, anodization, and
therefore, the choice of the technique depends on the chemical decomposition [56,57]. However, limited atten-
desired film properties such as thickness, uniformity, tion to these methods is given for the fabrication of gra-
and surface area. Generally, the direct coating method is phene-based biosensors as it involves limited direct
the first choice for biosensors; however, it involves multi- growth to the electrode surface.
step fabrication, material wasting, and is a time-con-
suming process [43–47]. Direct deposition-based methods
involve direct deposition of graphene and other nanoma-
terials on the electrode surface by dipping the electrodes in 2.2 Functionalization methods
a solution containing graphene and applying an electrical
voltage to control the coating. The electrospinning tech- Due to its unique electro- and thermomechanical proper-
nique involves the spinning of a nozzle jet containing the ties, applications of graphene are practically limitless. It
graphene nanomaterial onto the electrode surface. The showed excellent potential in transforming many areas
electrospray deposition method is similar to electrospin- ranging from electronics to healthcare such as touch
ning deposition; however, it offers control of droplet size, screens, sensors, biofunctionalized graphene, quantum
charge, and speed, and therefore, is utilized in industrial- dots, novel drug delivery, nanodevices for DNA sequen-
scale fabrication with high precision [48–51]. cing, etc. [58]. The unique molecular structure of gra-
Subsequently, printing-based fabrication methods were phene characterized by sp2-hybridized carbons, large
introduced with the revolution of 3D printers. Printing tech- specific-surface area (2,630 m2 g−1) as well as strong van
niques are very attractive because of many reasons including der Waal cohesive forces render graphene to agglomerate
large-scale fabrication, are cost-saving, and require low tem- easily and prevent its uniform dispersion [59]. Moreover,
peratures and solution ink. These methods fit properly with graphene sheets have poor water solubility because of
graphene-based biosensors and therefore are exploited exten- the strong π–π interactions between the sheets [60].
sively in the development of various biosensors [52–55]. In To address this, the functionalization of graphene is
Recent applications of graphene-based biosensing 99
performed in order to redesign its electronic, physical, and covalent surface modifications is also known as chemi-
chemical properties [61]. To date, functionalization is the sorption (grafting) of molecules on graphene lattice [68].
only effective technique that helps in reducing the cohe- This rehybridization of the π-conjugated carbon network
sive forces between graphene sheets and thus prevents its often forms hybrid-graphene materials with chromophores
agglomeration without losing its inherent properties [62]. or polymers that enhance the dispersibility of graphene
Functionalization involves the process of adding new [69]. Figure 2 illustrates different covalent functionaliza-
functions, characteristics, potentials, or properties to gra- tion techniques employed in the development of gra-
phene by changing its surface chemistry [63]. Functiona- phene-based biosensors.
lization modifies inert graphene sheets and is exception-
ally effective in fabricating sensors that have huge biomedical,
electrochemical, and diagnostic applications [64]. Functiona- 2.2.1.1 Free radical addition (FRA)
lization of pristine graphene through the covalent and non-
covalent functionalization provides a multitude of chemically Free radicals are extremely reactive uncharged chemical
activated, soluble, hybrid graphene [65] surfaces. Covalent species that contain an unpaired electron. They can react
functionalization forms a stable covalent bond, whereas the readily with the compounds containing multiple bonds to
noncovalent functionalization is formed through hydrogen produce another radical, which reacts further and goes
bonds, π–π interactions, and van der Waals interactions [66]. on [70]. FRA can be achieved through one or more syn-
thetic approaches such as aryl diazonium salts [71], per-
oxides, Bergman cyclization [72], and the Kolbe–Schmitt
2.2.1 Covalent functionalization reaction [73]. Among these, aryl diazonium salts are the
most studied method for stabilizing graphene layer with
Covalent functionalization of graphene involves rehybri- enormous applications in the development of semicon-
dization of sp2 C-atoms into the sp3-hybridized tetrahedral ducting nanomaterials, atom transfer radical polymerization,
configuration, chiefly at the edge [67]. This process of coupling reactions through click chemistry, grafting of
Figure 2: Covalent functionalization of graphene sheets through different synthetic protocols.100 Hassan A. Alhazmi et al.
heterostructures, and tuning of electrical conductivity [74]. substitutes the atom attached to the aromatic ring; usually,
The delocalized π-electrons of the graphene cage are shifted a hydrogen atom is replaced by an electrophile [89]. This
to electron-deficient diazonium electrophile and eliminate its reaction has gained much attention in the field of covalent
N2 molecule thereby forming a highly reactive aryl free functionalization of graphene because ESR can introduce a
radical that eventually reacts with the carbon atom of the wide range of functional groups on the graphene surface
graphene lattice and forms a covalent bond [75]. Functionali- and thus is a useful tool for tailoring the graphene proper-
zation through benzoyl peroxide (BPO) is another common ties [90]. Some of the hitherto reported ESR functionaliza-
approach for the FRA. BPO has been widely utilized as an tion includes halogenation [91], nitration [92], sulfonation
important organic peroxide initiator because of its easily acces- [93], Friedel–Crafts acylation, alkylation [94] reactions, etc.
sible benzoyloxy radicals after homolytic fission [76,77]. The
functionalization of graphene by Bergman cyclization has
many advantages including simple steps, superior effi- 2.2.1.5 Addition of chromophores
cacy, tailored structure, and catalyst-free procedure [78].
Organic compounds having extended π-system such as
2.2.1.2 Nucleophilic addition porphyrins [95], phthalocyanines [96], azobenzenes [97],
and other chromophores were identified as potential can-
The nucleophilic addition reaction is widely employed in didates for preparing hybrid graphene nanoplates with
the functionalization of pristine graphene sheets due to the superior optical limiting property. Covalently functio-
its electron acceptor properties [79]. Recently, a soluble nalized soluble hybrid porphyrin–graphene was prepared
charm-bracelet-type poly-(N-vinylcarbazole) functionalized by the reaction of 5-4(aminophenyl)-10,15,20-triphenyl
graphene sheet has been developed by the reaction of carba- porphyrin and GO in N,N-dimethylformamide (DMF). The
nion intermediate of poly(N-vinylcarbazole) and graphene [80]. amide linkage in the hybrid porphyrin–graphene signifi-
In another study, covalent modification of rGO was performed cantly improved the dispersion and thereby solubility of
through the nucleophilic addition reaction using nitrogen graphene in organic solvents. Moreover, this donor–ac-
anions, formed by sodium hydride. The π–π interaction and ceptor nanohybrid also exhibited superior optical limiting
“polymer wrapping” effect between the polymers and gra- performance due to photoinduced electron and/or energy
phene resulted in improved dispersion of graphene [81]. transfer [98]. Table 1 summarizes different covalent func-
tionalization methods used in the development of gra-
phene-based biosensors.
2.2.1.3 Cycloaddition reaction
The cycloaddition reaction involves the formation of a 2.2.2 Noncovalent functionalization
new ring by the σ bonds through the reaction of two
π-electron systems [82]. The aromatic properties of gra- Noncovalent interactions are reversible interactions between
phene is exploited through a number of cycloaddition the graphene and organic molecules or polymers without
reactions involving 1,3 dipolar cycloadditions, [2 + 2] disruption of the delocalized π-system of graphene and
cycloadditions, [2 + 1] cycloadditions, and Diels–Alder therefore its electronic properties [99]. Physical forces such
reaction [83–86]. Diels–Alder reaction is one of the pro- as hydrophobic, van der Waals, and electrostatic forces are
mising methods for the modification of pristine graphene the major forms of interactions utilized in the noncovalent
because of its click-type procedure, high efficiency, ver- functionalization [100]. Numerous studies reported the
satility, and efficiency [87]. Dihydronaphthalene-grafted immobilization of proteins, DNA–protein complexes, enzyme–
graphene was designed using cis-diene and the resulting drug complexes, functional nanomaterials, and organic
modified graphene showed a p-type doping effect with supramolecules using a noncovalent functionalization tech-
improved conductivity that can be used for making trans- nique [101]. In recent years, enormous advancements were
parent electrodes [88]. made in terms of functionalization of graphene through π–π
stacking with polyaromatic compounds such as naphthalene
[102], pyrene, 4-n-octyl-4′-cyanobiphenyl, tetrafluoro-tetra-
2.2.1.4 Electrophilic substitution reactions (ESRs) cyanoquinodimethane [103], 3,4,9,10-perylenetetracarboxylic
diimidebisbenzenesulfonic acid [104], pyridinium-functiona-
Electrophilic aromatic substitution reactions are very fas- lized porphyrin, [105] etc. Noncovalent immobilization of
cinating versatile organic reactions where an electrophile enzymes on graphene surfaces was also reported to haveRecent applications of graphene-based biosensing 101
References
improved biocatalytic efficiency; for instance, immobilization
[83,84]
of glucose oxidase and glucoamylase enzyme for one-pot
[76,77]
conversion of starch into gluconic acid [106]. Highly stable
[98]
[94]
[78]
[74]
[79]
and water-soluble gold nanoparticles (Au-NPs) of DNA-deco-
High temperature required that is often
rated graphene nanosheets were also reported to be a pro-
Variations in the reactivity of polymers
incompatible with organic substrates
Localized functionalization and a low
mising approach to design a 2D-conductance device for DNA
Polyalkylated products are formed
sequencing (Figure 3) [107].
and their dispersion stability
3 Biomedical applications of
Self-polymerization
graphene-based biosensors
Disadvantages
efficiency
Traditional sensing methods are expensive, require high-
precision equipment and costly reagents, and the majority
—
—
of reactions are not quantifiable in real-time. Graphene-
p-Type doping effect and improved conductivity to graphene,
Solar-energy conversion materials for optoelectronic devices
based sensors are now being used as an alternative method
The electrical conductivity of graphene decreased and the
for the identification of disease-related biomolecules and
Atom transfer radical polymerization, voltammetric
they offer a wide range of biomedical applications.
In the preparation of graphene–polymer interface
Graphene biosensors are easy-to-use, cost-effective, non-
immunosensor, antistatic coatings electrode,
supercapacitor, photoactive graphene sheet,
Carbon-rich nanoparticles/networks, carbon
toxic, and are equipped with excellent sensing properties
[108]. Generally, a sensor consists of two elements: receptor
(linked with target molecule) and transducer (converts che-
Photonic and optoelectronic devices
nanomembranes, and nanodevices
mical information into signals). A graphene or GO-based
biosensor acts as a transducer converting the receptor–
hole-doping level increased
target molecule interaction into a detectable signal. Biore-
transparent electrodes
ceptors such as antibodies, enzymes, or nucleic acid are
usually immobilized to the transducers in order to allow
nanocomposites
Table 1: Summary of functionalization methods of graphene through covalent bonding
target molecules to interact (Figure 4) [12,109].
Applications
3.1 Detection of microbes
chlorobenzene, or polystyrene
Antibodies, nucleic acids, proteins, and enzymes are
Reaction condition/reagents
prophyrin (TPP-NH2), DMF
Aryl diazonium molecules
immobilized to graphene biosensors using various
Nucleophile and base
Amine-functionalized
Ar-ion laser beam
Methylbenzene,
cis-Diene
Enediyne
Addition of chromophores
Friedel–Crafts reaction
Nucleophilic addition
Aryl diazonium salts
Diels–Alder reaction
Bergman cyclization
Types of covalent
functionalization
BPO
Figure 3: Some common noncovalent functionalization showing
S.No.
pyrene–graphene, glucose oxidase–graphene, and
ssDNA–graphene hybrids.
6
4
2
3
7
5
1102 Hassan A. Alhazmi et al.
Figure 4: Applications of biomolecules-immobilized graphene-based biosensors in the detection of target molecules.
processes, which can be identified using spectroscopic typhimurium antibody for S. typhimurium bacteria, and
methods [15]. Attachment of antibodies to the graphene anti-E. coli O157:H7 antibody for the detection of E. coli.
surface is mainly applied for the detection of infectious Moreover, the Dengue virus and rotavirus have also been
diseases caused by viruses and bacteria. Table 2 summarizes identified using antibodies immobilized to GO biosensors
the studies on graphene-based biosensors used for the [122–124]. Rotavirus and G2 monoclonal antibodies were
detection of microbes including bacteria and viruses. Gra- used in these techniques that bind to the graphene nano-
phene biosensors have been successfully applied for the material using a carbodiimide-assisted amidation reac-
detection of Ebola virus [110,111], Escherichia coli [112], and tion and an electrostatic bond. Another promising break-
Zika virus [113], whereas graphene biosensors modified with through was achieved when graphene quantum dots
silver and gold nanoparticles were developed for the detec- were prepared that were successfully applied for the
tion of Salmonella typhimurium [114], hepatitis-C virus (HCV) highly sensitive detection of hepatitis B and adenovirus
[115,116], and avian influenza virus H7 [117]. Modifications of [125–127].
graphene biosensors have further been proven to be effective
in the detection of diseases as dendrimers, polymers, and
cyclodextrin modifications could all be used to diagnose 3.2 Detection of nucleic acids and genes
celiac disease [118], human immunodeficiency virus (HIV)
[119,120], and cholera toxin [121]. The DNA-based graphene biosensor helps in the detec-
Different types of antibodies are immobilized on the tion of various types of biomarkers (DNA, RNA, small
graphene surface and are being used for the detection of molecules, proteins), viruses, and genes using electro-
target molecules. For instance, PAC1 is used for the diag- chemical and fluorescent detection techniques (Table 3).
nosis of cardiovascular diseases, anti-GHRL and anti-PYY In the electrochemical approach, the immobilization of
antibodies for hormone detection, an anti-tTG antibody DNA is achieved using covalent bonds, π–π interactions,
for celiac diseases, an anti-HCV antibody for Hepatitis C or EDC/NHS chemistry on the surface of graphene biosen-
virus, anti-CT for cholera toxin, anti-rotavirus antibodies sors. Electrochemical signals are generated when DNA is
for rotavirus detection, monoclonal antibodies (H5N1, hybridized or oxidized. Differential pulse voltammetry, CV,
H1N1, H7) for avian influenza virus H7 and influenza and electrochemical impedance spectroscopy were used to
A virus, anti-Zika NS1 antibody for Zika virus, anti-S. quantify voltage and current shifts triggered by a numberRecent applications of graphene-based biosensing 103
Table 2: Graphene-based biosensors for the detection of pathogens
Target Immunosensor design Detection methods Detection limit Refs
−1
Ebola virus rGO Electrochemical 2.4 pg mL and [110,111]
1 μg mL−1
E. coli rGO Electrical 103 CFU mL−1 [112]
Zika virus Graphene Electrical 0.45 nM [113]
S. typhimurium GO–AgNPs nanocomposite Cyclic voltammetry (CV) 10 CFU mL−1 [114]
Hepatitis C virus (HCV) rGO, rGO/CuNPs Optical, electrochemical 10 fM, 0.4 nM [115,116]
Influenza A virus Graphene oxide–MB–chitosan Electrochemical 9.4 and 8.3 pM [117]
Celiac disease GQDs on AuNPs with polyamidoamine Electrochemical 0.1 fg per 6 µL [118]
dendrimer embedded on MWCNTs
HIV G/CVD, GO/PANi Electrochemical 0.1 ng mL−1, 100 aM [119,120]
Cholera toxin Graphene–polypyrrole Surface plasmon 4 pg mL−1 [121]
resonance
Dengue virus rGO, rGO/PAMAM Optical 0.08 pM [122,123]
Rotavirus GO Photoluminescence 105 PFU mL−1 [124]
Hepatitis B virus rGO/AuNPs, Gquantum dots Electrochemical 3.8 ng mL−1, 1 nM [125,126]
Adenovirus Graphene quantum dots Optoelectronic 8.75 PFU mL−1 [127]
Human MWCNT-NH2-IL-rGO Electrochemical 1.3 nM [128]
papillomavirus (HPV)
HCV Magnetic rGO–CuNCs Electrochemical 405.0 pM [129]
E. coli O157 APTMS–ZnO/c-GO Electrochemical 0.1 fM [130]
B. anthracis GO Fluorescence 0.625 µM [131]
S. aureus Graphene–AuNPs Surface acoustic wave 12.4 pg mL−1 [132]
Porcine epidemic AuNP–MoS2–rGO Electrochemical — [133]
diarrhea virus
E. coli O157 AuNPs–Graphene Electrochemical 102 CFU mL−1 [134]
E. coli K12 AuNPs–Graphene Electrochemical 12 CFU mL−1 [135]
of factors, such as conductivity changes or electron deple- peroxide, caffeic acid, glucose, bilirubin, and 17β-estradiol
tion caused by oxidation or hybridization. In the fluores- have been detected by enzyme-immobilized graphene biosen-
cence approach, the immobilization of DNA can be achieved sors (Table 4).
through π–π interaction (direct adsorption of the DNA probe
on the biosensor). This is based on the hybridization of two
single-stranded DNA (ssDNA); one strand being fluores- 3.4 Detection of severe acute respiratory
cently labeled while the other is the complementary DNA syndrome-coronavirus-2 (SARS-CoV-2)
to the target DNA.
Recently, graphene biosensors were applied in the devel-
opment of point-of-care (POC) testing (POCT) devices for
3.3 Detection of enzymes and other the detection of coronavirus disease-19 (COVID-19), an
molecules ongoing pandemic the world is suffering from. The rapid
and sensitive detection of SARS-CoV-2, the causative
Similarly, a number of enzymes and related biomolecules pathogen of COVID-19, is an ongoing global challenge
such as horseradish peroxidase, laccase, glucose, and that is associated with large-scale diagnosis in order to
bilirubin oxidase were also immobilized onto the graphene downregulate its spread within the communities. It demands
biosensor via covalent bonding, physical entrapment, early identification of infection in presymptomatic and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/ asymptomatic individuals [162,163]. There remains an urgent
N-hydroxy sulfosuccinimide (EDC/NHS) chemistry, and adsorp- need for a rapid and precise diagnostic method for the
tion methods. These types of sensors are based on two detection and screening of the disease. The graphene-
main mechanisms: catalytic properties of the enzymes based biosensors have attracted much attention in the out-
and enzyme activity inhibition/moderation. Different break owing to their extraordinary properties [164] and
molecules such as phenols (2,6-dimethozyphenol), hydrogen have emerged as a successful application tool to detect104 Hassan A. Alhazmi et al.
[139,140]
COVID-19. Antibody-conjugated graphene sheets could
[144,145]
rapidly detect target viral proteins and were used for
[148]
[146]
[149]
[138]
[120]
[136]
[142]
[143]
[147]
[150]
[137]
[141]
Refs
large-scale screening and development of biosensors [165].
The available conventional technology such as RT-PCR is
4.28 × 10−19 M and 1.58 × 10−13 M
time-consuming, labor-intensive, and scarcely available in
remote areas. These POC biosensors are typically low-cost,
user-friendly, and have remarkable potential as medical
diagnostics [166]. These biosensors are now a future diag-
600 zM and 20 aM
nostic approach for COVID-19 as clinical diagnostics [167]
Detection limit
3.2 × 10−21 M
1.0 × 10−16 M
owing to their accuracy, affordability, and portability
0.00625 nM
200 nM and
[168]. The introduction of nanomaterials unquestionably
2.02 µM
14.3 nM
600 zM
enhanced the performance of graphene biosensors, and
32 nM
0.1 fM
24 fM
15 fM
the sensing abilities of these biosensors are of a peerless
—
level of ultrasensitivity [169]. Table 5 summarizes the gra-
Electrochemical detection
Electrochemical detection
Electrochemical detection
Electrochemical detection
Electrochemical detection
Electrochemical detection
Electrochemical detection
phene-based biosensors developed so far in the point-of-
Fluorescence detection
Fluorescence detection
Fluorescence detection
Fluorescence detection
care testing (POCT) for COVID-19.
Detection methods
The breakthrough in the discovery of graphene-based
Electrochemical
biosensors for the diagnosis of COVID-19 was made when a
Electrical
Electrical
graphene-based electrochemical biosensor coupled with
an electrical readout setup was developed, which was
selectively able to detect the SARS-CoV-2 genetic material
[162]. The biosensor was made selective by integrating
thiol-modified antisense oligonucleotides (ssDNA), which
Thionine functionalized rGO (Thi–rGO) and rGO-graphene double-layer
were specific for the N-gene (nucleocapsid phosphopro-
tein) of SARS-CoV-2. The sensitivity of the detector was
further enhanced when the thiol-modified ssDNA-capped
gold nanoparticles (AuNPs) were applied on the gold elec-
Zirconia reduced graphene oxide–thionine (ZrO2–rGO–Thi)
trode. Similarly, a wireless graphene-based telemedicine
Glassy carbon electrode modified with rGO (GCE/RGO)
Pencil graphite electrode modified with GO (GO/PGE)
platform known as the SARS-CoV-2 RapidPlex was devel-
Table 3: Graphene-based biosensors for the detection of nucleic acids and genes
Armchair graphene nanoribbon-Au electrode- FET
oped, which could successfully detect the nucleocapsid
protein (NP) as well as specific immunoglobulins against
Graphene field-effect transistor biosensor
the spike protein (S1) (S1-IgG and S1-IgM), in both blood
Deformed monolayer graphene channel
and saliva of the patients [163]. The detector consisted of
laser-engraved graphene sensor arrays that were proved to
Polyaniline/graphene biosensor
be a highly convenient, rapid, accurate, and stage-specific
AuNPs–GO nanocomposite
GO ethidium bromide (EB)
tool for the detection of the virus. 1-Pyrene butyric acid
Crumpled graphene FET
(PBA) was utilized as the linker to attach the receptors
Immunosensor design
onto the graphene surface.
GO–DNA sensor
In another study, the SARS-CoV-2 spike protein anti-
body was immobilized onto the fabricated graphene-based
electrode
device using 1-pyrene butyric acid N-hydroxysuccinimide
ester (PBASE) as an interface coupling agent [170]. The
GO
GO
developed biosensor showed an excellent LOD of 1 fg mL−1
DNA and exonuclease activity
Hepatitis B virus (HBV) gene
T antigen gene of SV40 DNA
of the viral spike protein. The sensitivity of the biosensor
Staphylococcus aureus DNA
Amelogenin gene (AMEL)
was assessed using a control experiment that showed that
the spike protein was essential for specific binding with
DNA hybridization
the viral antigen. The selectivity was confirmed when the
RNA HIV-1 gene
Nucleic acids
Nucleic acids
developed COVID-19 FET did not exhibit any response to
miRNA let-7b
HIV-1 gene
HIV-1 gene
MERS-CoV spike proteins. Recently, another graphene-
ssDNA
ssDNA
Target
based FET device was developed as a portable bifunctional
electrical detector through either nucleic acid hybridizationRecent applications of graphene-based biosensing 105
or antigen–antibody protein interaction [178]. The devel-
[153,154]
[156,157]
oped biosensor showed ultra-low detection limits in the
[160]
[158]
[159]
[152]
[161]
[155]
[151]
Refs
range of 0.1–1 fg mL−1 and was able to detect the pathogen
in real samples. The biosensor was fabricated using PBASE
0.3–6 mM and 0.07–1.10 mM
in acetonitrile and was exposed to ssDNA probe or antigen
protein.
0.011, 0.006, 0.31 µM
0.2 μM to 1.1 mM and
0.085–209.7 µM
Detection limit
0.38–100 μM
0.01–12 mM
respectively
4 Patents and clinical trials on
0.9–11 pM
124.19 µM
1 fg mL−1
7.5 mM
graphene-based biosensors
A number of patents were granted worldwide on gra-
Electrochemical detection
Electrochemical detection
Electrochemical detection
Electrochemical detection
Amperometric detection
phene-based biosensor devices and the methods of manu-
facturing. The first patent was granted in the year 2010, a
Detection methods
Electrochemical
Electrochemical
Electrochemical
Electrochemical
few years after the discovery of graphene in which the
configuration of graphene device was described [179].
The patented device consisted of two electrodes, three
layered structures (conductive, insulating, and graphene),
and a superconducting dopant island. The graphene layer
Lactate dehydrogenase immobilized on the active graphene surface using
was adsorbed to the electrodes. In between the electrodes,
3D graphene/methylene blue-carbon nanotubes and calcium carbonate
the dopant island was coupled to an exposed surface of
CVD-graphene on SiO2/Si substrate followed by deposition of Nafion
the graphene layer. This device was applied in the che-
mical detection by applying a voltage to the conductive
(CaCO3) microspheres encapsulated with graphene capsule
layer and observing the voltage response of the dopant
island. Subsequently, in 2011, a worldwide patent was
Molybdenum disulfide and graphene quantum dots
granted on the graphene-based biosensor in which the
L-Aspartic acid-modified CVD graphene electrode
Table 4: Graphene-based biosensors for the detection of enzymes and other molecules
method of preparation of the graphene biosensor was
described that helped to detect biological molecules [180].
3D graphene and 3D GO and polyanilline
This sensor consisted of the graphene structure (thickness
Nafion, chitosan, and glutaraldehyde
of about 10 atomic layers of graphene), two electric contacts
or micro electrodes (that determine the conductivity), and at
Graphene/cellulose microfiber
least one linker (one of the linkers has a biological molecule
GO–rhodium nanoparticles
GO–NP embodied complex
binding affinity). The patterned graphene structure was
Immunosensor design
composed of at least one (three-dimensional) channel of
about 100 μm and two microelectrodes were arranged on
opposite sides of the channel. The linker could be an aniline
group, amino group, or a diazonium salt that could be
changed according to the biological sample to be tested.
The change in conductivity in the prepared sensor mea-
sured the biological molecule.
Hydrogen peroxide with horseradish
This led to an increase in the number of patents in the
following years on graphene-based biosensors owing to a
Lung cancer biomarker (CD59)
Glucose with glucose oxidase
Acetaminophen, Epinephrine,
much expected attraction of scientists worldwide. In
17β-Estradiol with laccase
Caffeic acid with laccase
2013, a US patent was granted in which the graphene
Catechol with laccase
Target with enzymes
biosensor was prepared using graphene-coated electrodes
that were linked to one to six layers of the flexible
Glucose oxidase
substrate through the enzyme lactate oxidase (biosensing
L-Lactic acid
peroxidase
element) [181]. Graphene electrodes consisted of two term-
tyrosine
inals: negative (second end) and positive (first end) term-
inals. Once the analyte in the sample is in contact with theTable 5: Portable biosensors in POCT for COVID-19
106
Type of graphene Technology/nanotechnology used Time of detection Limit of detection (LOD) Outcomes of the study Reference
biosensor
Quantitative paper-based AuNPs capped with highly specific Less than 5 min Significant amplification in the The developed sensor showed [162]
electrochemical antisense oligonucleotides (ssDNA) output signal in the presence of almost 100% accuracy, specificity,
sensor chip aimed to target nucleocapsid SARS-CoV-2-RNA; Sensitivity: 231 and sensitivity
phosphoprotein (N-gene) (copies μL–1)−1; LOD: 6.9 copies μL−1
Multiplexed, portable, Laser engraved graphene electrodes — — The developed sensor could detect [163]
wireless electrochemical viral antigen N-protein, as well as
biosensor IgM and IgG antibodies along with
Hassan A. Alhazmi et al.
the C-reactive protein (CRP), the
inflammatory biomarker
FET-based biosensor Graphene sheets of the FET were — FET device could detect SARS-CoV-2; Successful fabrication of the FET [170]
coated with specific SARS-CoV-2 LOD (in the culture medium): 1.6 × biosensor as a highly sensitive
monoclonal antibodies against S- 101 Pfu mL−1; LOD (in clinical immunological diagnostic method
protein samples): 2.42 × 102 copies mL−1
A novel biosensor based on Human chimeric spike S1 antibody Ultra-rapid manner (3 min) 1 fg mL−1 Configured biosensor could be [171]
bioelectric recognition immobilized on membrane- applied as a ready-to-use tool in
assay engineered mammalian cells the mass screening of SARS-CoV-2
antigens. No sample processing
step is required
CRISPR-based assay Based on CRISPR complex (Cas12a/ ∼50 min LOD of 2 copies per sample Results demonstrated rapid [172]
gRNA) attached to a fluorescent probe analytical sensitivity and robust
that could detect targeted amplicons diagnostic performance to improve
produced by RT-PCR the current COVID-19 screening.
Simple immunosensor Utilizes the dual-labeled magnetic Rapid quantification (Table 5: Continued
Type of graphene Technology/nanotechnology used Time of detection Limit of detection (LOD) Outcomes of the study Reference
biosensor
Voltammetric genosensor Hexathia-18-crown-6 (HT18C6) — Concentration range: 1.0 pM to Detection of SARS-CoV-2 in human [176]
modified with Ag + ions used as redox 8.0 nM; LOD: 0.3 pM. Against sputum
probe; carbon paste electrode (CPE) SARS-CoV-2 RdRP
coated with HT18C6-Ag and modified
further using chitosan and PAMAM
dendrimer-coated silicon quantum
dots (SiQDs-PAMAM)
Label-free paper-based Immunosensor designed for the — — Opens new possibilities for [177]
electrochemical biosensor detection of immunoglobulin against diagnosing COVID-19
SARS-CoV-2 with high specificity and
sensitivity
FET Portable bifunctional electrical Exhibited rapid detection — Efficient and accurate tool for high- [178]
detector based on graphene FET for speed (∼10 min for nucleic throughput point of care testing of
the detection of SARS-CoV-2 via acid detection and ∼5 min for COVID-19
nucleic acid hybridization or Ag–Ab immunoassay)
interaction
Recent applications of graphene-based biosensing
107108 Hassan A. Alhazmi et al.
graphene electrode, the voltage might be applied and the infarction was under clinical trial but is not yet recruiting
concentration was measured via the electric current response. [189]. They aimed to examine 100 people above the age of
In the same year, another graphene-based biosensor device 18 who complained of chest pain to see if they had an
received the patented status where the graphene nanosheet acute myocardial infarction.
modified with chitosan were coated on glassy carbon elec-
trodes and were used as sensing agents for polynucleotide
and mutation detection using voltammetry [182]. Another
breakthrough in the research on graphene-based biosen- 5 Biomedical graphene biosensors –
sors came when a patent on FET was developed for the
detection of biomolecular samples [183]. This sensor con-
market trends, opportunities, and
sisted of a graphene layer (semiconductor material) bound future perspectives
with ligand-binding protein that helped to detect protein-
based substances. Graphene often heralded as the wonder material is an
Another graphene-based FET biosensor device was advanced 2D carbon-based material of the 21st century
developed by modifying their edges with functional groups having exceptional properties of light-weightedness, high
that helped in detecting the sample using an analyzer by strength, super flexibility, excellent superconductivity, and
measuring the change in the current generated by the having a paramount potential in improving the human life
sample connected to the functional group. Thin-film gra- quality. Today, biosensors have made a huge impact in
phitic layers made up of many graphene layers were added various biosensing platforms as electrochemical biosensors,
to a base in the desired pattern and altered for better elec- fluorescent biosensors, and graphene biosensors for enzy-
trode connection [184]. A novel strategy for the detection matic biosensing and immunosensing applications. The
of samples present in food was developed using electro- superconductive material graphene is a low-cost ideal
chemical DNA graphene or biochip biosensor. Their detec- material for the construction of sensors and biosensors-
tion was based on the mechanism of DNA (present in the based devices for biomedical applications [190]. Graphene
sample)–redox (ruthenium hexamine molecule) electro- is considered one of the most advanced materials in
static complex, and their subsequent nonspecific adsorp- human healthcare as biosensing and diagnostic tool and
tion on the graphene surface. The developed biosensor this has accelerated medical diagnosing to new dimensions.
was able to detect meat samples using isothermal amplifi- It has successfully revolutionized our understanding of the
cation of DNA and electrochemical detection via square treatment of deadly diseases through its derivatives and
wave voltammetry [185]. Two Chinese patents were granted hybrids-based biosensors and their march toward the mar-
recently in the years 2018 and 2019 using graphene-based ketplace is at a high pace and more of these are expected to
biosensors for the detection of guanine ribose [186] and come into the market in the next decade as high-end diag-
cancer marker microRNA [187] using 3D graphene biosen- nostic tools in POCT [191].
sors. These biosensors consisted of a graphene layer over a As research and development are growing each year,
glass substrate where indium tin oxide was arranged on the biosensors are becoming a commercial reality. But, before
sides of the sensor. This 3D biosensor showed a number of becoming a commercially significant device, the graphene-
advantages including ease of operation and use, improved based biosensors are bound to face initial and primary chal-
specificity at lower operating voltages, and even great safety. lenges such as quality control, scalability, and durability
Very recently, a graphene biosensor was designed and man- issues that should be resolved transparently. The intrinsic
ufactured using GNP ink, which was deposited on the sur- properties of graphene, their integration with other func-
face of the substrate to provide at least one sensing layer. tional materials, fabrication of devices, and processing
This layer was processed via photonic curing to generate a steps need close considerations [192]. The market forecast
graphene electrode vertically arranged graphene sheets and reports on biosensors were shared for understanding more
used for plasma treatment for functionalization, which was about their market trends and opportunities. The Biome-
used for the identification of biological molecules [188]. dical Sensor Market - Forecast Report (2021–2026) stated
Various nongraphene biosensors are being tested in that these biosensors adjusted themselves according to
clinical trials for the identification of various diseases, the genetic make-up of each patient and are programmed
but graphene-based biosensors are only a few. A new to send alarm signals when anomalies or unexpected body
approach based on the photoelectrochemical immuno- readings are registered in real-time [193]. Biomedical sensor
sensor employed graphene quantum dots in conjunction devices greatly improved early detection of health problems
with Si nanowires for the early detection of myocardial by measuring blood pressure remotely, detection of toxinsRecent applications of graphene-based biosensing 109
in blood, analysis of glucose, lactate, and glutamine in The understanding of physics and nanotechnology
aqueous media, and analysis of other complex biological goes hand-in-hand and it holds potential in improving
media. the quality, cost of biological testing, speed, and capability
Similarly, the Frost and Sullivan Analysis Report to analyze the resulting complex data. Biosensors enable
(2018–2023) on biosensor market forecast expected growth biotechnicians to control the precise biological data [198].
in the biosensor market at a 12% compound annual growth Based on their pharmaceutical applications, the biome-
rate during 2018–2023 from $17.7 billion in 2018 to reach up dical biosensors’ market has been segmented as cardiac
to $31.2 billion by the end of 2023 [194]. The key application care, pain management, drug discovery, diagnostics, and
areas and segments marked by them included point-of-care genomics. The global market trends and opportunities
home diagnostics in the healthcare system, food, water, and show that wearable biosensors are in an upward trend,
air quality monitoring, agriculture and security, etc. Wear- especially in diabetics and cardiovascular diseases [199–201].
able biosensors for the noninvasive monitoring of heart
rate, breathing rate, glucose, disease diagnostics, and
detection are also mentioned in their report. The US
National Institute of Health would encourage and fund
6 Conclusions and future
biosensor-related research activities. The nano-biosensors perspectives
have promising applications in the biomedical sector and
diagnostics because of their improved specificity and selec- A vast variety of graphene-based nanomaterials con-
tivity. Other than the upward market trend of nano-biosen- sisting of pristine graphene, GO, rGO, and graphene
sors and wearable biosensors, the digestible biosensors, quantum dots are used for the preparation of graphene-
silicon photonic biosensors, implantable biosensors, and based biosensors. These biosensors showed a plethora
touch-based in-vehicle sensors are also having promising of applications in biomedical and nonbiomedical fields
future in this area of biosensing [194]. for the detection of target molecules. The uncountable
The Graphene Market and 2D Materials Assessment unique properties of graphene including mobility, large
Report (2021–2031) forecasted for 18 key applications surface area, transparency, nontoxicity, tensile strength,
areas and the movement of graphene biosensors from and superior electrical and thermal conductivity allowed
the laboratory to the commercial market in the coming the functionalization and immobilization steps leading to
decades. According to this assessment report, the fore- the development of sensitive and accurate biosensors.
cast was made that the graphene market will continue to Graphene-based materials can be employed and inte-
grow from110 Hassan A. Alhazmi et al.
such as type, molecular weight, surface properties, etc. [5] Lindsley MD, Mekha N, Baggett HC, Surinthong Y,
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