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Graphene Oxide-Based Biosensors for Liquid Biopsies in Cancer Diagnosis

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Abstract and Figures

Liquid biopsies use blood or urine as test samples, which are able to be continuously collected in a non-invasive manner. The analysis of cancer-related biomarkers such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA, and exosomes provides important information in early cancer diagnosis, tumor metastasis detection, and postoperative recurrence monitoring assist with clinical diagnosis. However, low concentrations of some tumor markers, such as CTCs, ctDNA, and microRNA, in the blood limit its applications in clinical detection and analysis. Nanomaterials based on graphene oxide have good physicochemical properties and are now widely used in biomedical detection technologies. These materials have properties including good hydrophilicity, mechanical flexibility, electrical conductivity, biocompatibility, and optical performance. Moreover, utilizing graphene oxide as a biosensor interface has effectively improved the sensitivity and specificity of biosensors for cancer detection. In this review, we discuss various cancer detection technologies regarding graphene oxide and discuss the prospects and challenges of this technology.
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nanomaterials
Review
Graphene Oxide-Based Biosensors for Liquid
Biopsies in Cancer Diagnosis
Shiue-Luen Chen 1, 2, , Chong-You Chen 1,2, , Jason Chia-Hsun Hsieh 3, , Zih-Yu Yu 1,
Sheng-Jen Cheng 1,2, Kuan Yu Hsieh 1,2 , Jia-Wei Yang 1,2, Priyank V Kumar 4,
Shien-Fong Lin 1,2 and Guan-Yu Chen 2,5,*
1
Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan;
g199500@gmail.com (S.-L.C.); ericchen161206@gmail.com (C.-Y.C.); viviyou0228@gmail.com (Z.-Y.Y.);
shengjen@nctu.edu.tw (S.-J.C.); Kuan.Yu@ibm.com (K.Y.H.); jiawei@nctu.edu.tw (J.-W.Y.);
linsf5402@nctu.edu.tw (S.-F.L.)
2Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao
Tung University, Hsinchu 300, Taiwan
3Division of Haematology/Oncology, Department of Internal Medicine, Chang Gung Memorial
Hospital (Linkou), Taoyuan 333, Taiwan; wisdom5000@gmail.com
4School of Chemical Engineering, University of New South Wales, Sydney, NSW 2052, Australia;
priyank.kumar@unsw.edu.au
5Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan
*Correspondence: guanyu@nctu.edu.tw; Tel.: +886-3-573-1920; Fax: +886-3-573-1672
These authors contributed equally to this work.
Received: 4 November 2019; Accepted: 25 November 2019; Published: 3 December 2019
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Abstract:
Liquid biopsies use blood or urine as test samples, which are able to be continuously
collected in a non-invasive manner. The analysis of cancer-related biomarkers such as circulating
tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA, and exosomes provides important
information in early cancer diagnosis, tumor metastasis detection, and postoperative recurrence
monitoring assist with clinical diagnosis. However, low concentrations of some tumor markers,
such as CTCs, ctDNA, and microRNA, in the blood limit its applications in clinical detection
and analysis. Nanomaterials based on graphene oxide have good physicochemical properties and are
now widely used in biomedical detection technologies. These materials have properties including good
hydrophilicity, mechanical flexibility, electrical conductivity, biocompatibility, and optical performance.
Moreover, utilizing graphene oxide as a biosensor interface has eectively improved the sensitivity
and specificity of biosensors for cancer detection. In this review, we discuss various cancer detection
technologies regarding graphene oxide and discuss the prospects and challenges of this technology.
Keywords:
liquid biopsy; circulating tumor cells; circulating tumor DNA; exosome; graphene oxide
1. Cancer and Diagnosis
Cancer is caused by abnormal cell proliferation and further forms of tumors. Cancer cells are
mostly derived from functional changes in genetic materials in the normal cell caused by oncogenic
factors and, in a few cases, are caused by parental inheritance of abnormal genes. Activation of
oncogene or loss of function of tumor suppressor genes can lead to uncontrolled cell division rates [
1
];
continued cell growth and division leads to tumor formation [
2
], which can threaten life by causing
organ failure or metastasizing to other sites [
3
]. Cancer is first diagnosed from medical imaging
technologies, such as X-ray, computed tomography, and magnetic resonance imaging. In most cases,
physicians will further employ a tissue biopsy to evaluate clinical TNM staging (Classification of
Malignant Tumors, T: the size of the primary tumor, N: nearby lymph nodes, M: distant metastasis),
Nanomaterials 2019,9, 1725; doi:10.3390/nano9121725 www.mdpi.com/journal/nanomaterials
Nanomaterials 2019,9, 1725 2 of 17
depending on the degree of tumor development, lymph node involvement, and distant metastasis [
4
].
However, traditional detection techniques can often only detect advanced cancers, and general tissue
section analysis cannot present complete information about cancer due to the heterogeneity of tumor
tissue [
5
,
6
]. Yet, physicians require more information to assess treatment strategies, and the detection of
specific genes can eectively determine the degree of damage to oncogenes and estimate the future type,
and possible target, of metastasis of tumors. Therefore, eective detection of cancer-related biomarkers
is an important objective for the clinical diagnosis of cancer. In addition, early diagnosis of cancer and
immediate treatment can significantly reduce patient mortality and improve the recovery rate [7].
2. Liquid Biopsies
Liquid biopsies have recently emerged as a method for the early detection of cancer [
8
,
9
]. It has
the advantages of being non-invasive and having a short detection time. Compared to traditional
tissue sectioning, which takes several weeks, liquid biopsies require a shorter time, and the collection
of only 7–10 mL of blood can screen whether cancer markers are present in the blood. Liquid biopsies
primarily screen the blood; because blood circulates through the whole body, the information that
can be provided by it is far more clinically significant than results from sections that represent only a
certain location [
10
]. This novel technology is a valuable tool in clinical diagnosis and treatment [
11
].
Moreover, it also plays an important role in monitoring prognosis, which can be used for regular
follow-up [
12
], and can be processed in a timely manner after recurrence. In addition, it also greatly
reduces the discomfort and surgical risk in patients due to repeated sampling. This method can also be
used to assess the appropriate dose of medication to a patient [
13
]. However, current technologies are
not yet mature and the sensitivity of detection may also miss these rare but important tumor markers
in the blood. Therefore, improving detection sensitivity and developing high-throughput detection are
urgently needed to overcome the shortcomings of liquid biopsies [14].
3. Circulating Tumor Cells (CTCs)
The concept of a liquid biopsy is derived from circulating tumor cells (CTCs), which, as the name
suggests, are tumor cells that circulate in the blood. Due to intrinsic or extrinsic factors, the cancer cells
of the primary tumor will penetrate the tissue’s basement membrane, enter the bloodstream, and then
circulate throughout the body via the blood, thus resulting in metastasis [
13
]. By monitoring and
tracking these cells, it is possible to monitor the course of disease in the patient at any time and adjust
the direction of treatment in a timely manner. CTCs play an important role in cancer research and
treatment; they originate from a researcher’s “seed and soil” theory published in 1889 [
13
], where the
seeds represent cancer cells and the soil represents the microenvironment preferred by the cancer
cells for growth. When the cancer begins to metastasize, it releases many signaling factors similar
to fertilizer, which travel through the bloodstream to specific tissues and organs, and then these
factors will attract tumors to settle and grow. This phenomenon also explains why cancer metastasis
does not occur randomly. The most essential mechanism during the process of cancer metastasis is
epithelial–mesenchymal transition (EMT) [
15
,
16
], which describes the transformation of the phenotype
of some cells in the tumor from densely connected epithelial tissue to more flexible and invasive
interstitial cells. This causes normal cells to loosen and provides an opportunity for tumor cells to invade
the blood vessels and circulate to the whole body through the blood. When cancer cells reach a suitable
environment for growth, they undergo mesenchymal–epithelial transition (MET), the mechanism that
is the reverse of EMT, in which cells revert to the original epithelial cells. However, this mechanism of
EMT does not represent an absolute negative, it is also an indispensable mechanism for embryogenesis
and organogenesis [17].
Due to the extremely low content of CTCs in the blood, a high purity, highly sensitive purification
method is required to separate CTCs from a blood sample for detection and subsequent analysis.
The current sorting and purification methods can be divided into biochemical separation methods
and physical separation methods [
18
,
19
]. Biochemical separation methods primarily utilize capturing
Nanomaterials 2019,9, 1725 3 of 17
specific protein markers expressed on the surface of CTCs by antibodies, to separate CTCs from the
blood [
19
]. Physical methods are based on the dierences in size and density between CTCs and other
blood cells or small molecules in the blood by mechanical or electrical methods for separation, including
density centrifugation, filtration, and microfluidic chips [
20
]. Currently, both purification methods
have been applied in clinical experiments, but there are still many issues that must be overcome,
especially the loss during purification methods due to the heterogeneity of CTCs. However, combining
the two will be a future trend in the development of CTC purification methods [20].
4. Circulating Tumor DNA (ctDNA)
The unbalanced cell cycle of cancer cells causes cancer cells to excessively proliferate and die.
When the rate of debris clearance of macrophage is lower than the rate of tumor cell death, the content
of cell-free DNA (cfDNA) will increase greatly and continue to circulate in the blood [
21
]. Many studies
have shown that in the development of cancer, the concentration of cfDNA in the blood of cancer
patients is much higher than in normal people [
22
]. In 1989, Stroun and colleagues indicated that a
small proportion of cfDNA in the serum of cancer patients is released by dying cancer cells. The cfDNA
containing cancer genes are also known as circulating tumor DNA (ctDNA) [
23
]. In recent years,
analyzing mutation information in ctDNA from the blood of patients has been used for early diagnosis,
formulating follow-up treatment strategy, and assessing prognosis, and it can also be used to monitor
treatment ecacy, analyze drug resistance, and discover possible metastasis targets at early stages [
24
].
The methods currently used to analyze ctDNA can be broadly divided into two categories. The first
category is Polymerase chain reaction (PCR)-based techniques [
24
27
], which are commonly used
to detect single or multiple hotspot mutations in a single gene [
24
,
26
28
]. One of the most common
techniques is droplet digital PCR (ddPCR) [
27
,
29
31
], in which a sample is separated into multiple
independent droplets through dierent sample isolation methods, such as emulsified droplet or
micro-channel techniques [
32
,
33
]; each droplet undergoes PCR, then statistical analysis is performed to
quantify fluorescent signals in each droplet for absolute quantitation of the target sequence. This method
can eectively improve the ctDNA sensitivity of conventional PCR technology and the diculty
in quantitation, and it is currently the primary method for monitoring clinical ctDNA content [
24
].
The second category of methods uses various next-generation sequencing (NGS) techniques for whole
genome sequencing [
34
,
35
] and deep sequencing of target regions [
33
,
36
38
]. These techniques are
commonly used to confirm genovariation in patients, to detect potential chromosomal mutations, to
evaluate the ecacy of medication and treatment from genotypes, to predict cancer development trends,
and to provide timely treatment. However, these ctDNA analysis techniques usually require PCR for
sample amplification—this increases background cfDNA concentration and dilutes the detection target,
which aects the sensitivity and quantitative evaluation of the assay [
30
]. In other words, ultra-low
concentration ctDNA used in early cancer detection needs a high-sensitivity, high-specificity probe
detection platform.
5. Exosome
In 1980, Johnstone et al. found that certain transferrin receptors and membrane-associated
substances in the reticulocytes of adult mammals selectively release multi-vesicular bodies into
the circulatory system, and named them exosomes [
39
41
]. Exosomes are extracellular vesicles
released by cells that range in size from 30 to 200 nm [
42
44
] and serve as a medium for intercellular
communication [
45
47
]. They are primarily responsible for transporting biologically active molecules
such as nucleic acids, proteins, lipids, RNAs (mRNA, miRNA, long non-coding RNA), and DNAs
(mtDNA, ssDNA, and dsDNA) [
48
,
49
]. Exosomes originate from cancer cells and establish a tumor
microenvironment by immune system suppression, angiogenesis promotion, and EMT induction [
50
],
which gives surface markers of exosomes great potential in early cancer diagnosis, metastasis,
and monitoring of cancer treatment.
Nanomaterials 2019,9, 1725 4 of 17
The main bottleneck in the clinical application of exosomes is the lack of ecient isolation methods.
Traditional separation methods include dierential ultracentrifugation, density-gradient separation,
and anity [
51
]. Dierential centrifugation is based on the dierence in size and buoyancy between
exosomes and other extracellular vesicles (EVs), but it requires a long centrifugation time and the
recovery and specificity is low [
52
,
53
]. Density-gradient centrifugation has a higher recovery and
purity than dierential centrifugation, but the buoyancy and density of exosomes are similar to those of
shed microvesicles (sMVs) and viruses [
54
]. The above-mentioned methods cannot separate exosomes
eectively. As a result, many research groups in the past decade have attempted to apply microfluidics
to the separation of exosomes. As the name implies, reducing the fluid motion to the micrometer
(
µ
m) level in microfluidics, inertia, and gravity in the fluid mechanics at this small scale is negligible.
Instead, the viscosity of the fluid becomes a key factor. The microfluid is also closer to the flow pattern
of human body fluids in blood vessels. The experimental procedure is shrunk to the size of a chip,
which greatly decreases detection time and increases accuracy, while reducing the need for a sample,
revealing prospects for precision medicine [55].
Currently, the isolation of exosomes in microfluidic chips mainly relies on anity [
51
], in which
an appropriate antibody is selected to capture the surface markers of the exosome. The antibody is
modified on the substrate or on the surface of magnetic beads to enhance the interaction between
the capture probe and the exosomes by taking advantage of the large surface area. Although this
technology is very mature, it often faces the bottlenecks of low exosome recovery eciency and the
need for a large amount of sample volume (or cell number) preparation. If these problems can be
eectively solved, then breakthroughs in the development of exosome-based early diagnosis will
be possible.
6. Biomedical Diagnostic Applications of GO
Nanomaterials are a research trend that has begun to be applied in interdisciplinary studies in recent
years, especially in biomedical fields. Nanomaterials such as gold nanoparticles, carbon nanotubes,
graphene, and graphene oxide (GO) have been mentioned in the literature as having applications in
biological probes, tissue engineering, and cancer treatment studies [
56
66
]. Of these, graphene and GO
are especially popular topics of research in the field of biosensors [
67
71
]. GO possesses oxygen-rich
functional groups on the surface. It is easily oxidized, acidified, and easily forms covalent bonds, so it is
quite suitable for chemical modification. Moreover, due to the good physicochemical and hydrophilic
properties of GO, there have been studies in recent years that have begun to use GO for probes,
biological reagent analysis, and biological imaging [
72
,
73
], demonstrating its great potential in the
biomedical field. In addition, GO has high surface capacity, water solubility, and biocompatibility due to
its rich functional groups, which facilitate protein modification as biological probes. Existing literature
has indicated that GO can be used as a probe substrate to eectively capture many small biological
molecules, DNA, bacteria, and cells [74,75].
7. GO-Nanointerface for CTC Diagnosis
Among the cell capture methods using GO substrates, a popular topic nowadays is the linkage
of intact antibodies to GO substrates to capture rare circulating tumor cells or viruses in blood
samples [
72
,
76
]. Li et al. used a high-temperature method to eliminate the oxygen functional groups
on GO to prepare reduced graphene oxide (rGO) in order to capture CTCs with modified antibodies
(Figure 1A–D). Because of the rough texture and low stiness of the rGO surface, the interaction
between cells and the biological interface may be enhanced. In addition, because rGO carries a
negative charge and is highly hydrophilic, it can prevent non-specific cell adhesion, which eectively
improves the capture eciency of CTCs and reduces background noise. CTCs have been specifically
and successfully captured from whole blood containing 10 CTCs/mL [
77
]. However, there are still
some problems with the use of intact antibodies for cell capture that limit their eciency. For example,
the production of intact antibodies is costly, and the production process is also cumbersome. In addition,
Nanomaterials 2019,9, 1725 5 of 17
in order to improve target capture eciency, additional modification of the GO substrate surface with
gold nanoparticles (Figure 1E) [
76
]. Traditional methods for immobilizing intact antibodies to GO
surfaces typically require N-hydroxysuccinimide-lysine(NHS) ester or maleimide chemistries to label
free lysine or cysteine residues, respectively, which typically results in intact antibodies immobilized in
random orientations and causes lower specificity of the biological probe [
72
,
78
]. Using this linkage
method also limits subsequent applications; for example, N-hydroxysuccinimide–lysine (NHS) and
maleimide–cysteine covalent bonding prevents further linkage of fluorescent groups, which greatly
limits fluorescence quantification in the preparation of biological probes. In addition, studies have
been conducted to immobilize antibodies to the substrate in a uniform orientation. A common method
is to use biotin-streptavidin/NeutrAvidin. However, such methods usually require biotinylation
at lysine or cysteine residues. The intermediate proteins required (such as biotin) tend to cause
biological probes to have reduced activity [
76
,
79
,
80
], and also causes lower capture eciency in CTCs.
Therefore, related research topics need to not only develop new antibody production technologies to
replace intact antibodies but also develop new linkage methods. Another issue in CTC diagnosis is
to release the CTCs from the capture nanointerface for subsequent analysis. Yoon et al. developed a
thermo-responsive polymer and functionalized GO composite film for the capture/release of CTCs
(Figure 1F). They immobilized antibodies to the functionalized GO for capturing CTCs from breast
cancer patients’ samples. Then, the captured CTCs could release from the polymer matrix with a
lower critical solution temperature (LCST) of 13
C. The ecient release of captured CTCs from the
polymer–GO microfluid make it ideal for various downstream analyses and also shows the potential
for liquid biopsies [81].
8. GO-Nanointerface for Gene Probe Diagnosis
Among the literature describing the application of GO in DNA assays, there are many that
demonstrate the superiority and sensitivity of GO-based DNA-based sensors that take advantage
of the optical, electrical, mechanical, and chemical properties of GO and use the unique features of
GO nanostructures and chemical properties [
68
,
82
]. These GO sensors can be roughly divided into
two types (Table 1). The first type uses GO as a superior receptor for DNA-derived fluorescent probe
sensors in Förster resonance energy transfer (FRET) [
82
,
83
]. The bases of the DNA probe form a
hexagonal honeycomb structure, which interacts with the graphitic (sp2) domains on the GO surface
and, through
π
π
stacking, adsorbs the DNA probe to the GO surface, where the fluorescence is
quenched by Förster resonance energy transfer (FRET) (Figure 3A) [
69
,
84
,
85
]. After the DNA probe
binds to the target molecule, the binding force between the DNA probe and the target molecule is
greater than the binding force between the DNA probe and GO surface, which separates it from the GO
surface (Figure 3B). At the same time, a fluorescence recovery signal is generated due to the reduced
FRET eect [
84
]. Many researchers have used this GO as a substrate to develop fluorescent biosensor
systems to assay for metal ions, DNA [
84
,
86
], RNA [
87
], small-molecule organic matter [
88
], peptides,
proteins [89], and even cells, and most studies have proven that GO is advantageous for applications
related to biological probes. Among them, He et al. used multi-color fluorescent probes to detect
specific target sequences and rapidly obtained highly specific and sensitive detection results in complex
environmental solutions. They also used this technology to eectively distinguish sequences with
single-base errors [
84
], demonstrating the potential of GO for cancer gene detection. Eftekhari-Sis et al.
successfully used this technique to detect exon 19 deletions in the estimated Glomerular filtration
rate (EGFR) gene, which is a gene mutation that plays a very important role in lung cancer and
is used clinically to evaluate the use of targeted drugs in patients with non-small cell lung cancer
(Figure 3D) [
90
]. In addition, it has a linearity of R
2
=0.9992 for the detection of the target exon
19 deletion sequence at dierent concentrations between 0 and 80 fmol/
µ
L, demonstrating that it
can detect extremely low concentrations of the target sequence (Figure 3E) [
91
]. However, there are
currently few studies on the application of GO-DNA fluorescent probe optical sensors using FRET for
cancer gene detection, because the limits of many such detection techniques fail to detect extremely
Nanomaterials 2019,9, 1725 6 of 17
low concentrations of cancer genes [
84
]. Thus, improving the limit of detection is a major issue for
future research.
Nanomaterials 2019, 9, x FOR PEER REVIEW 5 of 17
antibodies to GO surfaces typically require N-hydroxysuccinimide-lysine(NHS) ester or maleimide
chemistries to label free lysine or cysteine residues, respectively, which typically results in intact
antibodies immobilized in random orientations and causes lower specificity of the biological probe
[72,78]. Using this linkage method also limits subsequent applications; for example, N-
hydroxysuccinimidelysine (NHS) and maleimidecysteine covalent bonding prevents further
linkage of fluorescent groups, which greatly limits fluorescence quantification in the preparation of
biological probes. In addition, studies have been conducted to immobilize antibodies to the substrate
in a uniform orientation. A common method is to use biotin-streptavidin/NeutrAvidin. However,
such methods usually require biotinylation at lysine or cysteine residues. The intermediate proteins
required (such as biotin) tend to cause biological probes to have reduced activity [76,79,80], and also
causes lower capture efficiency in CTCs. Therefore, related research topics need to not only develop
new antibody production technologies to replace intact antibodies but also develop new linkage
methods. Another issue in CTC diagnosis is to release the CTCs from the capture nanointerface for
subsequent analysis. Yoon et al. developed a thermo-responsive polymer and functionalized GO
composite film for the capture/release of CTCs (Figure 1F). They immobilized antibodies to the
functionalized GO for capturing CTCs from breast cancer patients’ samples. Then, the captured CTCs
could release from the polymer matrix with a lower critical solution temperature (LCST) of 13 °C.
The efficient release of captured CTCs from the polymerGO microfluid make it ideal for various
downstream analyses and also shows the potential for liquid biopsies [81].
Figure 1. Example of antibody-modified graphene oxide for capturing CTCs. (A) A reduce graphene
oxide film efficiently captures circulating tumor cells (CTCs) from clinical blood samples. (B)
Environmental Scanning Electron Microscope (ESEM) image of the reduced graphene oxide (rGO)
layer-by-layer structure, and (C) an anti- Epithelial cell adhesion molecule (EpCAM) -rGO film after
capture CTCs. (D) The modification steps of anti-EpCAM-rGO film. (E) Schematic of CTC capture
system using functionalized graphene oxide (GO) nanosheets on a patterned gold surface. (F)
Figure 1.
Example of antibody-modified graphene oxide for capturing CTCs. (
A
) A reduce
graphene oxide film eciently captures circulating tumor cells (CTCs) from clinical blood samples.
(
B
) Environmental Scanning Electron Microscope (ESEM) image of the reduced graphene oxide (rGO)
layer-by-layer structure, and (
C
) an anti- Epithelial cell adhesion molecule (EpCAM) -rGO film after
capture CTCs. (
D
) The modification steps of anti-EpCAM-rGO film. (
E
) Schematic of CTC capture
system using functionalized graphene oxide (GO) nanosheets on a patterned gold surface. (
F
) Schematic
of a polymer–GO microfluidic device. Figures (
A
D
) reproduced with permission of [
77
], Wiley
©
, 2015;
(E) [76], Springer Nature©, 2013; (F) [81] Wiley©, 2016.
The second type of GO sensors are electrochemical DNA-based sensors designed to utilize the
excellent electrochemical properties of the nanomaterial [
67
,
82
,
92
]. An advantage of electrochemical
sensors is that they are label free sensors. By analyzing the electrical signal dierences during
the interaction between the analyte and the probe, the concentration of the analyte can be derived
from analysis of the relevant data [
67
]. Many studies have indicated that biological detection
interfaces using GO as an electrochemical sensor can eectively improve sensitivity. For example,
Chu et al. combined MoS
2
and GO materials using hydrothermal and ultrasonic methods to prepare
MoS
2
/graphene nanosheets to improve the electrical conductivity and electrochemical activity of
electrochemical DNA-based sensors (Figure 2A,B) [
93
]. Intermolecular
π
π
stacking between DNA
nucleobases and GO is also beneficial to the sensitivity of single-stranded DNA immobilization
on the surface of MoS
2
/graphene nanosheet-modified electrodes, and they can be used to detect
ctDNA with a limit of detection of 1
×
10
17
M (Figure 2C) [
93
]. In addition, the combination of
gold nanoparticles and GO has been widely used as electrodes for electrochemical sensors [
68
,
94
].
Nanomaterials 2019,9, 1725 7 of 17
Abdul Rasheed et al. used this technique to combine a capture probe (DNA-c) with a reporter probe
(DNA-r) to hybridize with target DNA (DNA-t) in a sandwich structure (Figure 2D,E) [
95
]. Oxidation
of the gold nanoparticle modifications of the reporter probe were used for the specific detection of
the BRCA1 gene associated with cancer. The results showed that this sensor had a stable limit of
detection of 1 fM [
95
]. Although there have been many studies reporting that the application of
GO to electrochemical sensors can eectively improve sensitivity, the interaction between GO and
probes and analytes has not yet been fully elucidated, and the intermolecular forces and electrical
properties between them are expected to be confirmed in the future, further enhancing the sensitivity
and specificity of this technology and extending its application to ctDNA monitoring and detection at
early stages of cancer.
Nanomaterials 2019, 9, x FOR PEER REVIEW 8 of 17
Figure 3. GO-based DNA-based optical sensors. (A) Schematic of MoS2/graphene nanosheets
electrode for ctDNA detection. (B) Scanning Electron Microscope (SEM) image of MoS2/graphene
composites. (C) The Differential Pulse Voltammetry (DPV) plots change after hybridization of various
concentrations of ctDNA. (D) Schematic of sensing steps of graphene-DNA electrochemical sensor
with AuNPs functionalized report DNA. (E) SEM image of sensor without adding DNA-r AuNPs
(left) and adding DNA-r AnNPs (right). Figures (AC) reproduced with permission of [93], RSC©,
2016; (D,E) [95], Elsevier©, 2014.
9. GO-Nanointerface for Exosome Diagnosis
Due to GO’s nano-parameter structure and high-compatibility, this material has high potential
as an interface of exosome biosensors. Mei Heb et al. modified a GO substrate with a layer of
polydopamine (PDA) and used protein G to immobilize antibodies on GO for exosome capture [101].
Chae et al. used oxygen plasma treatment to enhance the reduction of a reduced graphene oxide
(rGO) sensor surface for exosome diagnosis in Alzheimer disease patients and found that rGO
reduced by oxygen plasma treatment showed a 3.33-fold higher target specificity compared to before
treatment (Figure 4AD). [102]. Wang et al. used DNA aptamers to design a new signal amplification
platform for colorectal cancer exosome surface markers CD63 and EpCAM. This method requires
only 5 µL of serum sample for the detection of colorectal cancer exosomes. It has significant diagnostic
capabilities, confirming that the platform could not only be used for colorectal cancer exosomes, but
also for other cancer exosomes [103]. Hyungsoon Im et al. designed a nanoplasmonic (NPS) platform
for high-throughput EV analysis. The combination of GO-based interface and heatmap means that
EV markers analysis can quickly and sensitively measure 7 biomarkers in 100 samples, as shown in
Figure 4E [104]. Cancer-derived circulating exosome play an important role in cancer diagnosis, and
moreover, people have tried to use exosomes as an innovative clinical treatment [105]. However, the
current exosome detection methods are low recovery or non-specific. The combination of materials
and interface modification for exosome detection is indispensable.
Figure 2.
GO-based DNA-based electrochemical sensors. (
A
) Schematic of MoS2/graphene
nanosheets electrode for ctDNA detection. (
B
) Scanning Electron Microscope (SEM) image of
MoS2/graphene composites. (
C
) The Dierential Pulse Voltammetry (DPV) plots change after
hybridization of various concentrations of ctDNA. (
D
) Schematic of sensing steps of graphene-DNA
electrochemical sensor with AuNPs functionalized report DNA. (
E
) SEM image of sensor without
adding DNA-r AuNPs (left) and adding DNA-r AnNPs (right). Figures (
A
C
) reproduced with
permission of [93], RSC©, 2016; (D,E) [95], Elsevier©, 2014.
Table 1. Performance comparison between GO-based DNA sensors for DNA detection.
Type of GO-Based
DNA-Based Sensors Description of Method Sensitivity References
Optical
Multicolor fluorescent DNA nanoprobe
100 pM [84]
Fluorescein amidites (FAM) labeled
DNA probe 1 nM [91]
Molecular beacon 2 nM [96]
DNA probe and DNA-intercalating
dyes (SYBR Green I) 0.31 nM [97]
Electrochemical
MoS2/graphene nanosheet-modified
electrodes 0.01 fM [93]
Gold nanoparticle labeled reporter DNA
(DNA-r.AuNP) and DNA-c modified
glassy carbon electrode (GCE)/Gr
1 fM [95]
PP3CA/ERGO/GCE 3 fM [98]
cDNA2 modified AuNPs with catalyzed
silver staining and GCE-GR/cDNA1 72 pM [99]
Nanomaterials 2019,9, 1725 8 of 17
Nanomaterials 2019, 9, x FOR PEER REVIEW 7 of 17
and analytes has not yet been fully elucidated, and the intermolecular forces and electrical properties
between them are expected to be confirmed in the future, further enhancing the sensitivity and
specificity of this technology and extending its application to ctDNA monitoring and detection at
early stages of cancer.
Table 1. Performance comparison between GO-based DNA sensors for DNA detection.
Description of Method
Sensitivity
References
Multicolor fluorescent DNA nanoprobe
100 pM
[84]
Fluorescein amidites (FAM) labeled DNA
probe
1 nM
[91]
Molecular beacon
2 nM
[96]
DNA probe and DNA-intercalating dyes
(SYBR Green I)
0.31 nM
[97]
MoS2/graphene nanosheet-modified
electrodes
0.01 fM
[93]
Gold nanoparticle labeled reporter DNA
(DNA-r.AuNP) and DNA-c modified glassy
carbon electrode (GCE)/Gr
1 fM
[95]
PP3CA/ERGO/GCE
3 fM
[98]
cDNA2 modified AuNPs with catalyzed
silver staining and GCE-GR/cDNA1
72 pM
[99]
Figure 2. GO-based DNA-based optical sensors. (A) Schematic of fluorescent sensors using DNA-
functionalized graphene oxide. (B) Molecular dynamics simulation of FAM-tagged singlestranded
DNA (ssDNA) absorbed on the surface of GO (left) and doublestranded DNA (dsDNA) detached
from the surface of GO (right). (C) Photographs showing GO and rGO had strong fluorescence
quenching ability. (D) Schematic of using a DNA-functionalized graphene oxide sensor for deletion
mutation in the EFGR gene in lung cancer. (E) Fluorescence spectra for fDNA after the detection of
various concentrations of cDNA. Figures (A,B) reproduced with permission of [84], Wiley©, 2010; (C)
[100], ACS©, 2010; (D,E) [91], Elsevier©, 2016.
Figure 3.
GO-based DNA-based optical sensors. (
A
) Schematic of fluorescent sensors using
DNA-functionalized graphene oxide. (
B
) Molecular dynamics simulation of FAM-tagged
single—stranded DNA (ssDNA) absorbed on the surface of GO (left) and double—stranded DNA
(dsDNA) detached from the surface of GO (right). (
C
) Photographs showing GO and rGO had strong
fluorescence quenching ability. (
D
) Schematic of using a DNA-functionalized graphene oxide sensor
for deletion mutation in the EFGR gene in lung cancer. (
E
) Fluorescence spectra for fDNA after the
detection of various concentrations of cDNA. Figures (
A
,
B
) reproduced with permission of [
84
], Wiley
©
,
2010; (C) [100], ACS©, 2010; (D,E) [91], Elsevier©, 2016.
9. GO-Nanointerface for Exosome Diagnosis
Due to GO’s nano-parameter structure and high-compatibility, this material has high potential
as an interface of exosome biosensors. Mei Heb et al. modified a GO substrate with a layer of
polydopamine (PDA) and used protein G to immobilize antibodies on GO for exosome capture [
101
].
Chae et al. used oxygen plasma treatment to enhance the reduction of a reduced graphene oxide
(rGO) sensor surface for exosome diagnosis in Alzheimer disease patients and found that rGO
reduced by oxygen plasma treatment showed a 3.33-fold higher target specificity compared to before
treatment (Figure 4A–D). [
102
]. Wang et al. used DNA aptamers to design a new signal amplification
platform for colorectal cancer exosome surface markers CD63 and EpCAM. This method requires only
5
µ
L of serum sample for the detection of colorectal cancer exosomes. It has significant diagnostic
capabilities, confirming that the platform could not only be used for colorectal cancer exosomes,
but also for other cancer exosomes [
103
]. Hyungsoon Im et al. designed a nanoplasmonic (NPS)
platform for high-throughput EV analysis. The combination of GO-based interface and heatmap
means that EV markers analysis can quickly and sensitively measure 7 biomarkers in 100 samples,
as shown in Figure 4E [
104
]. Cancer-derived circulating exosome play an important role in cancer
diagnosis, and moreover, people have tried to use exosomes as an innovative clinical treatment [105].
However, the current exosome detection methods are low recovery or non-specific. The combination
of materials and interface modification for exosome detection is indispensable.
Nanomaterials 2019,9, 1725 9 of 17
Nanomaterials 2019, 9, x FOR PEER REVIEW 9 of 17
Figure 4. Application of GO-based biosensors for exosome detection. (A) Schematic of antibody
immobilization on rGO surface. (B) Atomic Force Microscope (AFM) image (5 × 5 µm2) of antibody-
immobilized surface. (Scale bar is 1 µm). (C) Efficiency of antibody immobilization on different rGO
surfaces. (D) Resistance change (Rab-R)/R before and after immobilization and counting the number
of immobilized antibodies with a particular size on the AFM image (79 nm). (E) New nanoplasmonic
sensor (NPS) platform and heatmap analysis. Figures (AD) reproduced with permission of [102],
Elsevier©, 2017; (E), [104], Elsevier©, 2018.
10. GO-3D Printing and Micropatterning for Diagnosis
Currently, some research groups are utilizing GO to enhance cancer-related molecule capture
by improving the problem of low reproducibility caused by an insufficient sample number in the
blood. It can be combined with micropatterning, including soft lithography [106], electron beam
lithography [107], microcontact printing [108], self-assembled monolayer [109], and inkjet printing
[110], as a novel technique to help capture cancer-related cells in the biosensor system. However,
when safety, time, and cost are considered, inkjet printing is simple, fast, inexpensive, and non-
contact at the same time, which is conducive to large-scale production [111]. Also, it is applied in low-
temperature environments, so changes in the material properties of printing are avoided and printing
is possible on a variety of biomaterials. In addition, the inkjet distance, content, and size are
automatically calculated by a computer program, which ensures high sensitivity and reproducibility
of experiments and makes it suitable for related biochip research [112].
11. GO-3D Printing and Micropatterning in CTC, ctDNA, and Exosome Diagnosis
As mentioned above, many researchers have used micropatterning to increase the capture
efficiency of CTC [113,114], DNA [115], and exosomes [116]. For example, in addition to increasing
the contact surface area, microposts can be used to generate turbulence to increase the uniformity of
sample mixing [117]. There are also some studies that used different printing patterns of GO mixtures
calibrated to detect cancer-related markers [118,119]. Yoon et al. used GO modified with EpCAM
antibodies deposited on flower-shaped gold microposts on a flat surface to capture CTCs expressing
EpCAM that are present in the blood at early stages of cancer (Figure 5AC) [76]. The results of cell
capture tests using human breast cancer cell lines (MCF-7) showed that the micropatterned silicon
substrate had an MCF-7 cell recovery rate of 48% and the GO pattern microposts had a minimum
MCF-7 recovery rate of 73%, and even reached 100%. In addition, Zhang et al. used a photo-etching
technique to create a Y-shaped mold, and then a Polydimethylsiloxane (PDMS) molding technique
was used to produce a special nano-interfaced microfluidic exosome platform (nano-IMEX) pattern
(Figure 5DF) [101]. Because of the three-dimensional Y-shaped microposts, it not only allows fluid
to periodically mix with the surrounding liquid, and the asymmetric flow caused by the Y-shaped
structure allows larger vesicles to flow to the surface then be captured, but it can also increase mixing
Figure 4.
Application of GO-based biosensors for exosome detection. (
A
) Schematic of antibody
immobilization on rGO surface. (
B
) Atomic Force Microscope (AFM) image (5
×
5
µ
m
2
) of
antibody-immobilized surface. (Scale bar is 1
µ
m). (
C
) Eciency of antibody immobilization
on dierent rGO surfaces. (
D
) Resistance change (Rab-R)/R before and after immobilization and
counting the number of immobilized antibodies with a particular size on the AFM image (7–9 nm).
(
E
) New nanoplasmonic sensor (NPS) platform and heatmap analysis. Figures (
A
D
) reproduced with
permission of [102], Elsevier©, 2017; (E), [104], Elsevier©, 2018.
10. GO-3D Printing and Micropatterning for Diagnosis
Currently, some research groups are utilizing GO to enhance cancer-related molecule capture
by improving the problem of low reproducibility caused by an insucient sample number in
the blood. It can be combined with micropatterning, including soft lithography [
106
], electron beam
lithography [
107
], microcontact printing [
108
], self-assembled monolayer [
109
], and inkjet printing [
110
],
as a novel technique to help capture cancer-related cells in the biosensor system. However, when safety,
time, and cost are considered, inkjet printing is simple, fast, inexpensive, and non-contact at the
same time, which is conducive to large-scale production [
111
]. Also, it is applied in low-temperature
environments, so changes in the material properties of printing are avoided and printing is possible on
a variety of biomaterials. In addition, the inkjet distance, content, and size are automatically calculated
by a computer program, which ensures high sensitivity and reproducibility of experiments and makes
it suitable for related biochip research [112].
11. GO-3D Printing and Micropatterning in CTC, ctDNA, and Exosome Diagnosis
As mentioned above, many researchers have used micropatterning to increase the capture
eciency of CTC [
113
,
114
], DNA [
115
], and exosomes [
116
]. For example, in addition to increasing
the contact surface area, microposts can be used to generate turbulence to increase the uniformity of
sample mixing [
117
]. There are also some studies that used dierent printing patterns of GO mixtures
calibrated to detect cancer-related markers [
118
,
119
]. Yoon et al. used GO modified with EpCAM
antibodies deposited on flower-shaped gold microposts on a flat surface to capture CTCs expressing
EpCAM that are present in the blood at early stages of cancer (Figure 5A–C) [
76
]. The results of cell
capture tests using human breast cancer cell lines (MCF-7) showed that the micropatterned silicon
substrate had an MCF-7 cell recovery rate of 48% and the GO pattern microposts had a minimum
MCF-7 recovery rate of 73%, and even reached 100%. In addition, Zhang et al. used a photo-etching
technique to create a Y-shaped mold, and then a Polydimethylsiloxane (PDMS) molding technique
was used to produce a special nano-interfaced microfluidic exosome platform (nano-IMEX) pattern
(Figure 5D–F) [
101
]. Because of the three-dimensional Y-shaped microposts, it not only allows fluid
Nanomaterials 2019,9, 1725 10 of 17
to periodically mix with the surrounding liquid, and the asymmetric flow caused by the Y-shaped
structure allows larger vesicles to flow to the surface then be captured, but it can also increase mixing
eciency, thereby increasing the eciency of specific immunological exosome capture, while reducing
non-specific exosome capture. After modifying the GO on the microposts, polydopamine (PDA) is
directly added to the GO, which will self-synthesize and form a nano-structural interface that increases
surface area and antibody binding eciency. Labeled exosomes can be specifically captured using
auxiliary CD63, CD81, and EpCAM antibodies. The authors also mentioned that nano-IMEX can be
used to directly quantify circulating exosomes in 2 µL of untreated blood.
Nanomaterials 2019, 9, x FOR PEER REVIEW 11 of 17
Figure 5. Examples of microfluidic devices with microposts for capturing tumor cells. (A) Schematic
of circulating tumor cell capturing system with microposts. (B) SEM image of flower shaped
microposts. (C) SEM image of flower shaped micropost with captured tumor cell. (D) Schematic of
nano-interfaced microfluidic exosome platform and Graphene oxide/polydopamine (GO/PDA)
coated interface. (E) SEM image of Y shaped microposts with GO/PDA coating. (F) SEM image of
GO/PDA-coated channel. Figures (AC) reproduced with permission of [76], Springer Nature©, 2013;
and (DF) [101], RSC©, 2016. This article is licensed under a Creative Commons Attribution-
NonCommercial 3.0 Unported Licence.
Figure 6. Examples of inkjet printing utilized in tumor-related molecule sensing. (A) Schematic of
inkjet printing graphene oxide. (B) Uniformly inkjet-printed different sizes of graphene oxide
micropattern. (C) Schematic of graphene oxide support system (GOSS) and its detection mechanism.
(D) Electrical performance of original pentacene field-effect transistor (FET) and inkjet-printed
pentacene FET. (E) Schematic of paper-based electrochemical biosensor and its sensing mechanism.
Figures (A,B) reproduced with permission of [127], Wiley©, 2018; (C,D) [120], RSC©, 2017; and (E)
[122], Elsevier©, 2017.
12. Outlook
With the advancements in hardware technology and the development of nanomaterials, the
combination of GO and biosensors provides great benefits in the detection of clinical biomarkers. Not
only does it provide faster and easier detection methods, it also reduces analysis time compared to
traditional biological analysis. With advances in various fields, interdisciplinary collaboration is
indispensable in order to meet clinical needs, including electrodes, nanomaterials, signal processing,
Figure 5.
Examples of microfluidic devices with microposts for capturing tumor cells. (
A
) Schematic of
circulating tumor cell capturing system with microposts. (
B
) SEM image of flower shaped microposts.
(
C
) SEM image of flower shaped micropost with captured tumor cell. (
D
) Schematic of nano-interfaced
microfluidic exosome platform and Graphene oxide/polydopamine (GO/PDA) coated interface. (
E
) SEM
image of Y shaped microposts with GO/PDA coating. (
F
) SEM image of GO/PDA-coated channel.
Figures (
A
C
) reproduced with permission of [
76
], Springer Nature
©
, 2013; and (
D
F
) [
101
], RSC
©
, 2016.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
In addition, some of the characteristics of inkjet printing, such as its stability and reproducibility,
have been applied in a case using GO to detect cancer. Lee et al. used the principle of the field-eect
transistor to create a GO support system (GOSS) for pentacene-based field eect transistor (FET)s
to detect target DNA and Circulating tumor microemboli (CTM) (Figure 6C,D) [
120
]. Using the
malleable and biocompatible pentacene as the active layer in the FET system, inkjet printing was
used to inject materials such as GOSS, which can increase the linkage between antibodies and target
DNA, onto the pentacene. Finally, GO was modified with probe DNA or breast cancer-specific
antibody (HER2) to capture target DNA and CTM (SkBr3). The electrical analysis will dier when
the system is hybridized with DNA or CTM. In addition, the DNA of HPV (human papilloma virus),
which is associated with cervical cancer, can be used to determine the presence of cervical cancer [
121
].
Teengam et al. developed an inexpensive, disposable HPV-DNA detection and monitoring device
using inkjet printing with graphene-polyaniline (G-PANI) conductive ink to modify a paper-based
electrochemical biosensor (Figure 6E) [
122
]. The device has advantages for large-scale production,
stability, and reproducibility and has reduced demand for samples. In addition, it is modified with
a synthetic anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) to
detect papillomavirus (HPV) type 16 DNA. The current decreases greatly after the target is added,
and the degree of capture is determined. In addition to inkjet printing, there have been reports of a
device using screen printing of GO for determining blood
α
-amylase concentration to detect pancreatic
and lung cancer [
123
]; blood
α
-amylase concentration may be associated with pancreatic [
124
] and lung
Nanomaterials 2019,9, 1725 11 of 17
cancer [
125
]. Teixeira et al. developed an
α
-amylase immunosensor platform by electropolymerization
of aniline on a screen-printed graphene electrode, forming a layer of polyaniline film. This film can
transfer electrons to the underlying graphene layer, immobilization of
α
-amylase antibody allows
capture of
α
-amylase molecules, and the
α
-amylase signal is quantified using the principles of
electrochemical impedance spectroscopy (EIS). The signal has a linear response when the concentration
of
α
-amylase is between 1 and 1000 international units/L (IU/L). Similarly, screen printing has been
used to print graphene, which was used by Haque et al. for the diagnosis and evaluation of DNA
methylation to distinguish between dierent types of cancer.
With respect to specific epigenetic indicators of the degree of DNA methylation, disease diagnosis
and prognosis can be estimated by the degree of methylation, because dierent cancers have dierent
degrees of methylation in specific body sites [
126
]. Through the anity between DNA bases and
graphene, the authors obtained and processed single-stranded DNA from cells and added it to the
surface of graphene-modified screen-printed carbon electrodes (g-SPCE), along with
Fe(CN)6(3/4)
.
If there is single-stranded DNA with a high degree of methylation, the DPV current will be lower
on conventional dierential pulse voltammetry analysis. The authors used the above methods with
esophageal squamous cell carcinoma (ESCC) cell lines and esophageal squamous cell carcinoma
cells to determine the degree of FAM134B promoter gene methylation and the degree of FAM134B
gene expression.
Nanomaterials 2019, 9, x FOR PEER REVIEW 11 of 17
Figure 5. Examples of microfluidic devices with microposts for capturing tumor cells. (A) Schematic
of circulating tumor cell capturing system with microposts. (B) SEM image of flower shaped
microposts. (C) SEM image of flower shaped micropost with captured tumor cell. (D) Schematic of
nano-interfaced microfluidic exosome platform and Graphene oxide/polydopamine (GO/PDA)
coated interface. (E) SEM image of Y shaped microposts with GO/PDA coating. (F) SEM image of
GO/PDA-coated channel. Figures (AC) reproduced with permission of [76], Springer Nature©, 2013;
and (DF) [101], RSC©, 2016. This article is licensed under a Creative Commons Attribution-
NonCommercial 3.0 Unported Licence.
Figure 6. Examples of inkjet printing utilized in tumor-related molecule sensing. (A) Schematic of
inkjet printing graphene oxide. (B) Uniformly inkjet-printed different sizes of graphene oxide
micropattern. (C) Schematic of graphene oxide support system (GOSS) and its detection mechanism.
(D) Electrical performance of original pentacene field-effect transistor (FET) and inkjet-printed
pentacene FET. (E) Schematic of paper-based electrochemical biosensor and its sensing mechanism.
Figures (A,B) reproduced with permission of [127], Wiley©, 2018; (C,D) [120], RSC©, 2017; and (E)
[122], Elsevier©, 2017.
12. Outlook
With the advancements in hardware technology and the development of nanomaterials, the
combination of GO and biosensors provides great benefits in the detection of clinical biomarkers. Not
only does it provide faster and easier detection methods, it also reduces analysis time compared to
traditional biological analysis. With advances in various fields, interdisciplinary collaboration is
indispensable in order to meet clinical needs, including electrodes, nanomaterials, signal processing,
Figure 6.
Examples of inkjet printing utilized in tumor-related molecule sensing. (
A
) Schematic of inkjet
printing graphene oxide. (B) Uniformly inkjet-printed dierent sizes of graphene oxide micropattern.
(
C
) Schematic of graphene oxide support system (GOSS) and its detection mechanism. (
D
) Electrical
performance of original pentacene field-eect transistor (FET) and inkjet-printed pentacene FET.
(
E
) Schematic of paper-based electrochemical biosensor and its sensing mechanism. Figures (
A
,
B
)
reproduced with permission of [
127
], Wiley
©
, 2018; (
C
,
D
) [
120
], RSC
©
, 2017; and (
E
) [
122
],
Elsevier©, 2017.
12. Outlook
With the advancements in hardware technology and the development of nanomaterials,
the combination of GO and biosensors provides great benefits in the detection of clinical biomarkers.
Not only does it provide faster and easier detection methods, it also reduces analysis time compared
to traditional biological analysis. With advances in various fields, interdisciplinary collaboration is
indispensable in order to meet clinical needs, including electrodes, nanomaterials, signal processing,
and biomedicine. We expect that the combination of GO and biosensors mentioned in this paper will
be developed and further help liquid biopsies; this non-invasive and novel diagnosis method can be
Nanomaterials 2019,9, 1725 12 of 17
used in clinical detection. Moreover, semiconductor materials will no longer be limited to their original
form, but will be combined with other biomaterial interfaces to develop fast and convenient detection
platforms with high sensitivity and high biocompatibility.
Author Contributions:
All authors contributed toward conceptualization, preparation, and validation of
the manuscript.
Funding:
G.Y.C. would like to acknowledge financial support from National Chiao Tung University (108W204,
108W211), the Ministry of Science and Technology (MOST 107-2622-E-009-023-CC1, MOST-107-EPA-F-007-002,
MOST-108-2636-E-009-007-), the National Health Research Institutes (NHRI-EX108-10714EC), and the Higher
Education Sprout Project of the National Chiao Tung University and the Ministry of Education, Taiwan. This work
was supported in part by the Novel Bioengineering and Technological Approaches to Solve Two Major Health
Problems in Taiwan sponsored by the Taiwan Ministry of Science and Technology Academic Excellence Program
under Grant Number: MOST 108-2633-B-009-001.
Conflicts of Interest: The authors declare no conflicts of interest.
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... Moreover, it is well known that the sensitivity and efficiency of electrochemical sensors are strongly associated with electrode materials [285]. Thus, GO with graphene-like structure and containing oxygen-based functional groups have many advantages over graphene, including higher solubility and the possibility for surface functionalization, making it an appropriate material for biosensor applications [270,289]. Remarkably, the use of GO as an electrode has been increasing very fast, owing to its outstanding biocompatibility, great hydrophilicity, huge specific surface area and abundant functional groups [286]. Like GO, rGO-based biosensors exhibit huge attention as a material with both graphene and GO advantages. ...
... The use of microfluidics to develop portable, easy-to-use and sensitive LOC devices for real-time detection presents several advantages over conventional analytical tools [347]. The modest consumption of liquid samples and the small dimensions of structures are distinguishing properties of microfluidics, which have a considerable influence on the development of biosensors [245,289]. Graphene is one of the best materials to design microfluidic devices among several nanomaterials owing to its eccentric structural characteristics and remarkable performances [246]. The single-layer graphene has superior transparency that can transmit 98% of visible light, it has impressively terrific electron mobility and high thermal conductivity, ultra-thin (about 0.35 nm) and ultra-light structure and low planar density (0.77 mg/m 2 ) [37,348]. ...
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Nowadays, cancer is increasingly becoming one of the foremost threats to human being life worldwide, and diagnosing this deadly disease is one of the major priorities of researchers. Described as a monolayer-thin-sheet of hexagonally patterned carbon atoms, ‘graphene’ is considered an innovative evergreen carbon material ideal for a wide array of sensing applications and nanotechnologies. Graphene-based materials have acquired a huge share of interest in the scope of biosensor fabrication for early and accurate cancer diagnosis. Herein, we have insights reviewed the various routes and technologies for synthesized graphene, and graphene-based materials including 3D graphene (i.e., hydrogels, foams, sponges, porous), and 0D graphene (i.e., quantum dots). Moreover, we have introduced the different types of graphene/graphene-based materials biosensors (i.e., electrochemical biosensors, optical biosensors, field-effect transistors biosensors, electrochemiluminescence biosensors, and microfluidics biosensors) and their merits and applications for cancer pre-stage detection.
... Moreover, it is well known that the sensitivity and efficiency of electrochemical sensors are strongly associated with electrode materials [285]. Thus, GO with graphene like structure and containing oxygen-based functional groups has many advantages over graphene, including higher solubility and the possibility for surface functionalization, making it an appropriate material for biosensor applications [270,289]. ...
... The use of microfluidics to develop portable, easy-to-use, and sensitive LOC devices for real-time detection presents several advantages over conventional analytical tools [349]. The modest consumption of liquid samples and the small dimensions of structures are distinguishing properties of microfluidics, which have a considerable influence on the development of biosensors [245,289]. ...
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Nowadays, cancer is increasingly becoming one of the foremost threats to human being life worldwide, and diagnosing this deadly disease is one of the major priorities of researchers. Described as a monolayer-thin-sheet of hexagonally patterned carbon atoms, 'graphene' is considered an innovative evergreen carbon material ideal for a wide array of sensing applications and nanotechnologies. Graphene-based materials have acquired a huge share of interest in the scope of biosensor fabrication for early and accurate cancer diagnosis. Herein, we have insights reviewed the various routes and technologies for synthesized graphene, and graphene-based materials including 3D graphene (i.e., hydrogels, foams, sponges, porous), and 0D graphene (i.e., quantum dots). Moreover, we have introduced the different types of graphene/graphene-based materials biosensors (i.e., electrochemical biosensors, optical biosensors, field-effect transistors biosensors, electrochemiluminescence biosensors, and microfluidics biosensors) and their merits and applications for cancer pre-stage detection.
... Undoubtedly interesting in terms of research and practical approach is the combination of graphene oxide and gold foil to enrich the isolation of circulating new cells [32,36]. Yoon et al. [32] presented the results of a study of a system for the sieving of circulating tumor cells, which also allowed for the storage and multiplication of isolated cells. ...
... The method for isolating cancer cells differed from the methods described in publications [16,17], which used mainly systems (chips) made of partially or fully transparent polymer materials. Graphene and graphene derivatives are rarely used, although studies have shown that the screening efficiency of circulating tumor cells has been improved [33][34][35][36]. The main purpose of these devices is to separate and diagnose cells. ...
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This work shows the effect of graphene oxide deposition on microsieves’ surfaces of gold and nickel foils, on DU 145 tumor cells of the prostate gland. The sieves were made by a laser ablation process. The graphene oxide (GO) deposition process was characterized by the complete covering of the inner edges of the microholes and the flat surface between the holes with GO. Electron microscanning studies have shown that due to the deposition method applied, graphene oxide flakes line the interior of the microholes, reducing the unevenness of the downstream surfaces during the laser ablation process. The presence of graphene oxide was confirmed by Fourier infrared spectroscopy. During the screening (sieving) process, the microsieves were placed in a sieve column. Gold foil is proven to be a very good material for the screening of cancer cells, but even more so after screening as a substrate for re-culture of the DU 145. This allows a potential recovery of the cells and the development of a targeted therapy. The sieved cells were successfully grown on the microsieves used in the experiment. Graphene oxide remaining on the surface of the nickel sieve has been observed to increase the sieving effect. Although graphene oxide improved separation efficiency by 9.7%, the nickel substrate is not suitable for re-culturing of the Du 145 cells and the development of a targeted therapy compared to the gold one.
... The long range pi conjugation in graphene leads to outstanding electrical, thermal, and mechanical properties [184]. These properties have been recently used for molecular detection and analysis of circulating biomarkers including EVs [185]. The electrical property of graphene and graphene oxide provide an excellent opportunity to design field-effect-transistor (FET)based biosensors for EV detection [186][187][188][189][190]. For example, Tsang et al. fabricated a backgated graphene FET biosensor by depositing a graphene monolayer onto a microfluidic device by chemical vapor deposition [186]. ...
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Extracellular vesicles (EVs) have emerged as a novel resource of biomarkers for cancer and certain other diseases. Probing EVs in body fluids has become of major interest in the past decade in the development of a new-generation liquid biopsy for cancer diagnosis and monitoring. However, sensitive and specific molecular detection and analysis are challenging, due to the small size of EVs, low amount of antigens on individual EVs, and the complex biofluid matrix. Nanomaterials have been widely used in the technological development of protein and nucleic acid-based EV detection and analysis, owing to the unique structure and functional properties of materials at the nanometer scale. In this review, we summarize various nanomaterial-based analytical technologies for molecular EV detection and analysis. We discuss these technologies based on the major types of nanomaterials, including plasmonic, fluorescent, magnetic, organic, carbon-based, and certain other nanostructures. For each type of nanomaterial, functional properties are briefly described, followed by the applications of the nanomaterials for EV biomarker detection, profiling, and analysis in terms of detection mechanisms.
... The development of graphene-based materials has also been studied. Graphene-based materials have been also studied for the generation of new imaging agents for the in vitro and in vivo diagnosis [20] of various cancer as well as for the development of biosensors for the detection of specific cancer biomarkers [53] . ...
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Cancer is a pathologically complex illness. In present times, selectivity, cytotoxicity, induction of multidrugresistance, and the production of stem-like cells are side-effects associated with chemotherapy. Due to the crucialand pharmacokinetically unique environment that occurs in tumour tissue, targeted delivery of therapeuticmolecules to tumour tissue is one of the most exciting and hard attempts in the clinical and pharmaceuticalsector. Nanomaterials are small molecules with unique optical, magnetic, and electrical properties (1-100 nm).To overcome toxicity and lack of selectivity, improved drug efficacy and bioavailability, and target cancer cells,the tumour microenvironment, and the immune system, these nanomaterials have been tailored for a widespectrum of cancer medications. Nanoparticle-based drug delivery has several advantages over traditional drugdelivery, including increased stability and biocompatibility, increased permeability and retention effect, andprecision targeting. Hybrid nanoparticles, which combine the capabilities of many nanoparticles, have taken thissort of drug-carrier system to the next level. Furthermore, it has been demonstrated that nanoparticle-based drugdelivery systems can help overcome resistance to cancer-related medication. Overexpression of drug effluxtransporters, faulty apoptotic pathways, and a hypoxic environment are some of the factors that lead to cancerdrug resistance. Multidrug resistance can be improved by using nanoparticles that target these processes. Despitean increase in the number of studies, the number of approved nano-drugs has remained scanty over time. Moreresearch on targeted drug delivery using nano-carriers to reduce toxicity, improve permeability and retentioneffects, and minimize the shielding impact of protein corona is needed to improve the clinical translation. Thisreview describes innovative nanomaterials manufactured in research and clinical usage in recent times. Thereview also summarizes the advancements of use of nanomaterial in cancer diagnosis and therapy. Moreover, thearticle attempts to forecasts the prospects and perspectives of diagnostic and therapeutic potency (theranostic) ofdifferent nanoparticles in cancer management.
... But, issues of photostability, quantum yield, and high cost displayed the need for materials that can provide better diagnosis at a lower cost than conventional organic dyes [16]. Many researchers have extensively studied GNs and their derivatives: graphene oxide (GO) and reduced graphene oxide (rGO) modified for various applications, functionalization with biomarkers for diagnosis [17,18] and for targeted drug delivery [19][20][21]. The capability of graphene-based nanomaterials for biosensing applications has been reviewed by several groups [22][23][24]. ...
Article
The rising demand for early-stage diagnosis of diseases such as cancer, diabetes, neurodegenerative can be met with the development of materials offering high sensitivity and specificity. Graphene quantum dots (GQDs) have been investigated extensively for theranostic applications owing to their superior photostability and high aqueous dispersibility. These are attractive for a range of biomedical applications as their physicochemical and optoelectronic properties can be tuned precisely. However, many aspects of these properties remain to be explored. In the present review, we have discussed the effect of synthetic parameters upon their physicochemical characteristics relevant to bioimaging. We have highlighted the effect of particle properties upon sensing of biological molecules through ‘turn-on’ and ‘turn-off’ fluorescence, and generation of electrochemical signals. After describing the effect of surface chemistry and solution pH on optical properties, an inclusive view on application of GQDs in drug delivery and radiation therapy has been given. Finally, a brief overview on their application in gene therapy has also been included.
... Hence, GO can be assumed as a superior acceptor for DNA-containing fluorescent probes in RET. The bases of this mechanism is that these probes can be caught by the GO surface by π-π stacking, which leads to fluorescence quenching by RET and then, after the DNA probes bind to the target sequence, fluorescence is switched on due to separation of the DNA from the GO surface in situ [234]. ...
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Cancer theranostics is a new concept of medical approach that attempts to combine in a unique nanoplatform diagnosis, monitoring and therapy so as to provide eradication of a solid tumor in a non-invasive fashion. There are many available solutions to tackle cancer using theranostic agents such as photothermal therapy (PTT) and photodynamic therapy (PDT) under the guidance of imaging techniques (e.g., magnetic resonance-MRI, photoacoustic-PA or computed tomography-CT imaging). Additionally, there are several potential theranostic nanoplatforms able to combine diagnosis and therapy at once, such as gold nanoparticles (GNPs), graphene oxide (GO), superparamagnetic iron oxide nanoparticles (SPIONs) and carbon nanodots (CDs). Currently, surface functionalization of these nanoplatforms is an extremely useful protocol for effectively tuning their structures, interface features and physicochemical properties. This approach is much more reliable and amenable to fine adjustment, reaching both physicochemical and regulatory requirements as a function of the specific field of application. Here, we summarize and compare the most promising metal-and carbon-based theranostic tools reported as potential candidates in precision cancer theranostics. We focused our review on the latest developments in surface functionalization strategies for these nanosystems, or hybrid nanocomposites consisting of their combination, and discuss their main characteristics and potential applications in precision cancer medicine.
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The response to neoadjuvant chemotherapy (NAC) is highly correlated with survival in breast cancer (BC) patients. The early prediction of the response to NAC could facilitate treatment adjustments on a patient-by-patient basis, which would improve patient outcomes and survival. Conventional techniques used for detecting circulating microRNAs (miRNAs), which act as biomarkers for the early prediction of NAC efficacy in BC patients, are associated with limitations such as low sensitivity and specificity. We designed a highly sensitive graphene oxide (GO)-based qRT-PCR method for detecting miRNAs associated with the chemotherapeutic response in BC patients. The results showed that miRNA levels at both the baseline and end of the first NAC cycle could help distinguish NAC responders from NAC nonresponders; BC patients with lower plasma miRNA levels were more likely to achieve pathological complete remission. Thus, GO-based qRT-PCR could facilitate early prediction of NAC efficacy in BC patients.
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Two-dimensional (2D) nanomaterials have been widely used in pharmaceutical applications due to their advantages of higher drug loading, controllable drug release, high stability, biodegradability, biocompatibility, and generally lower toxicity. This review presents state-of-the-art advancement in the context of utilizing 2D nanomaterials for the diagnosis and treatment of Alzheimer's disease (AD). The core pathological features of AD include amyloid plaques formed by amyloid beta protein (Aβ) aggregation in the cerebral cortex and hippocampus, and the self-assemble of Aβ peptides into β-sheet-rich aggregates. Early and accurate detection of AD biomarkers provide potential means for its diagnosis and treatment. Studies have indicated that 2D nanomaterials can significantly improve the detection accuracy and specificity of AD biomarkers (such as Aβ protein, tau protein, acetylcholinesterase (AChE), Acetylcholine (ACh), MicroRNAs, and S100β protein). In the treatment of AD, inhibiting the aggregation of Aβ and transforming the β-sheet structure of Aβ aggregates into harmless structures has been proved to be the effective strategies. 2D nanomaterials have application potential in the treatment of AD because they can degrade Aβ peptides or inhibit Aβ peptide aggregation through photodynamic or photothermal effects. However, most of the in vivo cytotoxicity mediated by nanomaterials has not yet been resolved.
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The performance of current microfluidic methods for exosome detection is constrained by boundary conditions, as well as fundamental limits to microscale mass transfer and interfacial exosome binding. Here, we show that a microfluidic chip designed with self-assembled three-dimensional herringbone nanopatterns can detect low levels of tumour-associated exosomes in plasma (10 exosomes μl ⁻¹ , or approximately 200 vesicles per 20 μl of spiked sample) that would otherwise be undetectable by standard microfluidic systems for biosensing. The nanopatterns promote microscale mass transfer, increase surface area and probe density to enhance the efficiency and speed of exosome binding, and permit drainage of the boundary fluid to reduce near-surface hydrodynamic resistance, thus promoting particle–surface interactions for exosome binding. We used the device for the detection—in 2 μl plasma samples from 20 ovarian cancer patients and 10 age-matched controls—of exosome subpopulations expressing CD24, epithelial cell adhesion molecule and folate receptor alpha proteins, and suggest exosomal folate receptor alpha as a potential biomarker for early detection and progression monitoring of ovarian cancer. The nanolithography-free nanopatterned device should facilitate the use of liquid biopsies for cancer diagnosis. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
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The high purity of target cells enriched from blood samples plays an important role in the clinical detection of diseases. However, non-specific binding of blood cells in the isolated cell samples can complicate downstream molecular and genetic analysis. In this work, we report a simple solution to non-specific binding of blood cells by modifying the surface of microchips with a multilayer nanofilm, with the outmost layer containing both PEG brushes for reducing blood cell adhesion and antibodies for enriching target cells. This layer-by-layer (LbL) polysaccharide nanofilm was modified with neutravindin and then conjugated with a mixture of biotinylated PEG molecules and biotinylated antibodies. Using EpCAM-expressing and HER2-expressing cancer cells in blood as model platforms, we were able to dramatically reduce the non-specific binding of blood cells to approximately 1 cell/mm2 without sacrificing the high capture efficiency of the microchip. To support the rational extension of this approach to other applications for cell isolation and blood cell resistance, we conducted extensive characterization on the nanofilm formation and degradation, antifouling with PEG brushes and introducing functional antibodies. This simple, yet effective, approach can be applied to a variety of microchip applications that require high purity of sample cells containing minimal contamination from blood cells.
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Recently, implantable artificial subretinal chips using electronic components have replaced photoreceptors to serve as the most feasible treatment for retinal diseases. As such a chip that is meant to be implanted and used for very long periods, growing retinal cells on it to improve the electrical stimulation efficiency and attraction of neuronal elements remains a challenge. Here, an inkjet printing technology is employed to create graphene oxide (GO) micropatterns onto microelectrodes of a photovoltaic‐powered implantable retinal chip. These GO micropatterns allow human retinal pigment epithelium (RPE) cells to specially attach and grow in each microelectrode. In addition, the cell proliferation, viability, and tight junction of RPE cells are improved during culturing. The development of a simple surface‐coating technology would pave the way for the development of the first fully integrated and encapsulated retinal prostheses with biocompatible on‐chip microelectrodes for long‐term implantation, which could be effectively applied in retina tissue engineering and therapy.
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Background: Eukaryotic cells release vesicles of different sizes under both physiological and pathological conditions. On the basis of the respective biogenesis, extracellular vesicles are classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are considered tools for innovative therapeutic interventions, especially when engineered with effector molecules. The delivery functions of exosomes are favored by a number of typical features. These include their small size (i.e., 50-200 nm), the membrane composition tightly similar to that of producer cells, lack of toxicity, stability in serum as well as other biological fluids, and accession to virtually any organ and tissue including central nervous system. However, a number of unresolved questions still affects the possible use of exosomes in therapy. Among these are the exact identification of both in vitro and ex vivo produced vesicles, their large scale production and purification, the uploading efficiency of therapeutic macromolecules, and the characterization of their pharmacokinetic. Objective: Here, we discuss two key aspects to be analyzed before considering exosomes as tool of delivery for the desired therapeutic molecule, i.e., techniques of engineering, and their in vivo biodistribution/pharmacokinetic. In addition, an innovative approach aimed at overcoming at least part of the obstacles towards a safe and efficient use of exosomes in therapy will be discussed. Conclusion: Several biologic features render exosomes an attractive tool for the delivery of therapeutic molecules. They will be surely part of innovative therapeutic interventions as soon as few still unmet technical hindrances will be overcome.
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Breast Cancer (BRCA) is the most common threat in women worldwide. Increasing death rate of diagnosed cases is the main leading cause of designing specific genosensors for BRCA − related cancer detection. In the present study, an ultrasensitive label − free electrochemical DNA (E − DNA) sensor based on conducting polymer/reduced graphene − oxide platform has been developed for the detection of BRCA1 gene. An electrochemical method was applied as a simple and controllable technique for the electrochemical reduction of graphene oxide and also, electro − polymerization of pyrrole − 3 − carboxylic acid monomer. The results of the present work show that the polymer − coated reduced graphene − oxide provide more active sites compare to polymer − coated glassy carbon electrode for the DNA probe immobilization. The fabricated signal − off E − DNA sensor employs Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS) techniques for monitoring the electrochemical behavior of the redox probe. To survey the morphological pattern and surface structural characterizations, the Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) have been applied, respectively. The applicability of the modified surface layer toward DNA sensing was evaluated by both DPV and EIS measurements. This platform permits quantitative determination of BRCA1 in the range of 10 fM−0.1 μM with a limit of detection as low as 3 fM. The proposed genosensor showed a perfect discriminatory power between complementary, non − complementary and mismatched DNA sequences. The described method combined the outstanding features in terms of selectivity, sensitivity, repeatability, reproducibility and remarkable reusability, without demanding for labor − intensive labeling steps. Moreover, the modified electrode was successfully used for accurate determination of trace amounts of DNA target in blood plasma samples.
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Exosomes have proved to be an effective cancer biomarker with significant potential, and several cell-specific molecules have been found in colorectal cancer (CRC) exosomes. Nevertheless, it is challenging to use exosomes in clinical lab diagnostics due to their nanoscale and the lack of a convenient and effective detection platform. Here, we developed a DNase I enzyme-aided fluorescence amplification method for CRC exosome detection, based on graphene oxide (GO)-DNA aptamer (CD63 and EpCAM aptamers) interactions. The fluorescence of fluorophore-labeled aptamers quenched by GO, recovered after incubation with samples containing CRC exosomes. The DNase I enzyme digested the single-stranded DNA aptamers on the exosome surface and the exosomes were able to interact with more fluorescent aptamer probes, resulting in an increase of signal amplification. The limit of detection for CRC exosomes is 2.1 × 10⁴ particles/μl. Consequently, a rapid and effective method with high sensitivity was established. The method was verified in 19 clinical blood serum samples to distinguish healthy and CRC patients, showing significant diagnostic power. Moreover, it can be expanded to other kinds of cancer exosomes, in addition to CRC.
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Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.