Chapter 13 Cytotoxicity of graphene and
Graphene-based Biosensors
13.1 Anti-bacterial graphene-based materials
13.2 Cytotoxicity of graphene and graphene-related materials
13.3 Graphene-based biosensors
1
13.1 Anti-bacterial graphene-based materials [13-1]
Preparation of GO and rGO
GO nanosheets were prepared according to a modified Hummer method,
resulting in a brown colloidal suspension. The thickness of the GO sheets was 1.1
nm as measured via atomic force microscopy (AFM), suggesting the formation of a
single-layer 2-D nanomaterial (Figure 1a). Hydrazine reduction of GO led to a black
rGO suspension, a more conductive version of GO nanosheets with less surface
defects. AFM measurements revealed that rGO had a reduced sheet thickness of
1.0 nm (Figure 1b), which was possibly attributed to partial removal of oxygen
functional groups on the surface of GO nanosheets during the reduction process.
Figure 1
2
Cellular uptake and cytotoxicity of GO nanosheets (85 g/mL) with a mammalian
cell line, A549. TEM studies demonstrated that GO nanosheets were inside the
endosome of the cytoplasm (Figure 2a), suggesting that GO nanosheets could be
internalized within A549 cells via endocytosis. GO nanosheets (20 g/mL) exhibited
no cytotoxicity to A549 within 2 h incubation and a slight decrease in cell viability
(20%) within 24 h. GO nanosheets of higher concentration (85 g/mL) led to an
increased cytotoxicity (50%) within 24 h (Figure 2b).
※
Fig. 2c depicts the
distribution of A549
cells without GO
nanosheet
treatment (left) and
treated with 20
g/mL (middle) and
85 g/mL (right) GO
for 24 h.
3
The number of untreated cells was 3.11-fold that of seeded cells (Table S1),
while the number of cells treated with GO nanosheets of 85 g/mL proliferated 2.78fold. The results implied that the observed small decrease in cell viability might
arise from GO-retarded cell cycles and thus slightly decreased proliferation rates,
rather than from apoptosis or death of cells. Therefore, we concluded that GO
nanosheets were relatively biocompatible nanomaterials with mild cytotoxicity.
Table S1*
人類周邊
血中的
顆粒球(G)，
單核球(M)
Flow cytometry 技術主要是為了快速偵測一顆接著一顆流動於液體水柱（fluid
stream）中的顆粒或細胞，因此flow cytometry 所偵測的訊號是以一個（而非一群）
細胞或顆粒，被雷射光激發後產生的光學訊號，再轉換電子訊號由電腦分析細胞
或顆粒的特性。
*流式細胞技術 Flow Cytometry 謝世良
4
Antibacterial Activity of GO Nanosheets
The metabolic activity of E. coli DH5 cells in the presence of GO nanosheets was
measured via a luciferase-based ATP assay kit. (Luciferase is a term for the class of
oxidative enzymes used in bioluminescence)
After 2 h incubation with GO nanosheets of 20 g/mL at 37 °C, the cell metabolic
activity for E. coli deceased to 70% and to 13% at a GO nanosheet concentration of 85
g/mL (Figure 3a), suggesting the strong inhibition ability of GO nanosheets to E. coli.
A classic colony counting method was to measure the microbial viability of E. coli
treated with 85 g/mL GO for 2 h. Significantly, GO almost completely suppressed the
growth of E. coli, leading to a viability loss up to 98.5% (Figure 3b).
TEM studies revealed that E. coli largely lost cellular integrity, with the cell
membrane being severely destroyed and the cytoplasm flowing out (Figure 3c,d).
5
Figure 4. shows antibacterial activity and cytotoxicity of rGO nanosheets. (a)
Metabolic activity of E. coli treated with 85 g/mL GO and rGO nanosheets,
respectively. (b) Antibacterial activity of 85 g/mL GO and rGO nanosheets against E.
coli. (c) TEM image of E. coli exposed to 85 g/mL rGO nanosheets at 37 °C for 2 h;
TEM studies revealed that rGO nanosheets destroyed the membrane of E. coli in a
way similar to that of GO nanosheets (Figure 4c). (d) Viability of A549 cell incubated
with 20 and 85 g/mL rGO nanosheets, respectively.
Figure 4.
rGO nanosheets possessed antibacterial
properties that were only slightly lower than those
of GO nanosheets while their cytotoxicity was
significantly higher than GO’s. Such difference in
cytotoxicity might arise from different surface
charges and functional groups of GO and rGO6
nanosheet surfaces.
Fig. 5 Photographs of E. coli
growth on GO (a) and rGO (b)
paper (overnight incubation at
37 °C). SEM images of E. coli
attached to GO (c) and rGO
(d) paper (12 h incubation at
37 °C).
After overnight incubation at 37 °C, we could not find any cell growth on the GO
paper (Figure 5a) and only a limited number of E. coli colonies on the rGO paper
(Figure 5b), implying the superior antibacterial effect of such graphene-based
papers. In contrast, control studies in the absence of either GO or rGO paper led
to a great number of colony-forming units (CFU). SEM studies further confirmed
that E. coli cells on the paper lost the integrity of membranes (Figure 5c,d), which
was responsible for the bacteria-killing effect of the graphene-based paper.
7
13.2 Cytotoxicity of graphene and graphene-related materials [13-2]
Most reports show that GO materials, including GO films (paper), are superior
biocompatible materials that allow the effective proliferation of human and
mammalian cells with limited or no cytotoxicity. Such characteristics seem to
indicate that GO materials may be used in tissue engineering, tissue implants,
wound therapy, and drug delivery applications.
Recently, several reports have shown that GO paper promotes the adhesion
and proliferation of L-929 cells, osteoblasts, kidney cells, and embryonic cells.
However, additional studies have shown that cellular internalization of GO
nanosheets applied to the culture media at a concentration of 20 μg/mL can cause
a 20% decrease in mammalian cell viability, while a concentration of 50 μg/mL can
lead to a 50% loss in cell viability, indicating that some inhibitory effect can be
observed if a GO suspension is applied to the growth media.
A recent study showed that graphene and graphene oxide materials are cytotoxic
to human erythrocytes and skin fibroblasts. Another study showed that films
developed from a suspension of reduced graphene oxide and polyoxyethylene
sorbitan laurate (TWEEN) were noncytotoxic to three different types of mammalian
cells. These combined results appear to support that GO materials are biocompatible
with mammalian cells by promoting cell adhesion and proliferation as effectively as
commercial polystyrene tissue culture materials. On the other hand, colloidal
8
GO solutions appeared to be mildly cytotoxic at high concentrations.
On the other hand, recent studies have also indicated that GO is not cytotoxic and
also lacks any antibacterial effect. Das et al. showed that, when GO was placed in
the center of a nutrient media plate previously inoculated with bacteria, a growth
inhibition zone was not formed. Alternatively, when silver decorated GO was used, a
clear inhibition zone was formed.
It shows that graphene oxide materials do not adversely impact microbial and
mammalian cell growth. Furthermore, graphene oxide materials tend to produce a
dramatic increase in microbial and mammalian cell proliferation, indicating that
graphene oxide is not a bactericidal or bacteriostatic material, but instead a general
growth enhancer that acts as a scaffold for cell surface attachment and proliferation.
The results showed that the GO-containing
samples achieved an average absorbance of 1.7
in 16 h of incubation while the bacteria growing in
LB broth only achieved an absorbance of 1.3
(Figure 1a) These results indicated that bacteria
in the presence of GO grew faster than bacteria
in LB media and were able to achieve cell
saturation sooner.
9
The culture tubes containing graphene oxide did not
visually show any apparent reduction in bacterial growth
(Figure 1b). Furthermore, they appeared more turbid than
the control culture (Figure 1c), and a dense dark precipitate
was observed at the bottom of the tube (Figure 1b). The
dark precipitate was not produced in the control cultures
without GO (Figure 1c).
We proceeded to determine growth level in the bacteria
cultures by measuring the absorbance at 600 nm. Samples
were taken from the supernatant without disturbing the
dark precipitates at the bottom of the samples containing
GO. It was possible that the dark precipitate observed in
samples containing GO was responsible for enhancing
bacterial growth in the media or harboring bacterial growth
itself.
SEM analysis showed
that the dark precipitate was
formed by a thick bacterial
biofilm(Fig.1f,g) containing a
large mass of aggregated
cells (Fig. 1g) and
extracellular polymeric
material (Fig. 1f).
10
The results showed that the precipitation of GO in the culture media may be
acting as a scaffold for bacterial attachment, proliferation, and biofilm formation.
Studies have shown that carbon nanomaterials could act as attachment surfaces
where small colonies grow around tubular carbon nanostructures. Further, it
seems that precipitated GO induced massive cell growth, aggregation, and
secretion of extracellular polymeric substance (EPS) (Figure 1f,g).
Quantitative real-time PCR was used to assess bacterial growth in filters
with and without GO (a). PVDF filters coated with 0 (c), 25 (d), and 75 μg (e) of
GO were inoculated with E. coli and incubated at 37 C for 18 h.
The results shown in Figure 2a indicate that GO not only lacks antimicrobial
properties, but that it actually enhances microbial growth when coated onto
another surface.
11
Small ∼1 cm2 pieces of PVDF filter (f), GO film (g), and Ag-GO film were inoculated
with E. coli and culture for 18 h at 37 C. Bacterial growth was quantified by real-time
PCR (b).
An area of dense bacterial growth in the LB media was produced around all neat
filter replicates (Figure 2c). This halo of cells was not observed in any of the GOcoated filters (Figure 2d,e). This was an interesting observation that implied that
there is an inherent preference by bacteria to attach and grow in areas containing
GO, especially those areas containing the highest GO levels (Figure 3).
Bacteria growth was observed with the naked eye in all samples, but the filters
containing GO presented large bacteria colonies around specific areas that seem
to contain more GO (Figure 3a-d).
12
Figure 3 Bacteria interaction with graphene oxide. Black arrows indicate some of
the areas with increased bacterial growth observed on filters coated with 25 (a,b)
and 75 μg (c,d) of GO. Bacterial colonies can be easily observed as elongated
features in GO-coated filters but not in a neat PVDF filter.
Bacteria Interaction with GO and Ag-GO Films.
Graphene oxide films were placed onto LB culture plates that were previously
inoculated with 1 x106. Then, Then, 1 x106 E. coli cells were directly inoculated on
top of the film pieces and allowed to dry. The purpose of this type of inoculation
was to observe growth over the GO film and also to determine if any growth
inhibition zone was formed around the GO film. Growth inhibition zones around
GO film have been reported in the past. Inhibition areas would indicate that the
material has some toxic effect on the bacteria.
13
Figure 4 shows graphene oxide and silver-coated graphene oxide
characterization. (a) TEM image of neat GO, (b) TEM image of Ag decorated GO, (c)
XRD spectrum of Ag-decorated GO and ICDD 00-004-0783 card data for facecentered cubic Ag, and (d) size distribution
studies performed using TEM for Ag-decorated
GO.
Results showed that growth inhibition zones
were not detected in the plate containing either
GO film or filter paper (Figure 2f,g). However,
Ag-decorated GO showed large growth
inhibition zones characterized by a clear area
with no cell growth (Figure 2h). These results
clearly demonstrate that GO does not have
any antimicrobial effects capable of producing
a toxic effect in the area surrounding the GO
film.
Figure 4
14
Mammalian Cell Attachment and
Proliferation onto GO Film.
A study was performed to test the role of GO film on mammalian cell attachment and
proliferation. Control glass slides and glass slides coated with 10 μg of GO (Figure 5a)
were placed onto a culture dish to which culture media and 6 x105 mammalian
colorectal adenocarcinoma HT-29 cells were added. The cells were allowed to attach
and develop on the slides. At various time intervals, cell attachment was assessed by
light microscopy. Shown in Figure 5b,c are representative images of cell morphology
after incubation for 6 h. The results indicated that the mammalian cells attached more
efficiently to the GO-coated glass slides and grew (Figure 5c).
The results of this study clearly demonstrate that graphene oxide does not have
antibacterial properties. Furthermore, graphene oxide lacks any bacteriostatic
property as shown by the prolific growth observed on all forms of GO tested. It
seems that GO acts as an enhancer of life, increasing not only mammalian cell
15
growth but also bacterial growth.
Cytotoxicity of Graphene Oxide and Graphene in Human Erythrocytes and Skin Fibroblasts
[13-4]
One method of toxicity assessment was based on measurement of the efflux of
hemoglobin from suspended red blood cells. At the smallest size, graphene oxide
showed the greatest hemolytic activity, whereas aggregated graphene sheets
exhibited the lowest hemolytic activity. Coating graphene oxide with chitosan nearly
eliminated hemolytic activity. Together, these results demonstrate that particle size,
particulate state, and oxygen content/surface charge of graphene have a strong
impact on biological/toxicological responses to red blood cells.
The compacted graphene sheets are more damaging to mammalian fibroblasts than the
less densely packed graphene oxide. Clearly, the toxicity of graphene and graphene oxide
depends on the exposure environment (i.e., whether or not aggregation occurs) and mode
of interaction with cells (i.e., suspension versus adherent cell
types).
16
Figure 4. (a) Percent hemolysis of RBCs incubated with different concentrations
(3.125 to 200 μg mL1) of GO (red), bGO (blue), pGO-5 (green), pGO-30 (purple),
and GS (black) for 3 h at 37 C with agitation. Data represent mean(SD from at least
five independent experiments. Also included is the percent hemolysis of RBCs
incubated with pGO-30/chitosan at 100 μg mL1 for 3 h at 37 C with agitation. (b)
Photographs of RBCs after 3-h exposure to GO, bGO, pGO-5, pGO-30, and GS at
different concentrations (3.125 to 200 μgmL1). The presence of red hemoglobin in
the supernatant indicates RBCs with membrane damage. (+) and () symbols
represent positive control and negative control, respectively.
17
Figure 5. Optical
microscographs of RBCs in
the presence of (a) PBS
(control), (b) pGO-5, (c) pGO30, and (d) GS at 25 μgmL1
for 3 h at 37 C with agitation.
Inset images are magnified
RBCs. Scale bars in the inset
images represent 10 μm. The
arrows in b and c show lysed
RBCs. The arrows in d
represent the
hemagglutination caused by
GS aggregates.
18
Figure 6. Cell viability of human skin fibroblast cells determined from MTT and WST8 assay after 24-h exposure to different concentrations of pGO-5 and GS. Data
represent mean ( SD.)
19
13.3 Graphene-based biosensors [13-5~8]
Biosensors comprise a selective interface in close proximity to or integrated with a
transducer, which relays information about interactions between the surface of the
electrode and the analyte either directly or through a mediator (Fig. 1).
Biosensors can be categorized depending on the transducing mechanism: (i)
resonant biosensors, (ii) optical-detection biosensors, (iii) thermal-detection
biosensors, (iv) ion-sensitive FET biosensors, and (v) electrochemical biosensors.
The existence of CNT with metallic impurity is
the main drawback while using in the modification
of electrode (Pumera, 2009, 2010). Such metallic
impurities are electrochemically active and can
dominate the electrochemistry of CNT. Impurities
present at 50 ppm can be toxicological hazards as
they can participate in redox reactions with the
biomolecules
Appropriate functionalization of graphene and the
immobilization of biomaterials on it are important, as
functional groups can create defects on graphene surfaces.
Fig. 1
20
It has been found that GO decreases cell adhesion when enters into
cytoplasm and Nucleus.
The growth of Gram-positive, Gram-negative, and Escherichia coli bacteria is
affected significantly by the sharp edges of the reduced graphene nanowalls.
Graphene-based enzymatic electrodes
1. Glucose oxidase biosensor
2. Cytochrome(細胞色素) c biosensor
3. NADH(nicotinamide adenine dinucleotide，菸醯胺腺嘌呤二核酸) biosensor
4. Hemoglobin (血紅素) biosensor
5. HRP(horseradish peroxide) biosensor
6. Cholesterol (膽固醇) biosensor
7. Catechol biosensor
Graphene-based non-enzymatic electrodes
1. Hydrogen peroxide;
2. Ascorbic acid, uric acid and dopamine
Graphene-based nano-electronic devices
1. DNA sensors;
3. Gas sensors;
2. Heavy metal ion detection
4. Field effect transistor
21
Graphene Oxide Sheet-Mediated Silver Enhancement for Application to Electrochemical
Biosensors [13-7]
Biosensors have emerged as extremely useful tools for bacterial cell detection in
the last decades. Various sensitive, reliable, and rapid methods have been reported
for the determination and monitoring of microorganisms and other biomolecules,
including surface plasma resonance, flow cytometry, epifluorescence microscopy,
quartz crystal microbalance, mass spectrometry, electrochemical techniques, and
insulator-based dielectrophoresis.
Other methods use gold nanoparticles, quantum dots, magnetic nanoparticles, or
carbon nanotubes as signal amplifiers combined with electrochemical or fluorescence
techniques. The use of nanomaterials as signal amplifiers has attracted increasing
interest in biosensor development, as well as in applications for detecting
biomolecules and bacterial cells. For example, silver (Ag) enhancement based on the
deposition substrate in gold nanoparticle probes has been used to detect and
visualize proteins,DNA, and bacteria.
This paper used functionalized graphene oxide (GO) sheets coupled with a signal
amplification method based on the nanomaterial-promoted reduction of silver ions for
the sensitive and selective detection of bacteria. The authors in this paper used an
ultrasensitive potentiometric stripping analysis (PSA) to develop an electrochemical
route combining with GO sheet-mediated Ag enhancement for biological/chemical
22
analyte detection.
They adopted antibody-functionalized GO sheet conjugates as biocatalytic probes,
in which the primary GO sheet-amplified immunoassays are coupled with Ag
enhancement. The sensitivity of the biosensor can be remarkably increased by
immersing the active GO sheets in an Ag enhancer solution.
Potentiometric stripping analysis (PSA，電位剝除法)：在經過預濃縮後 ，利用適當的
氧化劑、還原劑或提供適當氧化、還原電流，使分析物自電極表面剝除，紀錄剝除
時電位隨時間的變化，以電位對時間作圖即可得到電位剝除法圖譜。此時分析物的
濃度將會與剝除時間成正比。
23
成大化學系曾茂源碩士論文(2003)
Preparation of anti-SRB antibody (Ab)-labeled GO.
1. GO sheets were prepared from graphite according to the method reported by
Hummers.
2. The antibody-labeled GO conjugate was prepared through the following
approach:
(a) A 0.1 mL GO (1 mg mL-1) colloid solution was centrifugated at 15 000 rpm
for 20 min, and the supernate was removed.
(b) Then, 1 mL of aqueous solution of 100 mM 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide and 25mM N-hydroxysuccinimide was added to activate the
carboxylic groups onto the surface of the GO sheets for 1 h.
(c) The mixture was then centrifugated at 15 000 rpm for 20 min, and the
supernate was removed.
(d) Then, 100 μL of antibody solution (1 mg mL-1) was added into the centrifuge
tube containing the GO pellet.
(e) The final volume was adjusted to 200 μL by adding Milli-Q water and was
allowed to stand overnight at 4 C, followed by the addition of 800 μL of 1%
bovine serum albumin (BSA)-phosphate buffered saline (PBS) solution.
(f) The precipitate was then collected after a second centrifugation at 15 000
rpm for 20 min.
(g) Finally, the resulting conjugate was resuspended in 200 μL of PBS with 1%
BSA to stabilize the functionalized GO colloid and to minimize nonspecific
adsorption during the immunoassay.
24
PSA is a two-step technique consisting of an electrolysis step and a stripping
step. The electrolysis, performed using a mercury film-coated glassy carbon
electrode, is a preconcentration step in which the Ag ions are reduced to free metal
and electrodeposited as an amalgam onto the working electrode. The
measurements are made in the stripping step, during which the silver is reoxidized.
In electrochemical oxidation, an anodic current is imposed on the electrode to
reoxidize the electrodeposited metals.
The change in the electrode potential (E) over time (t) during the reoxidation
process is monitored. PSA experiments were carried out using a CHI760C
conventional three-electrode system that includes a platinum wire and a
saturated calomel electrode as the counter and reference electrodes, respectively.
The potential was first maintained at -0.5 V for 60 s to electrochemically
deposit metal silver onto the modified working electrode surface. All
electrochemical measurements were performed under stirring during the plating
and accumulation steps. After a 10 s rest, the electrochemical stripping was
carried out from 0 to 0.7 V with stripping current of 10-5 A. During stripping, the
solution was maintained under quiescent conditions.
25
Without With GO
GO sheets sheets
AFM image of the
GO (B) and Ab-GO (C)
sheets deposited on
a SiO2 substrate.
Ag nanoparticles formed
simultaneously without
(a) and with (b) the
addition of the GO sheets
TEM images (D) of AbGO sheets (a) and
Ag/Ab-GO sheet (b)
nanocomposite with
selected area electron
diffraction in the inset.
26
Figure 2. SEM micrographs of the SRB (A), the immune complex of GO-SRB (B), and
the GO-SRB after silver enhancement (C).
When the Ag enhancer solution was added, Ag metal was deposited onto the
surface of the GO sheets and the morphology of the SRB was destroyed due to the
low pH of the Ag enhancer solution (Figure 2C) .
The authors designed a PSA measurement and a colorimetric method (即色差
比對) for detecting SRB. Given that the sensor is Ag-dependent, it can be used to
detect Ag nanoparticles from the deposition substrate in the GO sheets.
Figure 3. Comparison in the response of immunosensor
based on the PSA analysis to 1.5 108 cfu mL-1 E. coli
(a) or 1.5 108 cfu mL-1 SRB (b).
(C)
Figure 4. Typical PSA spectra (A) based on GO-mediated immunosensor for antibody immobilization and
sample detection, calibration relationship (B) between the PSA responses and concentrations of SRB with
28
(9) and without (0) GO-mediated immunosensor, and detection of SRB based on immunoassay dot blot
analysis (C) using theGOsheets as deposition substrate for Ag enhancement.
In the absence of any functionalized GO sheets, there was no distinct detectable
signal even after 20 min of Ag enhancers using this method.
The stripping response in the immunosensor assay without any functionalized
GO sheets almost did not vary with the logarithmic value of the SRB concentration
over 7 orders of magnitude
The dot blot assay was used as a conventional immunoassay method for
comparison with the PSA method, as well as to observe the quality of the anti-SRB
Ab used in the immunosensor. Several concentrations of SRB (1.8 x102 to 1.8 x108
cfu/mL) were assayed using the dot blot assay, and the results are shown in Figure
4C. As expected, increasing the bacterial concentration (1.8 x102 to 1.8 x108 cfu/mL)
resulted in an increase in dot intensity. In addition, the blank (free bacteria) and
control sample (E. coli, 1.5 x108 cfu/mL) show a slight colorimetric response. The
detection limit of this dot blot assay was 1.8 x102 cfu/mL SRB.
29
A versatile graphene-based fluorescence “on/off” switch for multiplex detection
of various targets [13-8]
Molecular beacons (MBs) are dually labeled hairpin-structured oligonucleotides
that are internally quenched as a result of the proximity between a fluorophore
(donor) and quencher (acceptor) tagged at either end.
Molecular beacons are oligonucleotide hybridization
probes that can report the presence of specific nucleic
acids in homogenous solutions. The term more often
used is molecular beacon probes. Molecular beacons
are hairpin shaped molecules with an internally
quenched fluorophore whose fluorescence is restored
when they bind to a target nucleic acid sequence.
Oligonucleotide:寡核苷酸 are short, single-stranded
DNA or RNA molecules that have a wide range of
applications in genetic testing, research, and forensics.
GO with the photophysical feature made it a powerful dye quencher, thus
enabling its use in bioanalytical applications where MBs with an “on/off” switching
design would be highly desirable.
Quenching refers to any process which decreases the fluorescence intensity of a
given substance.
Two key features of graphene oxide can be exploited in this type of design. First,
GO can suitably serve as a “nanoscaffold” for single-stranded DNA (ssDNA)
because ssDNA can be spontaneously absorbed onto the surface of GO by means
of -stacking interactions between nucleotide bases and the GO sheet. Second, GO
can act as a “nanoquencher” for the fluorophore. By coupling these two
characteristics, we envisioned a MB-like probe design in which the ssDNA, which is
labeled with fluorophore only at one end, can self-organize onto the surface of GO
to form a stable ssDNA–GO architecture, while GO is used as the dye quencher.
Nucleotides (核苷酸) are biological molecules that form the building blocks of
nucleic acids (DNA and RNA) and serve to carry packets of energy within the cell.
However, in the presence of a target, competitive binding of the target and GO
with the ssDNA forces the dye-labeled ssDNA–GO architecture to undergo a
conformational alteration in response to interaction with the target, spontaneously
liberating the ssDNA from the surface of GO, which results in the restoration of dye
32
fluorescence.
Scheme 1. Schematic representation of the
ssDNA–GO architecture platform for
multiplex targets detection.
(a) In the presence of a complementary target
T3, P3 reacts with T3 to form a P3–T3
complex, which detaches from theGOsurface,
resulting in the fluorescence “on” state.
(b) P1 assumes the fluorescence “off” state by
the formation of Probe–GO architecture, but
switches to the “on” state by the interaction
with thrombin
(c) In the presence of Ag+ or Hg2+, P4 or P5 is self-folded to form the stable C–Ag+–C or T–Hg2+–T
structure, leading to the fluorescence “on” state. However, by continuing to add cysteine to the above solution,
the C–Ag+–C or T–Hg2+–T structure is disrupted by Ag–S or Hg–S bond between cysteine and Ag+ or Hg2+,
switching to the fluorescence “off” state again.
In this work, GO as a nanocarrier, was assembled with ssDNAs labeled with
different dyes to form a homogeneous ssDNA–GO architecture having the
capability of simultaneously detecting different targets. Here, we chose sequencespecific DNA, protein (thrombin), metal ions (Ag+ and Hg2+) and a small molecule
(cysteine) to test the feasibility of this method.
Multiplex GO-based MB probe for DNA detection and genotyping
Fig. 1. (A) The fluorescence emission spectra of P1/P2/P3–GO architecture in PBS buffer upon excitation
wavelength of 480 nm, 520nm and 630 nm. Fluorescence response of P1/P2/P3–GO solution (0.02mg/mL GO,
30nMDNAprobes) in the presence of differentDNAinputs including perfect-matched (pm) targets T1, T2, T3,
single-base-mismatched (sbMM) targets m1-T1, m1-T2, m1-T3, and double-base-mismatched (dbMM) targets
m2-T1, m2-T2, m2-T3. (B) Analysis of the loss of fluorescence restoration with the increased number of 33
mutations on the target T3.
Three probes, P1, P2 and P3, complementary to T1, T2 and T3, respectively
labeled with FITC, Cy3, and Cy5, were employed. As shown in Fig. 1A, the resulting
emission spectra from the three tested probes, P1, P2 and P3, were clearly
discriminated without interference, and the fluorescence from Probe–GO solution was
observed to have more than 95% fluorescence quenching by GO at three emission
wavelengths.
3.3. Multiplex analysis for Ag+ and Hg2+ detection
Hg2+ can specifically interact with thymine bases to form strong and stable
thymine–Hg2+ –thymine complexes (T– Hg2+ –T). Ag+ ion can exclusively captured by
cytosine–cytosine (C–C) in DNA duplexes to form Ag+–cytosine complexes.
Thymine(胸腺嘧啶):
The authors prepared two oligonucleotide probes of C-rich (P4) and T-rich (P5)
toward simultaneously detection of Ag+ and Hg2+ based on ssDNA–GO platform.
Specifically, they demonstrate the quantitatively analysis and selectivity of Ag+ and
Hg2+ ions. In their experiments, MOPS buffer (50mM NaNO3, 10mMMOPS, pH 7.0)
was used for multiplex detection due to that Cl−, and PO4 2− can form an insoluble
34
2
2+
product with Ag /Hg ions.
The ssDNA–GO
complex was prepared
by incubating the equal
amounts of P4 and P5
(each 15 nM) with 0.02
mg/mL GO solution for 5
min at room temperature.
The limit of
detection of Hg2+ was
estimated to be 5.7
nM,
Fig. 2. Sensitivity and selectivity of multiplexed analysis for Ag + and Hg2+ detection
using P4/P5–GO complex. Fluorescence response of P4/P5–GO to (A) Ag+ ion,
and (B) Hg2+ ion. The fluorescence emission spectra are shown for various ion
concentrations of 0, 0.02, 0.05, 0.1, 0.2, 0.5, 0.75, 1.0, 5.0 and 10.0M. Inset: Plot of
(F/F0 −1) as function of the metal ion concentrations; Selectivity analysis for (C) Ag+
detection, and (D) Hg 2+ detection.
35
3.4. Multiplex detection for various targets
It was reported that cysteine was a stronger Ag+ binder to grab the Ag+ ion from C–
Ag+–C base pairs. Based on the above finding, we rationally designed a cytosine-rich
P4 to simultaneously detect aqueous Ag+ ion and cysteine, taking advantage of the
competition reaction between P4 and cysteine for Ag+ ion. In the absence of Ag+,
theP4–GOcomplex is in the close state with fluorescence “off”. However, upon
incubation with Ag+, P4 self-folds into a hairpin structure of C–Ag+–C complex and is
liberated from the GO surface, leading to fluorescence “on”.
By addition of cysteine to the above system, Ag+ is grabbed from the C–Ag+–C
complex, resulting in the release of P4 from the Ag+–P4 complex and absorption by
GO to turn off the fluorescence.
The P1/P3/P4–GO complex solution
containing 10M Ag+ ions was prepared. After
adding cysteine with increasing concentrations,
a gradual fluorescence decrease in the
P1/P3/P4–GO solution was observed (Fig. 3A).
36
It was found that none of these amino acids,
except for cysteine, could lead an obvious
fluorescence decrease, meaning that only
cysteine could remove Ag+ from the Ag+–P4
hairpin structure (Fig. 3B).
Fig. 4 shows that targets of thrombin
(凝血脢), DNA (T3), Ag+ and cysteine can
be selectively detected simultaneously. For each target, we tested with two
different concentrations. As expected, the P1/P3/P4–GO complex offers a
versatile, sensitive, and robust platform, combining the features of Mbs and GOfor
multiplex analytical detection with exceptional target-binding selectivity and little
crosstalk interference between analytes.
The proposed ssDNA–GO architecture possesses some unique features over
currently available MB probes. First, GO can act as a novel universal fluorescence
quencher, showing an amazing superquenching effect (more than 95%) upon the
fluorophore. For the conventional MB probe, the selection of quenchers directly
influences the quenching efficiency upon the dyes, thus stable multiplex detection
cannot be satisfactorily met. The experimental results indicate that more than 95%
fluorescence intensities are quenched within 3 min for all tested dye-labeled probes,
37
which is faster than using SWNTs due to the surface effect.
Fig. 4. P1/P3/P4–GO (0.02mg/mL GO, each probe 10 nM) for multiplex detection of thrombin, T3, Ag+
and cysteine. Fluorescence response of P1/P3/P4–GO to (A) thrombin with three concentrations of 0,
100 and 200 nM, (B) T3 with three concentrations of 0, 10 and 50 nM, (C) Ag+ ion with three
concentrations of 0, 1 and 5M, and (D) cysteine (5MAg + ion added) with three concentrations of 0, 2
and 10M. Bars of the insets represent the fluorescence change (F/F0 −1).
38
Second, the ssDNA–GO complex demonstrates superior sensitivity and rapid
response. The ssDNA–GO architecture is based on the self-assembly of the
ssDNA onto GO by -stacking reaction between nucleotide bases and GO, whose
fluorescence “on/off” states can be controlled in response to added targets. This
“on/off” switching based on ssDNA–GO architecture exhibits smart and rigorous
discrimination for different targets and results in sensitive detection of targets by
changes in fluorescence. In addition, the time for the target detection is very
short of 10 min.
Third, the ssDNA–GO architecture design can be very versatile and easily
extended to develop a variety of MB-like probes for simultaneous detection of a
wide range of targets by simply changing the fluorophores and engineering
ssDNA sequences.
Aptamer是一種短片段單股的DNA、RNA或胜肽分子，利用其形成二級結構的特
性，可以專一性的與蛋白質的特定位置做鍵結，而對蛋白質的功能造成影響，
並且可以透過專一性aptamer來研究蛋白質的功能、交互作用，具有類似抗體的
功能。(成大蔡耀宗碩士論文，2010)
39
References
13-1 Wenbin Hu et al. ACS nano (2010) 4 7 4317
13-2 Oscar N. Ruiz et al. ACS nano (2011) 5 10 8100
13-3 Omid A et al. Biomaterials (2012) 33 8017
13-4 Ken-Hsuan Liao et al. ACS Applied Materials& Interfaces (2011) 3 2607
13-5 Yuyan et al. Electroanalysis (2010) 22 10 1027
13-6 Tapas Kuila et al. Biosensora and Bioelectronics (2011) 26 4637
13-7 Yi Wan et al. (2011) 83 648
13-8 Min Zhang (2011) Biosensors and Bioelectronics (2011) 26 3260.
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