pH-Responsive Fluorescence Enhancement in Graphene Oxide–Naphthalimide Nanoconjugates: A Fluorescence Turn-On Sensor for Acetylcholine

Article abstract of DOI:10.1002/chem.201702198

A pH-sensitive, fluorescence “turn-on” sensor based on a graphene oxide–naphthalimide (GO–NI) nanoconjugate for the detection of acetylcholine (ACh) by monitoring the enzymatic activity of acetylcholinesterase (AChE) in aqueous solution is reported. These

Full text of DOI:10.1002/chem.201702198

A Journal of
Accepted Article
Title: pH responsive fluorescence enhancement in graphene oxide -
naphthalimide nanoconjugates: A fluorescence turn-on sensor for
acetylcholine
Authors: Sreejith Mangalath, Silja Abraham, and Joshy Joseph
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To be cited as: Chem. Eur. J. 10.1002/chem.201702198
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pH responsive fluorescence enhancement in graphene oxide -
naphthalimide nanoconjugates: A fluorescence turn-on sensor
for Acetylcholine
Sreejith Mangalath,[a,b] Silja Abraham[a] and Joshy Joseph*[a,b]
Abstract: A pH sensitive, fluorescence ‘turn on’ sensor based on a
graphene oxide–naphthalimide nanoconjugate (GO-NI) for the
detection of acetylcholine (ACh) by monitoring the enzymatic activity
of acetylcholinesterase (AChE) in aqueous solution is reported.
These nanoconjugates were synthesized by covalently anchoring
picolyl substituted naphthalimide derivatives on GO/rGO surface via
EDC-NHS coupling strategy and the morphological and
photophysical properties were studied in detail. Synergistic effects of
- interactions between GO and naphthalimide chromophore, and
efficient photoinduced electron and energy transfer processes are
responsible for the strong quenching of fluorescence of these
nanoconjugates, which gets perturbed at acidic pH conditions
leading to significant enhancement of fluorescence emission. We
have successfully employed this nanoconjugate for the efficient
sensing of the pH change caused by the enzymatic activity of
properties.[4] Covalent and non-covalent functionalization of GO
and rGO with various organic and biomolecules is a key
requirement for tailoring the chemical and electronic properties
of these nanomaterials for specific applications.[5]
The complete absence of fluorescence in most of the
chromophores on GO surface is attributed to the 'super
quenching' ability of GO and its analogues due to the strong -
interactions and efficient energy and electron transfer processes
between the nanomaterial and the fluorophores.[6]
A key
property of the fluorescence quenching by graphene is its strong
distance dependence, which has been exploited by many
research groups in building fluorescence based sensors.[7]
Design of stimuli responsive fluorescence 'turn on' probes on
graphene surface is a challenging task, which requires a
stimulus which can perturb the strong interaction between the
fluorophore and graphene. Comparing non-covalent and
covalent approaches of surface modifications, the possibility of
non-specific binding is low in covalently linked systems and
hence sensor probes covalently attached to graphene are more
desirable. Although there are many reports on graphene-
chromophore covalent derivatives which exhibit better electronic
and optical properties,[8] very few reports are available on stimuli
responsive fluorescence enhancement of covalently linked
fluorophores on graphene surface without detaching the probes
to the bulk of the solution.[9]
Previously, graphene and its conjugates have been used
in the design of pH responsive probes via different strategies.[10]
For example, Palermo and co-workers reported a pH sensitive
terthiophene-grafted GO-nanohybrid, which exhibits a reversible
pH dependent photoemission.[9c] Another approach towards the
development of an efficient colorimetric pH sensor is based on
pH responsive polymer modified quantum dots, integrated on
graphene oxide via non-covalent interactions, which responds to
a wide range of pH changes.[11]
Acetylcholine (ACh) is an important neurotransmitter and
its abnormalities in brain are related to the neuropsychiatric
disorders, such as Alzheimer’s disease. The excessive
accumulation of ACh in synaptic clefts can cause fatal health
problems and its sensing is of greatest interest. Enzyme based
sensors are widely used owing to its specificity towards the
substrate, acetylcholine.[12] Enzymatic sensing on graphene
oxide nanosheets is highly desirable due to its excellent colloidal
stability in aqueous medium and also it provides a suitable
platform for enzyme immobilization.[13] More recently, Kim et al.
reported the pH sensitive enhancement of inherent fluorescence
in phenoxy-dextran modified GO where they demonstrate the
optical detection of enzymatic activity of acetylcholinesterase
(AChE) based on its fluorescence 'turn on' response upon
protonation.[14]
acetylcholinesterase, thereby demonstrating its utility as
fluorescence ‘turn-on’ sensor for acetylcholine in
a
the
neurophysiological range.
Introduction
Graphene and its chemically modified analogues are extensively
used for various applications ranging from electronics to drug
delivery due to their remarkable electronic, mechanical, optical,
and chemical properties.[1] Graphene oxide (GO), an oxidized
analogue of graphene is particularly useful for chemical and
biological applications due to its enhanced solubility in aqueous
media, colloidal stability and biocompatibility.[2] The presence of
various oxygen functionalities such as aldehyde, hydroxyl,
epoxide, carboxylic group, etc on the edges and basal planes
make GO
a
suitable candidate for facile chemical
functionalization via various strategies.[3] Reduced graphene
oxide (rGO), synthetically accessed through controlled reduction
of GO is another useful graphene analogue which interface
between GO and pristine graphene in its physical and chemical
[a]
[b]
Photosciences and Photonics Section, Chemical Sciences and
Technology Division, CSIR-National Institute for Interdisciplinary
Science and Technology, Thiruvananthapuram 695019, Kerala,
India
E-mail: joshyja@gmail.com
Academy of Scientific and Innovative Research (AcSIR), CSIR-
NIIST Campus, Thiruvananthapuram 695019, Kerala, India
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. Scheme for synthesis of GO-NI and rGO-NI nanoconjugates and sensing of ACh
Herein, we demonstrate a new strategy for the design of
pH responsive fluorescence 'turn on' sensors using
Modification of rGO was achieved via introduction of carboxylic
acid groups in the basal planes through 1,3 dipolar cycloaddition
reaction with a Boc-protected α-amino acid and formaldehyde
followed by EDC-NHS coupling with NI.[17] Both GO-NI and rGO-
NI form stable suspensions in water and in common organic
solvents such as acetonitrile, DMF and DMSO (Supporting
Information and Figure S1). These nanoconjugates were well
characterized using various spectroscopic and microscopic
techniques and their structural and photophysical properties
were investigated in detail.
a
picolylamine substituted naphthalimide derivative (NI) covalently
attached to GO and rGO surface (GO-NI and rGO-NI,
respectively; Figure 1) via two different coupling strategies.
These nanoconjugates exhibited negligible fluorescence
emission consequent to the synergistic quenching effects of the
- interaction with GO surface and intrinsic PET based
quenching pathways in the naphthalimide chromophore. At
lower pH conditions, both GO and the naphthalimide
chromophores gets protonated which perturb both the strong -
interactions between GO and the chromophore, and the PET
quenching pathways within the chromophore leading to
significant enhancement in the emission. This strategy has been
successfully demonstrated both in GO-NI and rGO-NI
nanoconjugates, with the former showing a higher extent of
fluorescence enhancement at lower pH conditions. The pH-
responsive fluorescence 'turn on' strategy was further exploited
for the detection of acetylcholine (ACh) in the neurophysiological
range by the enzymatic reaction with acetylcholine esterase
(AChE), which convert ACh to choline and acetic acid[15]
resulting in the reduction of the pH of the aqueous solution.
Spectroscopic evidence for the efficient covalent coupling
was obtained via FT-IR measurements which showed additional
peaks for GO-NI and rGO-NI corresponding to the NI
B)
A)
C)
D)
600
100
GO
NI
GO-NI
GO
GO-NI
500
400
300
200
100
0
Results and Discussion
80
60
40
20
0
The synthesis of few layer graphene oxide (GO) from graphite
powder was accomplished by modified Hummers method[16] and
reduction of exfoliated GO sheets using hydrazine hydrate
yielded reduced GO (rGO). The functionalization of GO and rGO
with a picolylamine substituted 1,8-naphthalimide fluorophore
(NI) was achieved through two different synthetic strategies as
shown in Figure 1. In the first approach, GO was directly
functionalized with NI chromophore via EDC-NHS coupling with
carboxylic acid present on the few layer GO nanosheets.
900 1200 1500 1800 2100
200 400 600 800
-1)
o
Wavenumber, (cm
Temperature ( C)
Figure 2. A) TEM & B) TEM images of GO-NI. C) Raman spectra of GO and
GO-NI, D) TG analysis of GO, NI and GO-NI
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A)0.6
450 nm - 700 nm range with a maximum around 540 nm and a
fluorescence quantum yield of 7.1% in water (Figure 3B).[20] On
the other hand, fluorescence spectrum of an optically matching
B)
30
GO-NI
NI
GO-NI
NI
GO
0.4
0.2
0.0
20
10
0
solution of the nanoconjugate GO-NI revealed
quenched emission (quantum yield of 0.42%) corresponding to
the NI chromophore. similar, but less pronounced
a highly
A
fluorescence quenching compared to NI chromophore was
observed in the case of rGO-NI nanoconjugate (Figure S7,
Supporting Information).
300
GO
450
600
750
900
500
600
700
C)
Wavelength (nm)
Wavelength (nm)
D)
60
45
30
15
The NI chromophore in aqueous solutions showed a bi-
exponential decay with an average fluorescence life time of 3.4
ns, whereas the nanoconjugates, GO-NI and rGO-NI showed
multi-exponential decays with significantly reduced fluorescence
lifetimes (Figure S8; Table S1, Supporting Information). The
strong quenching of NI chromophore fluorescence in GO-NI and
rGO-NI could be attributed to the possible energy and electron
transfer processes facilitated by the strong - interaction
between GO and the chromophores.[21] The intermolecular
interactions between GO and NI chromophores were probed by
titrating GO against the NI chromophore in aqueous solutions at
pH 7, which showed >90% quenching of the fluorescence of NI
chromophore upon addition of 30 μg/mL of GO (Figure 3C).
Corresponding Stern-Volmer plot shows an upward curvature
indicating the involvement of both dynamic and static quenching
pathways (Figure S9A, Supporting Information). This
observation was further confirmed by the fluorescence lifetime
quenching experiments which showed a gradual decrease of the
fluorescence lifetime corresponding to dynamic quenching with
an increase in the amplitude of the short lived component
representing the static quenching in the GO - NI chromophore
complex (Figure S9B; Table S2, Supporting Information). The
corresponding absorption spectra also showed signatures of
ground state interactions between GO and the NI chromophore
(Figure 3D).The fluorescence spectra of as synthesized nano-
conjugates were found to be highly sensitive towards the
changes in the pH of the aqueous solution, as shown in the pH
dependent fluorescence emission spectra of GO-NI (Figure 4)
and rGO-NI (Figure S10, Supporting Information). For example,
0.4
0.2
0.0
450
525
600
675
750
300 400 500 600 700
Wavelength (nm)
Wavelength (nm)
Figure 3. A) Absorption spectra of aqueous dispersions of GO, NI and GO-NI.
B) Fluorescence spectra of optically matching solutions of NI and GO-NI. C &
D) Absorption and fluorescence spectra of aqueous solutions of NI (22 μM)
upon addition of GO (0.2 mg/mL) up to a maximum concentration of 30 μg/mL.
(Excitation wavelength, λexc = 420 nm) at pH 7.
chromophore along with a change in the carbonyl stretching
frequency from 1740 cm-1 in GO to ~1640 cm-1 in the
nanoconjugates (Figure S2, Supporting Information). TEM and
AFM analysis of GO and its nanoconjugates showed micrometer
sized sheets with a height of 2-3 nm, which indicates the
formation of few layer nanosheets upon exfoliation (Figure 2A,
2B, S3 and S4, Supporting Information). The approximate NI
chromophore loading in these nanoconjugates after the coupling
reactions were estimated using thermogravimetric analysis
(TGA) and the values were found to be 6.2 wt% for GO-NI and
5.6 wt% for rGO-NI (Figure 2C and S5, Supporting Information).
Additional proof for covalent functionalization of GO with NI
chromophores was obtained from Raman spectroscopy (Figure
2D). GO-NI showed a slightly larger ID/IG ratio of 1.28 compared
to GO (1.15) due to the increased distortions on GO surface by
covalent functionalization with NI chromophores.[18]
300
8
7
The UV-visible absorption spectra of aqueous dispersions
of as synthesized GO showed characteristic features of few
layered GO nanosheets with an absorption maximum around
230 nm and a broad tail-end absorption extending up to 600 nm
(Figure 3A).[19] The nanoconjugate GO-NI exhibited absorption
features of both GO and NI chromophore with shoulder peak
maxima around 433 nm and 258 nm. The absorption maximum
around 433 nm corresponding to NI chromophore showed a red
shift of 8 nm compared to the absorption maximum of NI
chromophore alone (425 nm), which could be due to a possible
ground state interaction between GO and NI chromophore in the
nanoconjugate. UV-Visible spectroscopic analysis after a control
experiment with GO and NI in the absence of the coupling
reagents, under otherwise similar synthetic and subsequent
washing conditions, did not show typical NI absorption bands
(Figure S6, Supporting Information). The naphthalimide
derivative, NI showed a fluorescence emission spectrum in the
pH 2
6
5
4
3
2
1
250
200
150
100
50
8
7
6
5
4
3
2
pH 7
pH
pH < 7
450
500
550
600
650
700
Wavelength (nm)
Figure 4. Changes in fluorescence of GO-NI with decrease in pH from 7 to 2.
The inset shows a corresponding change in fluorescence intensity (I/Io) with
pH.
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A)
70
B)
7
6
5
4
5
4
3
2
1
60
50
40
30
20
10
60 mM ACh
0 mM
0
10
20
30
40
50
60
450
500
550
600
650
700
Concentration of ACh, mM
Wavelength, nm
Figure 5. A) Fluorescence spectra of GO-NI in presence of ACh at various concentrations from 1 to 60 mM. AChE (3 U/mL) was used for hydrolysis. Each
measurement has taken after incubation of the sample mixture at 37 0C for 10 mins. B) Fluorescence intensity changes (I/I0) of GO-NI – AChE mixture at 540 nm
(red squares) with various concentrations of ACh and pH change in the solution (blue stars). I0 and I are the fluorescence intensity of GO-NI – AChE mixture
before and after treatment with ACh.
the fluorescence intensity corresponding to the NI chromophore
of GO-NI doubles upon decreasing the pH of the aqueous
solution from 7 to 6 and upon further decreasing the pH to 2, a
~7.3 times fluorescence enhancement was observed. Similar
observations were made in the case of rGO-NI where a ~5 fold
enhancement in fluorescence intensity was observed upon
decreasing pH from 7 to 2. The observed pH dependent
enhancement in fluorescence emission in these nanoconjugates
could be explained as follows. At lower pH conditions, the N-
atoms in the NI chromophore and all the oxygen containing
functional groups on the GO surface gets protonated, which
perturb the strong interaction between GO and the NI
chromophore. In addition, protonation of N-atoms render the NI
chromophores more hydrophilic and solvated in aqueous media,
facilitating the dynamic dislocation of the chromophores from the
GO surface. Both these effects contribute to the observed
enhancement in the fluorescence emission. The lower extent of
interaction between GO and the protonated NI chromophores at
pH 2 was confirmed via titration experiments using GO and NI
which showed a lesser extent of fluorescence quenching (~29%)
upon addition of 30 μg/mL of GO, with no indication of ground
state complex formation in the corresponding absorption spectra
(Figure S11, Supporting Information). Another contributing factor
in the observed fluorescence enhancement is the change in the
intramolecular fluorescence quenching pathways in the NI
be attributed to the synergistic effects of reduced - interactions
between GO and NI chromophore and perturbation in the
internal electron transfer pathways within the chromophore.
The potential of these nanoconjugates in reporting
physiological pH changes was demonstrated using the
fluorescence response of GO-NI towards enzymatic hydrolysis
of acetylcholine (ACh) in presence of acetylcholinesterase
(AChE), which converts ACh to choline and acetic acid.[15]
Under optimized concentration of enzyme (3 U/mL; Figure S13,
Supporting Information), and reaction time (10 min), we have
observed a significant enhancement in the fluorescence intensity
of the nanoconjugate with increasing concentration (0 - 60 mM)
of ACh (Figure 5A). GO-NI exhibited nearly linear fluorescence
response at lower ACh concentrations (0 to 500 µM) and the
lower limit of detection is calculated to be 50 µM (Figure S14,
Supporting Information), which shows the ability of our sensor to
detect ACh in neurophysiological range. Although ACh
concentration in the extracellular fluid of brain is in nanomolar
range, its raises to low millimolar range during synaptic
transmission.[22] Our sensor assembly can detect this elevated
ACh concentration range, which makes it suitable for sensing
ACh within the synapse. The fluorescence response (I/I0) of GO-
NI towards ACh concentration and the corresponding pH
change are shown in Figure 5B, which directly correlate the
observed fluorescence enhancement with the pH changes of the
chromophore upon protonation of the picolylamine units. At pH 7, medium. Separate control experiments without the enzyme and
the possible photoinduced electron transfer from the
picolylamine moiety to naphthalimide fluorophore is responsible
for the reduction of the fluorescence quantum yield of NI. At
lower pH conditions, the protonation of N-atoms of the
picolylamine moiety block possible PET pathways, resulting in
an enhancement in the NI fluorescence (Figure S12, Supporting
Information). Thus, the observed enhancement in the
fluorescence of the nanoconjugates at lower pH conditions could
without the substrate under otherwise similar reaction conditions
showed negligible fluorescence response (Figure S15).
Conclusions
In summary, we report the design and synthesis of two novel
graphene nanoconjugates, GO-NI and rGO-NI with appended
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mmol) dissolved in 10 mL H2O was added to this mixture, slowly over 1
hr and the mixture was stirred for 24 hrs at room temperature. The
mixture was then centrifuged at 12000 rpm for 8 min and residue was
washed many times with water, DCM and water-acetone mixture (1:1) to
remove unreacted reagents and other by-products. Further, the product
was washed many times with aqueous HCl (2M) to achieve complete
removal of adsorbed chromophore. Finally, the residue is dispersed in 10
mL H2O and sonicated for 1hr and used for further characterization.
1,8-naphthalimide based fluorophores. In aqueous solutions,
both GO-NI and rGO-NI exhibited highly quenched fluorescence
emission consequent to the strong - interaction of the
chromophores with the GO surface and possible internal PET
processes. At lower pH conditions, these nanoconjugates
exhibited a unique fluorescence enhancement due to the
synergistic effects of reduced - interactions between GO and
NI chromophore and perturbation in the internal electron transfer
pathways within the chromophore. The potential of these
nanoconjugates in reporting real time pH reduction via a
fluorescence 'turn on' response was demonstrated using
enzymatic hydrolysis of acetylcholine and thus demonstrated a
fluorescence “turn-on” sensor for acetylcholine in the
neurophysiological range.
Sensing of ACh. In a typical experiment, the enzyme AChE (final
concentration: 3 U/mL) was added to 1 mL aqueous suspension of GO-
NI (final concentration: 25 µg/mL). Then ACh is added (concentrations
ranging from 0 to 60 mM) and fluorescence measurements were taken
after incubation of the sample mixture at 37 0C for 10 minutes.
Data Analysis: Responsiveness of the sensor was analyzed over a
range of ACh concentrations from 0 to 0.5 mM. Lower limit of detection is
determined based on standard deviation of the response (σy) of the curve
and the slope of calibration curve (m), according to the formula LOD =
3.3(σy/m). The standard deviation of the response can be determined
based on the standard deviation of y-intercepts of regression lines.
Experimental Section
Materials and Methods. The reagents and materials for synthesis were
purchased from Sigma-Aldrich, Alfa-Aesar and Spectrochem.
Acetylcholinesterase from Electrophorus electricus (electric eel) and
acetylcholine were purchased Sigma-Aldrich. 1H-NMR & 13C-NMR
spectra were recorded using DPX 500 MHz spectrometer using
tetramethylsilane (TMS) as the internal standard and deuterated solvents
were used for the measurements. Absorption spectra were recorded on a
Shimadzu UV-2600 UV-Visible spectrophotometer. Fluorescence spectra
were collected using SPEX-Fluorolog F112X Spectrofluorimeter
equipped with a 450 W Xenon arc lamp and spectra obtained were
corrected using the program supplied by the manufacturer. FT-IR spectra
were recorded on a Shimadzu IRPrestige-21 Fourier Transform Infrared
Spectrophotometer. Raman analysis were carried out in a WITec Raman
instrument (Witec Inc. Germany, alpha 300R) with a laser beam directed
to the sample through 60× water immersion objective and a Peltier-
cooled CCD detector. WITec Project Plus (v 2.1) software was used for
data evaluation.
Acknowledgements
This work is financially supported by the Council of Scientific and
Industrial
Research
(CSIR-12FYP-M2D-CSC-0134)
and
Department of Science and Technology, Government of India
(Ramanujan Fellowship Grant RJN-19/2012). Sreejith M.
acknowledge Council of Scientific and Industrial Research
(CSIR, Government of India) for his research fellowship.
Keywords: Graphene oxide • Naphthalimide • pH sensing •
Acetylcholine
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10.1002/chem.201702198
Chemistry - A European Journal
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Sreejith Mangalath,[a,b] Silja
Abraham[a] and Joshy
Joseph*[a]
Page No. – Page No.
"Turning it ON'' on Graphene Oxide surface: A pH sensitive, fluorescence
‘turn on’ sensor based on a graphene oxide-naphthalimide nanoconjugate for
the detection of acetylcholine (ACh) by monitoring the enzymatic activity of
acetylcholinesterase (AChE) in aqueous solution is reported. Synergistic effects
of - interactions between GO and naphthalimide chromophore, and efficient
photoinduced electron and energy transfer processes are responsible for the
strong quenching of fluorescence of these nanoconjugates, which gets perturbed
at acidic pH conditions leading to significant enhancement of fluorescence
emission.
pH responsive fluorescence
enhancement in graphene oxide -
naphthalimide nanoconjugates: A
fluorescence turn-on sensor for
Acetylcholine
This article is protected by copyright. All rights reserved.