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Solvent Effect on Supramolecular Self-Assembly of Chlorophylls a

on Chemically Reduced Graphene Oxide
Tejaswini R. B. Ramakrishna, Motilal Mathesh, Zhen Liu, Chunmei Zhang, Aijun Du, Jingquan Liu,
Colin J. Barrow, Min Chen, Mark J. Biggs, and Wenrong Yang*
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ABSTRACT: Solvent plays an important role in the surface

interaction of molecules. In this study, we use “chlorophyll a”, an
archetypical molecule, to investigate its supramolecular self-
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assembly with chemically reduced graphene oxide in three different

types of solvents: polar protic, polar aprotic, and non-polar. It was
observed that only a polar protic solvent that can donate protons
facilitates the hydrogen bonding between chlorophyll a and
chemically reduced graphene oxide nanosheets in a hybrid system.
The formation of hydrogen bonds further initiates the other non-
covalent interactions such as π−π stacking and hydrophobic interaction, which altogether play a key driving force for supramolecular
self-assembly of chlorophylls on chemically reduced graphene oxides. The experimental results are strongly supported by density
functional theory calculations, which show robust electron coupling between chlorophylls and chemically reduced graphene oxide.

■ INTRODUCTION
Many biological and chemical reactions are triggered in
believe that this is an important aspect that needs to be
considered while designing, as it can provide insights into
solutions in which solvents play a major role in the functional enhancing its stability and tuning its energy levels for
characteristics of the individual solutes.1−4 One such important photocatalytic applications.
feature influenced by solvents is the supramolecular assemblies Herein, we use 2D chemically reduced graphene oxide
between two different molecules in a hybrid system,5 essential (CRGO) as an archetypical molecule to understand and learn
for its stability during/after a reaction.6 It has been widely the supramolecular assembly behavior of Chl a. CRGO is a
shown that such solvent-assisted supramolecular self-assembly
architectures have been used in both research and practical derivative of GO and is used as a material with high potential
applications such as in biomedicines,7 environments,8 and for bio-catalytic applications.19 CRGO surfaces possess
energy storage devices.9 However, the supramolecular self- carboxylic groups at the edges, which aid in its dispersion in
assembly is not only limited due to interaction of molecules hydrophilic solvents. These unique properties of CRGOs are
with solvents. In some cases, amino acid and peptides also play useful in studying the supramolecular assembly of Chl a in
an important role in the construction of functional molecules solutions. In this study, both Chl a and CRGOs were dispersed
via controlled self-assembly.10−12 In this context, the supra-
in three representative types of solvents, namely, polar protic
molecular assemblies based on “chlorophyll a” (Chl a) with
novel materials such as nanomaterials are gaining much (methanol), polar aprotic (N,N,-dimethylformamide, DMF),
attention.13 Chl a, a magnesium-centered (Mg2+) metal- and non-polar (O-dichlorobenzene, O-DCB), to study their
loporphyrin biomolecule found as a photosynthetic pigment solvation effect and understand how they facilitate interactions
in bacterial and plant species, is gaining popularity along with with each other in these solvents. The solvent effect and its
its combination with two-dimensional (2D) nanomaterials role in charge transfer between solutes have been studied
such as graphene, graphene oxide (GO), and their derivatives,
previously by using UV−Vis and fluorescence spectroscopy.20
giving rise to novel hybrid materials. Due to its availability in
nature and wide optical coverage over solar energy spectrum,
the hybrid has been used for phototransistor application.14 Received: August 11, 2020
Furthermore, surface modification of graphene by Chl a has Revised: September 28, 2020
been studied in solar energy conversion, catalysis, and sensing
applications.15−18 However, a deep understanding of their
fundamental interactions at a molecular level through a self-
assembly process in different solvents is currently lacking. We

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RESULTS AND DISCUSSION any spectral shift, indicating weak or no interaction (Figure
In this study, the solvent effect on supramolecular assembly of 1b,c). The distinguished difference between the solvents is the
Chl a and CRGO was studied using a UV−Vis and proton-donating groups present in methanol, offering more
fluorescence spectrophotometer. A simple schematic repre- protons in solution and facilitating interaction, resulting in a
sentation showing the outline of this research and the material spectral red shift. Previously, it has been reported that GO with
system is shown in Scheme 1. surface functional groups such as −COOH and −OH
undergoes protonation in polar solvents.21 Protonation often
Scheme 1. Synthesis and Dispersion of CRGO and leads to changes in properties such as surface charge, mass,
Chlorophyll a Bound to CRGO in an Organic Solvent hydrophilicity, reduction potential, and optical properties.22,23
Since CRGOs have −COOH groups at the edges, it can be
expected that these groups undergo protonation in methanol.
As a result, CRGOs interact with Chl a through protons, which
form hydrogen bonds between the molecule and CRGO. In
addition, Chl a and CRGO undergo π−π stacking between the
porphyrin ring and hexagonal carbon rings present in Chl a
and CRGO, respectively. The red shift observed for Chl a at
665 nm in methanol confirms the π−π interaction between
CRGOs and Chl a, whereas DMF and O-DCB do not facilitate
such a configuration. As observed in Figure 1d, there is no
change in the peak position of Chl a in these solvents due to
lack of proton donors to facilitate the interactions. The change
in Chl a spectrum after titrating with smaller volume of
CRGOs showed similar data (Figure S1), in which the red shift
First, we used UV−Vis spectrophotometry to probe the of Chl a in methanol was observed at 200 μL of CRGO
interaction between Chl a and CRGO in the presence of addition.
methanol, DMF, and O-DCB solvents, and in each experiment, Second, fluorescence spectrophotometry was used to
both Chl a and CRGO stock solution were prepared in their understand the rate of photo-induced electron transfer
respective solvents (see the Supporting Information, S1). A between CRGO and Chl a in each solvent, namely, methanol,
simple titration of Chl a against CRGO in these solvents DMF, and O-DCB (Figure 2). During the measurement, the
resulted in characteristic band patterns, as shown in Figure 1. concentration of Chl a (300 μL) was kept constant while the
During the titration of Chl a against CRGOs in methanol, a concentration of CRGO was increased in the solvent. As
gradual decrease in intensity of Chl a (300 μL) at 665 nm with shown in Figure 2a, the fluorescence of Chl a at ∼675 and
addition of 100 and 200 μL of CRGOs was observed. Further ∼726 nm is significantly quenched when 100 μL of CRGO
titration with increased volume of CRGO led to a red shift in dispersed in methanol was added and essentially quenched at
the peak of Chl a from ∼665 to 687 nm, (Figure 1a), twice the CRGO volume. In contrast, as depicted in Figure
suggesting the solvent-assisted interaction between Chl a and 2b,c, the level of quenching is far less when the other two
CRGO. Similar titrations in DMF and O-DCB did not show solvents are used. These results together with the findings of

Figure 1. UV−visible spectra for chlorophyll a (Chl a, 300 μL from 0.1 mg/mL stock concentration) titrated with chemically reduced graphene
oxide (CRGO, 0.08 mg/mL stock concentration) in three different solvents: (a) polar protic solvent (methanol), (b) polar aprotic solvent (DMF),
and (c) non-polar solvent (O-DCB). (d) Red shift of the Chl a absorption peak at 665 nm against concentration of CRGO.

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Figure 2. Fluorescence spectra for chlorophyll a (Chl a, 300 μL was kept constant) titrated with increasing concentration of chemically reduced
graphene oxide (CRGO): (a) polar protic solvent (methanol), (b) polar aprotic solvent (DMF), and (c) non-polar solvent (O-DCB) (was excited
at 665 nm). (d) Peak shift of Chl a fluorescence intensity against concentration of CRGOs. Excitation wavelength, 665 nm.

UV−Vis measurements collectively reflect that methanol with Chl a. GO surfaces are known to be relatively more
facilitates close interaction between the porphyrin ring and hydrophilic than those of CRGO due to the oxygen groups
the electron-absorbing CRGO.24 Also, unlike UV−Vis, the (epoxy, carboxyl, and carbonyl) covering their carbon basal
fluorescence spectrophotometry was more sensitive in studying planes. As observed in experiments, there was no spectral shift
the interaction between the CRGOs and Chl a in these three or photo-induced electron transfer in both the UV−Vis and
different solvents. Along with changes in intensity, even the
fluorescence spectra of Chl a in methanol solvent (Figures S1.1
shift in Chl a fluorescence against the concentration of CRGOs
in methanol, DMF, and O-DCB can be clearly observed and S2). This could be explained by the fact that the oxygen
(Figure 2d). In methanol, the Chl a fluorescence shifted from groups of GO cover carbon hexagonal rings and prevent the
675 to ∼665 nm, whereas in DMF and O-DCB, the shift was π−π stacking between Chl a and GO.25 Therefore, there is a
far less to ∼669 and ∼673 nm, respectively. The comparatively lack of photo-induced electron transfer from Chl a to GO, thus
large blue shift of 10 nm in methanol is due to the polarity of inhibiting any interaction between the two molecules.
the solvent, suggesting the strong interaction of CRGOs with Furthermore, the phytol tail is hydrophobic,26 therefore
Chl a in methanol as compared to DMF or O-DCB. increasing the probabilities of hydrophobic interaction
Previously, studies have been carried out to investigate the between Chl a and CRGO to be more dynamic than with
effect of organic solvents on GO fluorescence, from which it GO. Hence, this study proves the availability of the carbon
was observed that in the polar solvent, the GO peak showed a
basal plane and surface hydrophobicity of CRGO as two
large red shift upon excitation. This was due to the protonation
of −COOH and −OH groups on GO in the polar solvent, important factors to form the CRGO/Chl a hybrid. From the
which slowed down the solvation dynamic process to the same UV−Vis and fluorescence spectra, it can be concluded that Chl
time scale as fluorescence emission/lifetime.21 In this study, we a undergoes supramolecular assembly on CRGOs in methanol
observed a similar trend where Chl a fluorescence undergoes a solvent via hydrogen bonding, π−π stacking, and hydrophobic
blue shift with an increase in the concentration of CRGOs. interactions, as shown in Scheme 2.
CRGOs also possess the abovementioned functional groups as
GO, which undergoes protonation in polar solvents. Therefore, Scheme 2. Supramolecular Self-Assembly of Chlorophyll a
we propose that due to facilitating protons in methanol, the on Chemically Reduced Graphene Oxides in Methanol
binding process of CRGOs to Chl a is fast, thereby resulting in Solvent
a blue shift together with quenching. The fast quenching
response directly relates to the photo-induced electron transfer
and binding between Chl a and CRGOs. These changes in
spectra showcase the interaction of CRGOs with Chl a through
the proposed π−π stacking and hydrogen bonding. Our results
correlate well with previous studies,20,21 and we can confirm
that solvents play an important role for a chemical reaction to
occur.
In order to investigate the influence of the structure and
surface hydrophobicity of CRGO on Chl a, a similar
interaction study was carried out using GO to conjugate
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Figure 3. (a) Top view and (b) and side view of 3D charge density difference for the CRGO/Chl a hybrid with respect to graphene and Chl a
fragments. Brown, blue, pink, red, purple, and orange atoms represent C (in CRGO), C (in Chl a molecule), N, O, H, and Mg atoms, respectively.
Yellow and cyan isosurfaces represent electron accumulation and depletion in the 3D space with an isovalve of 0.001e/A3.

Furthermore, the quenching efficiency of CRGOs on Chl a energy of the CRGO/Chl a system was calculated to be −3.24
fluorescence in methanol, DMF, and O-DCB solvents was eV using the following equation:
quantified using the Stern−Volmer method:27
Eads = Etot − ECRGO − EChl a (3)
I0/I = 1 + KSV[Q ] (1)
where Etot represents the total energy of the CRGO/Chl a after
where I0 represents the intensity of Chl a in the absence of geometry optimization, ECRGO is the energy of the relaxed
CRGOs, I is the intensity of Chl a in the presence of CRGOs, CRGO, and EChl a is the energy of the Chl a molecule. The
KSV is the Stern−Volmer quenching constant, and [Q] is the adsorption energy indicated a strong interaction between the
concentration of CRGOs. A Stern−Volmer plot of Chl a/ Chl a molecule and CRGO nanosheet and thus the enhanced
CRGO was plotted, as shown in Figure S3. It was observed stability of the CRGO/Chl a hybrid complex. The 3D charge
that with an increase in the concentration of CRGO, the density difference was obtained by subtracting the electronic
Stern−Volmer quenching constant increased in methanol and charge of the CRGO/Chl a hybrid from the CRGO surface
DMF at 675 and 726 nm, respectively. However, in DMF, the and Chl a molecule. Figure 3 shows that there is a significant
data points did not fit the curve and the Stern−Volmer charge distribution within the hybrid complex, suggesting a
quenching constant was relative lower than that in methanol. strong charge transfer, i.e., electron coupling reactions between
This signifies that in methanol, the fluorescence quenching the Chl a molecule and CRGO nanosheet. The apparent
mechanism between the Chl a and CRGO complex is dynamic. electron-rich area (below the Mg atom) on the graphene
Meanwhile, in O-DCB, the Stern−Volmer quenching constant surface and the hole-rich area on the chlorophyll molecule are
declined at 675 nm with an increase in CRGO concentration, caused by the transfer of electrons from the chlorophyll
while it increased at 726 nm, indicating a partial quenching molecule to the graphene.
effect at the far end of the Q-band region of Chl a in O-DCB, Subsequently, we confirmed the hybrid formation between
but the Stern−Volmer quenching constant was significantly CRGO and Chl a in methanol solvent by atomic force
lower when compared to the slope in methanol. Therefore, it microscopy (AFM), and structural characterization was carried
reveals that the interaction mechanism of fluorescence out by Raman spectroscopy and attenuated total reflectance
quenching is static both in DMF and O-DCB. The Stern− infrared spectroscopy (ATR-FTIR). AFM measurements were
Volmer quenching constant of CRGOs on Chl a fluorescence used to examine the topography and height profile of CRGO
in methanol, DMF, and O-DCB is also summarized in Table before and after interaction with Chl a, which confirmed the
S1. Based on the above results, the binding constant between hybrid formation between CRGO and Chl a. The height
CRGO and Chl a was calculated using the Scatchard profiling revealed the CRGO sheets to be ∼0.7 nm (Figure
method:28 4a), which is consistent with the literature.29 In the case of
r /c = nKb − rKb (2) CRGO/Chl a hybrids, the height observed was ∼1.8 nm
(Figure 4b), which suggests the Chl a assembly to be nearly
where r is the bound CRGO concentration, c is the molar planar to the CRGO surface. The thickness of monomeric
concentration of Chl a, Kb is the binding constant, and n is the forms of Chl a is found to be ∼0.55 nm.30 The increase in
number of binding sites. This method is simple and commonly height profile by ∼1 nm suggests that these monomeric forms
used to calculate the binding constant between molecules. We of Chl a were bound on both sides of the CRGO basal plane,
observed that Chl a in methanol was strongly bound to CRGO, forming a sandwich-like structure.
while it desorbed in DMF and O-DCB after a solvent wash In addition, Raman and ATR-FTIR data showed changes in
(data not shown). As a result, the binding constant was the spectrum of CRGO after interaction with Chl a. The
calculated between Chl a and CRGOs only in methanol using Raman spectra of CRGO (Figure S5) show two major bands
eq 2. The calculated binding constant of Chl a to CRGO in corresponding to the D (defect) and G (graphite) band,31 and
methanol was 10.76 × 104 M−1. To the best of our knowledge, Chl a exhibits many significant bands with respect to its surface
this is the first study that has quantitatively measured the functional moieties.32 Meanwhile, the addition of Chl a to
binding constant between Chl a and CRGO in methanol CRGO shows an increase in G band intensity with red shift.
solvent. Also, the linear curve obtained through the Scatchard Few peaks from 1300 to 1000 cm−1 were also observed,
plot (Figure S4) represents the extensive interaction between indicating the strong interaction of Chl a with CRGO and their
Chl a and CRGO in methanol solvent. successful self-assembly. Furthermore, deconvolution studies
Additionally, to understand the interaction between Chl a were performed to calculate the integrated area ratios (AD/AG)
and CRGO, we carried out theoretical calculations based on of the CRGO/Chl a hybrid. The deconvolution studies on the
density functional theory (DFT) (Figure 3). The adsorption Raman spectrum reveal the molecular structural change in the
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on the CRGO nanosheet. The main driving interaction is

hydrogen bond formation between CRGO and Chl a, which is
anticipated to also facilitate π−π stacking, hydrophobic
interactions, and robust electron coupling. Close proximity
interaction between Chl a and CRGO in methanol favors a
significant rate of electron transfer when Chl a is photo-excited.
We anticipate that this study will open a way to optimize the
photo-induced electron transfer and tune the optical properties
of the hybrid molecules by controlling the solvent polarity for
Chl a and Chl a-like molecules to be used on CRGO for
photocatalytic applications.

■ EXPERIMENTAL METHODS
Materials. Graphite (particle size, <45 μm), hydrazine hydrate
(50%), L-ascorbic acid, anhydrous N,N-dimethylformamide (DMF)
Figure 4. (a) Atomic force microscopy of single sheet CRGO of ∼0.7 (purity, ≥99.8%), and anhydrous O-dichlorobenzene (O-DCB)
nm. (b) Topography of Chl a on CRGO with a total height of ∼1.8 (purity, ≥99.9%) were used, and chlorophyll a (1 mg) was purchased
nm in the polar protic (methanol) solvent. (c) Topography of from Sigma-Aldrich (NSW, Australia). Sodium chloride, hydrogen
aggregated Chl a on CRGO with varied height from ∼5−10 nm in a peroxide (30%), and potassium permanganate were purchased from
mixture of methanol and water solvent. Chem Supply (NSW, Australia). Methanol of HPLC grade (purity,
≥99.9%) was purchased from Fisher Scientific (NSW, Australia);
CRGO carbon network when hybridized with other mole- ammonia solution (25%), hydrochloric acid (32%), and sulfuric acid
cule.33 Our studies showed that CRGOs after interaction with (98%) were purchased from Scharlab S.L (Barcelona, Spain).
Synthesis of Graphene Oxide (GO). First, GO was synthesized
Chl a had an increased AD/AG ratio of 1.73 as compared to by a modified Hummer’s method from graphite flakes.36 Two grams
CRGO with an AD/AG ratio of 1.15 (Figure S5.1), indicating of graphite flakes with 12 mL of concentrated sulfuric acid was heated
the Chl a interaction with CRGO, thereby increasing the at 80 °C on a hot plate for 4.5 h. The mixture was cooled to room
defects in CRGOs. Likewise, ATR-FTIR for the CRGO/Chl a temperature and subjected to ultrasonication for 5 h to break large
hybrid showed the presence of functional groups correspond- graphite flakes. After sonication, the mixture was diluted with 500 mL
ing to Chl a on CRGO surfaces (Figure S6). The functional of Milli-Q water and allowed to stand overnight. The graphite flakes
groups were observed at wavelengths of ∼1570, ∼1545, were filtered and dried in a hot air oven at 70 °C, which were then
∼1457, ∼1095, and ∼1021 cm−1,34 which confirmed the transferred into 120 mL of concentrated sulfuric acid. Fifteen grams of
binding of Chl a on CRGOs. potassium permanganate was slowly added with stirring for 2 h, and
this mixture was diluted with 200 mL of Milli-Q water and stirred for
Considering the importance of maintaining the solvent
2 h followed by additional dilution with 700 mL of Milli-Q water.
polarity in hybrid fabrication, we performed experiments by Furthermore, this mixture was incubated overnight with 20 mL of
changing the solvent polarity within the different polar protic hydrogen peroxide. The solution was again sonicated in a water bath
solvents, e.g., a mixture of water and methanol. The UV−Vis for 3−4 h until large graphitic crystals were broken into fine crystals.
and fluorescence spectra showed aggregation of Chl a in these The mixture was divided into 15 mL batches and was centrifuged at
solvent mixtures (Figures S1.2 and S2.1), which was confirmed 12,000 rpm for 30 min. The pellets were then washed with
by AFM that showed formation of chain-like self-assemblies hydrochloric acid (1:10, v/v) to remove any metal ions for 10 min
(polymers) on CRGO (Figure 4c). Our results are consistent at 12,000 rpm. The repeated washes with Milli-Q water were
with literature35 that have reported the aggregation behavior of continued until a light golden layer of GO started to float, which was
Chl a in the presence of water mixtures with different organic then collected.
Chemically Reduced Graphene Oxide (CRGO). The GO
solvents. The parameters influencing the aggregation of Chl a synthesized from modified Hummer’s method was chemically reduced
were studied by UV−Vis, fluorescence, and FTIR studies. First, using hydrazine hydrate.37 The weight of GO/hydrazine was in a ratio
the aggregation was evidenced through large red/blue shifts of 10:7 (W/V %) at pH 10 using ammonia solution. This solution
and fluorescence quenching, especially when there were more mixture is reduced in an oil bath for 1 h at 95 °C till the solution color
water molecules present in organic solvents. Later, studies turns from golden yellow to black dispersion.
showed that the dielectric constant, refractive index, hydrogen Dispersion of Graphene Oxide (GO), Chemically Converted
bonding, hydrophobic interaction, and interaction of solutes Graphene (CRGO), and Chlorophyll a in Organic Solvents. In
the Polar Protic (Methanol) Solvent. Graphene oxides can be
with solvents affected the aggregation of Chl a and it was
dispersed directly in methanol solvent due to their both hydrophilic
concluded that water plays an important role in self-assembly and hydrophobic surfaces,38 and they were dispersed first by
of Chl a. The presence of water offers more protons in the separating the GO dispersion from water through centrifugation at
solvent, which leads to the formation of intra-hydrogen 12,000 rpm for 20 min, and then the GO pellets were redispersed in
bonding within Chl a, resulting in aggregation. Therefore, it methanol solvent followed by sonication in a water bath for 1 h until
indicates that solvent polarity is an important parameter that they were completely dispersed.
needs to be considered to control the stability of the Chl a CRGO, which is more hydrophobic, was dispersed first by
molecule in solution. aggregating the CRGO sheets with NaCl salt, as reported in

■
literature.39 The addition of NaCl into CRGO resulted in flocculation
CONCLUSIONS of their sheets. These aggregates were separated from water through
vacuum filtration and redispersed in methanol solvent followed by 1−
In summary, solvent polarity plays an important role in 2 h of sonication. The water content in the polar solvent is <10%.
efficient formation and stability of such an organic hybrid In the Polar Aprotic (DMF) Solvent. GO and CRGO can be
system. Among solvents, methanol offers sufficient protons in dispersed in DMF solvent from the method reported by Park et al.40
the solution that facilitates Chl a supramolecular self-assembly In this method, the GO was dispersed in DMF solvent by dispersing 1

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mL of GO in 9 mL of DMF solvent with addition of 1 μL of hydrazine Samples of Chl a, CRGOs, and Chl a/CRGOs were prepared in
monohydrate, and the mixture was stirred at 80 °C for 12 h to reduce methanol solvent, washed by centrifugation at 12,000 rpm, and
graphene oxide into CRGO. redispersed in the same solvent. The samples were drop-casted on an
In the Non-Polar (O-DCB) Solvent. The dispersion of GO and ATR-FTIR crystal and air-dried until a thin layer was formed on the
CRGO in non-polar solvents are often unstable and only low crystal surface before measurement.
concentration can be obtained, and their dispersion in O-DCB solvent Note that the concentrations of Chl a, CRGOs, and Chl a/CRGOs
was according to the method by Konios et al.41 The method is to dry were all maintained and were the same as those used for the
GO and CRGO in a hot air oven at 70−80 °C and then disperse them spectroscopic study.

■
in O-DCB through ultrasonication in a water bath for 30 min.
All spectral measurements were carried out by using 0.08 mg/mL ASSOCIATED CONTENT
stock concentration of GO and CRGOs.
Chlorophyll Dispersion in Methanol, DMF, and O-DCB Solvents. *
sı Supporting Information

The chlorophyll a (Chl a) dried pellet from Sigma-Aldrich (1 mg) was The Supporting Information is available free of charge at
directly dispersed in each of the three solvents separately. These https://pubs.acs.org/doi/10.1021/acs.langmuir.0c02370.
solutions were further diluted to 0.1 mg/mL before titrating against
CRGOs.
UV−Vis spectra, fluorescence spectra, fluorescence
UV−Visible and Fluorescence Spectroscopy. A UV−Vis quenching, Stern−Volmer plot, Stern−Volmer quench-
Varian Cary 300 spectrophotometer in continuous mode from ing constant, binding constant, Scatchard plot, Raman
800−200 nm wavelength at a scan rate of 500 nm/min and a spectra, deconvolution data, and ATR-FTIR spectra
fluorescence Varian Cary 300 spectrophotometer in continuous mode (PDF)

■
from 600 to 800 nm range at a scan rate of 600 nm/min were used to
acquire sample spectra using a quartz cuvette of 1 mm path length.
Density Functional Theory (DFT) Calculations. The calcu- AUTHOR INFORMATION
lations were performed based on density functional theory (DFT) by Corresponding Author
using the plane-wave basis Vienna ab initio simulation package Wenrong Yang − School of Life and Environmental Science,
(VASP) code,42,43 implementing the projector augment wave (PAW) Deakin University, Geelong, Victoria 3216, Australia;
method.44 A dispersion correction of total energy (DFT-D3 orcid.org/0000-0001-8815-1951; Email: wenrong.yang@
method)45 was used to incorporate the long-range van der Waals
interaction. To study two-dimensional (2D) systems under the deakin.edu.au
periodic boundary condition, a vacuum layer with a thickness of 18 Å Authors
and a plane-wave basis set with an energy cutoff of 500 eV were set to
minimize artificial interactions between neighboring layers. The Tejaswini R. B. Ramakrishna − School of Life and
structures studied here were fully relaxed until energy and force were Environmental Science, Deakin University, Geelong, Victoria
converged to 10−6 eV and 0.005 eV/Å, respectively. Due to the large 3216, Australia
supercell, a single Gamma point (1 × 1 × 1) was used to sample the Motilal Mathesh − School of Life and Environmental Science,
2D Brillouin zone for optimizing the geometry and for calculating the Deakin University, Geelong, Victoria 3216, Australia
charge density. Zhen Liu − College of Materials Science and Engineering,
AFM. The topography images and the sample height profile data Institute for Graphene Applied Technology Innovation, Qingdao
were obtained using an AFM on multimode 8 from Bruker Bioscience University, Qingdao 266071, People’s Republic of China
corporation (U.S.A) in peak force quantitative nanomechanical
Chunmei Zhang − Institute of Modern Physics, School of
imaging mode. Chl a and CRGOs prepared in methanol solvent
were mixed and incubated for 5 min, and the samples were Physics, Northwest University, Xi’an 710069, People’s Republic
centrifuged at 12,000 rpm to pellet out Chl a and CRGOs. The of China
supernatant was discarded, and the pellets were resuspended in Aijun Du − Science and Engineering Faculty, Queensland
methanol solvent. These samples were then drop-casted on a mica University of Technology, Brisbane, Queensland 4000,
surface and air-dried. Also, as a control, Chl a and CRGO prepared Australia; orcid.org/0000-0002-3369-3283
individually in methanol were drop-casted on a mica surface and air- Jingquan Liu − College of Materials Science and Engineering,
dried. All samples were scanned at 512 scans/lines at a scan rate of Institute for Graphene Applied Technology Innovation, Qingdao
0.521 Hz with an aspect ratio of 1 at room temperature using an AFM University, Qingdao 266071, People’s Republic of China;
silicon tip on the nitride lever (Bruker) of a SCANASYST-AIR model. orcid.org/0000-0001-6178-8661
Nanoscope analysis software (version 8.1) was used to process AFM
image and height data.
Colin J. Barrow − School of Life and Environmental Science,
Raman Spectrophotometer. A Renishaw Invia Micro spec- Deakin University, Geelong, Victoria 3216, Australia
trometer (Renishaw plc, Glocestershire, UK) equipped with an Ar+ Min Chen − School of Biological Science, The University of
ion laser and CCD detector was used to obtain Raman spectra for all Sydney, Sydney, New South Wales 2006, Australia;
samples. Chl a, CRGOs, and Chl a/CRGOs prepared in methanol orcid.org/0000-0001-8070-3248
solvent were washed through centrifugation at 12,000 rpm and Mark J. Biggs − Heriot-Watt University, Edinburgh EH14 4AS,
redispersed in the same solvent. These samples were then drop-casted United Kingdom
on a glass slide covered with aluminum foil and air-dried before
measurement. The scanning resolution was set for a sample exposure Complete contact information is available at:
time of 10 s at 4 cm−1 spectral resolution with 5−10% laser power. https://pubs.acs.org/10.1021/acs.langmuir.0c02370
WiRE software (version 8.1) was used to perform the deconvolution
study. Author Contributions
ATR-FTIR Spectrophotometer. A Bruker Alpha ATR-FTIR The manuscript was written through contributions of all
spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany) authors. All authors have given approval to the final version of
equipped with a deuterated triglysine sulfate (DTGS) detector and the manuscript.
single reflection diamond ATR sampling module (Platinum ATR
Quik − Snap TM) was used. The sample scanning parameters were Funding
set at a scan rate of 256 nm/min and 4 cm−1 spectral resolution. The Australian Research Council (DP130101714) and the
OPUS.7 software suite was used to process the sample spectral data. NSF of China (21675075) are thanked for financial support.
F https://dx.doi.org/10.1021/acs.langmuir.0c02370
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Notes (16) Das, D.; Sarkar Manna, J.; Mitra, M. K. Electron Donating
The authors declare no competing financial interest. Chlorophyll-a on Graphene: A Way toward Tuning Fermi Velocity in

■
an Extended Molecular Framework of Graphene/Chlorophyll-a
Nanohybrid. J. Phys. Chem. C 2015, 119, 6939−6946.
ACKNOWLEDGMENTS (17) Xue, T.; Jiang, S.; Qu, Y.; Su, Q.; Cheng, R.; Dubin, S.; Chiu, C.
This work is financially supported by the Australian Research Y.; Kaner, R.; Huang, Y.; Duan, X. Graphene-Supported Hemin as a
Council (DP130101714), and the NSF of China (21675075) Highly Active Biomimetic Oxidation Catalyst. Angew. Chem., Int. Ed.
is thanked for financial support. T.R.B.R. acknowledges Deakin Engl. 2012, 51, 3822−3825.
University for her admission to the Master of Honours (18) Xu, Y.; Zhao, L.; Bai, H.; Hong, W.; Li, C.; Shi, G. Chemically
Converted Graphene Induced Molecular Flattening of 5,10,15,20-
(Biotechnology) program to pursue her studies.

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Tetrakis(1-methyl-4-pyridinio) Porphyrin and Its Application for
Optical Detection of Cadmium(II) Ions. J. Am. Chem. Soc. 2009, 131,
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O-dichlorobenzene; ATR-FTIR; attenuated total reflectance- (20) Bagchi, B.; Jana, B. Solvation Dynamics in Dipolar Liquids.
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