SUPPORTING INFORMATION
Probing the nature of electron transfer in metalloproteins on graphene-family materials as
nanobiocatalytic scaffold using electrochemistry
Sanju Gupta and Aline Irihamye
Experimental
Materials Preparation
The Py–1SO3 (1-pyrenesulfonic acid sodium salt) and Py–Me-NH2 (1pyrenemethylamine salt) yellowish solutions were prepared by dissolving 1 mg of Py–1SO3 and
Py–Me-NH2 powder (Sigma–Aldrich, P97.0% (HPLC)) in 10 mL of distilled water. The
dispersion was produced by sonicating 30 mg of graphite flakes (Graphene Supermarket, Grade:
99.5) with 10 mL of as-prepared Py–1SO3 and Py–Me-NH2 solution in ultrasonicator bath
(Fisher Scientific, MA). After 80 min sonication, the large clusters of non-exfoliated graphite
were removed by mild centrifugation at 1000 rpm for 20 min. The dispersion was then re-filled
with water and centrifuged at 12,000 rpm and 3,000 for 20 min for three times in order to wash
the excess Py–1SO3 and Py–Me-NH2 molecules. It was found that the smaller rpm of 3,000
provided relatively larger flakes as compared to larger 12,000 rpm. After washing the graphenebased materials collected at the bottom of the micro-centrifuge tubes, they were re-sonicated in
water for 1 min. In the last step, the final dispersion was obtained after removing the residual
graphite flakes further by mild centrifugation at 1000 rpm for 20 min (Fig. S1a). The resulting
dispersed suspension is light gray and stable over months. Shown in Fig. 1b is the suspensions
containing cuvettes from step 1 to 4 (final) for Py–1SO3 (top panel) and Py–Me-NH2 (bottom
panel), respectively. Suspension is obtained with nonionic surfactant Pluronic® P-123 (PEGPPG-PEG or Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)
polymer Sigma-Aldrich, MA) following the modified procedure described in Ref. Error! Bookmark
not defined.. Briefly, the natural graphite powder (75 mg mL−1) and Pluronic P-123 (0.75 wt%) were
ultrasonicated for 5 hours followed by centrifugation for 5 min at 5,000 rpm (Fig. S1a).
1
We also sonicated the dispersion for 3 days in order to achieve a high yield of singlelayer graphene (~ 50%), but the flakes were too small and it was tedious to find them under
optical microscope. For graphene laminate or paper shown in Fig. S1c is prepared by filtering
140 mL dispersion obtained using Pluronic® P-123 on an alumina membrane (pores size of 0.2
m). The film drying was carried out in air at room temperature for ~ 3 days. All of the
suspensions are well characterized using UV-Vis spectrophotometer and Raman spectroscopy
along with optical microscopy and scanning electron micrograph of precursor natural graphite
flakes. A typical TEM at two magnifications containing several graphene flakes is shown as an
example of mono- and bi-layer graphene layers (Fig. S1d). The concentration of dispersion
remaining after centrifugation is ~ 0.024 mg/mL for Py–1SO3 and Py–Me-NH2 and 0.8 mg/mL
for Pluronic® P-123, respectively, calculated using an extinction coefficient following LambertBeer’s behavior for all pyrene solvents, <660nm> = 2,460 L g-1 m-1Error! Bookmark not
defined. and <660nm> = 1,390 L g-1 m-1 2 in surfactant/water solutions.
GO is synthesized following modified Hummer and Offeman method described in
literature.3,4 Briefly, natural graphite powder (4 g, Flake Graphite, Graphene Supermarket, TX) is
oxidized by adding to a hot solution (80 oC) of concentrated sulfuric acid H2SO4 (25 mL)
containing K2S2O8 (8 g) and P2O5 (5 g). The resultant dark blue mixture is thermally isolated and
slowly brought to room temperature over a period of 6 h. The mixture is then diluted to 300 mL
and filtrated with a 0.22 m membrane. The filtered product is dried overnight at 60 oC. The preoxidized graphite powder (2 g) is added to 90 mL of cold H2SO4 (0 oC), and 12 g of KMnO4 as
strong oxidizing agents is gradually added under stirring in an ice bath. After being stirred for 15
min, 2 g of NaNO3 is added to the mixture, and the resulting mixture is further stirred at 35 oC
for 2 h and distilled water (200 mL) is added. The reaction is stopped with the addition of a
mixture of 560 mL of distilled water and 10 mL of 30% vol. H2O2. For purification, the mixture
is washed with 1:10 HCl and then with water. The GO product is re-suspended in water to a
brown dispersion, which is subjected to dialysis to remove residual metal ions and acids. The
purified GO dispersion is sonicated by centrifugation (3000 rpm, 5 min). The as-prepared GO
samples resulting in ratio of C:O (2:1) were then characterized with atomic force microscopy in
tapping mode and transmission electron microscopy (not shown). Likewise, to prepare rGO a
colloidal suspension of GO platelets in purified water (3 mg/ml) was sonicated in ultrasound bath
(Fisher Scientific TM Model 15-335-60) for 3 h. Hydrazine monohydrate (1 l for 3 mg of GO,
98%, Sigma-Aldrich for 3 mg of GO) was subsequently added to the suspension. Additional
stirring with Teflon coated stirring bar in an oil bath held at 80 oC for 12 h yielded a black
precipitation of rGO powder. After cooling to room temperature, the powder is filtered through a
fritted glass filter (medium pore size) followed by suction-drying under house vacuum for 12 h.
The resulting black material (reduced graphene oxide, rGO) is dried under vacuum using a
mechanical pump. Hydrazine is effective for removal of in-plane functional groups (epoxides and
hydroxyls) but leave the edge moieties (carboxyls and carbonyls) intact with C:O (12:1).Error!
Bookmark not defined. Through x-ray energy dispersive spectroscopy, we found a ratio of 8:1 for our
rGO. Also, it may introduce amine functional group and it is found that the metal particles bind
strongly to these functional moieties than oxygen containing groups on GO.
Water soluble metalloproteins used in the present study are cyt c (from equine heart,
cytochrome c, MW 12 kDa), Mb (from equine heart, myoglobin, MW 17,500 Da) and HRP (EC
1.11.1.7 type VI, horseperoxidase, MW 42 kDa), all of them are obtained from Sigma-Aldrich
(St. Louis, MO, USA) and they were used as purchased. The stock solution of these
metalloproteins is prepared in 25 mM phosphate buffer solution (25 mM NaCl, pH 7.4) and
stored at 4 oC. All of the solutions were prepared with Nanopure water (18 M cm-1) from a
Millipore (Milli-Q) system.
Methods and Characterization
A UV–Vis-NIR spectrophotometer (Perkin-Elmer 1050) in a range of 190-800 nm and
spectral resolution of 0.5 nm was used to measure optical absorption spectroscopy of the
graphene dispersions and of protein-graphene conjugates suspensions purged with nitrogen for
30 min. Then the prepared dispersion was drop casted on a SiO2/Si substrate for scanning
electron microscopy (SEM), Atomic Force Microscopy (AFM, not shown), transmission electron
microscopy (TEM) and Raman Spectroscopy (RS) characterization. SEM images were taken on
a JEOL (Model JEOL 5400LV, MA) instrument operating with thermionic emission gun
(Tungsten filament) at different primary electron acceleration voltage (Vacc) in secondary
electron imaging (SEI) mode and collected with an in-lens detector and equipped with an x-ray
ISIS EDS system. Samples for TEM were prepared by placing two drops on commercial lacey
carbon Cu 400 mesh grids (Ted Pella Inc. CA) and allowing it to dry in air giving several regions
and sufficient number of isolated graphene flakes. A FEI Tecnai (Model G2 F20) with 200 kV
accelerating voltage was used to determine lateral dimensions. Height and shape of the
nanoplatelets were determined using AFM and SEM. A PicoPlus instrument series 5500
(Molecular Imaging Inc. MI) operating in tapping mode for AFM imaging was used. The AFM
tips were of silicon with nominal resonance frequency and spring constant of 15 kHz and 0.4
N/m, respectively, and the scan speed varied depending on the image size and ranged from 1 to
10 m/s, lateral optical level sensitivity of 1 V and manufacturer’s stated tip radius of
curvature when first used of < 10 nm (BudgetSensors, ContAl, Bulgaria). The Raman spectra
were recorded using a micro-Raman spectrometer (Model InVia Renishaw plc, UK) equipped
with excitation wavelength of 632.8 nm (1.92 eV) and 532 nm (2.32 eV) and maximum power
18 mW (~ 5-7 mW incident at the sample), with edge filters cutting at ~ 100 cm-1 to remove the
laser excitation. The scattered light from the sample is collected in backscattering geometry,
transmitted by a beam splitter and detected by CCD camera. An objective lens of 50x was used
providing spot size of ~ 2 m. Extreme care is taken to avoid sample damage or laser induced
thermal degradation and desorption of the molecules.
All of the electrochemical measurements were performed with a CH Instruments (Model
CHI 760 E, Austin, TX). A conventional three-electrode cell consists of a modified polished
glassy carbon (GC) disk electrode as working electrode (2.5 mm in diameter), a Pt wire counter
electrode and Ag/AgCl (saturated with 3M KCl) reference electrode were used. Working
electrodes were polished by 0.3 and 0.05 m of alumina polishing compounds, rinsed with
excess water and ethanol, and briefly sonicated prior to each experiment. Phosphate buffer
solution (25 mM NaCl, pH 7.4) was used as aqueous electrolyte purged with high-purity N2 gas
prior to measurements. All of the metalloproteins (cyt c, Mb and HRP) of 2 mg/mL and monographene, Gr_LPE, rGO, GO (both 0.3 mg/mL) were prepared in de-ionized water and they were
mixed for 5-7 min at room temperature. Then 10 L of the mixed solution was drop-cast on GC
electrodes allowed to dry at room temperature for electron transfer kinetic experiments
investigated by electrochemistry. Figure 1 shows schematic of the graphene-family
nanomaterials supported / modified heme-containing metalloproteins at the surface of GC
electrodes.
Characterization
Graphene flakes in solution
The high surface energy of water does not allow dispersion and exfoliation of graphite in
this solvent. Pyrene, by itself, is not soluble in water. Different groups are attached to tune its
solubility such as sulphonic groups (SO3), amine groups (NH2), carboxylic acid groups (COOH)
and more complex groups generating aromatic amphiphiles. The nature and number of the
functional groups is critical because the molecule should have a good affinity with the solvent.
Thus the easiest solution is to use a mixed solvent such as methanol and water: the first allows
dispersion of the molecule in the solvent, while the second allows interactions between the
molecule and graphene. To provide quantitative assessment and comparison of the dispersing
ability of various solvents and surfactant used in this study, we measured the UV-Vis optical
absorption of the suspensions at a specific wavelength of 660 nm and estimated the
corresponding concentrations or yields therefrom in addition to the absorption spectra in the 190800 nm spectral window shown in Fig. S3 for Py-1SO3 and Py-Me-NH2 displaying the
characteristic peaks between 200 and 300 nm albeit producing graphene without toxic chemical
reduction.5 Compared to the absorption spectrum of Py–1SO3 and Py-Me-NH2, the baseline of
the absorption of the graphene-based dispersion absorbs over the whole frequency range, as it is
expected for graphene dispersions. The original peaks of the Py–1SO3 and Py-Me-NH2 solution
are still visible at around 340 nm, but they are broader and split: this is fingerprint of the –*
interaction between Py–1SO3 (and Py-Me-NH2) and graphene observed when single-walled
CNTs were dispersed by pyrene-based molecules.6 The exfoliation of graphite to graphene sheets
with sonication dramatically increased the surface area for pyrene-molecule adsorption thus the
concentration of free pyrene molecules in the solution is decreased. A peak at 233 nm
corresponds to -* transition belonging to C=C bond and a shorter peak around 270 cm
corresponding to -* transition revealing that -conjugation of graphene sheets is largely
retained during the direct LPE process. In fact fluorescence spectroscopy has shown that the use
of Py-1SO3 resulted in a shorter period of sonication to diminish the excimer peak at 501 nm.
Additionally, fewer washing cycles were needed to remove sulfonated and amine monomers.7 As
a result, these subtle differences may be due to the different solubility of the two large planar
aromatic pyrene-based molecules. Collectively, these observations confirm the presence of both
pyrene-based solvent molecules and graphene, i.e. the molecules interacting with the graphene
(hydrophobic) sheets through –* interactions thus exfoliating and stabilizing graphene in
water. The negative and positive charges in both dispersion molecules act as stabilizing species
to maintain steric repulsion force between the charged graphene sheets. This also means that
there is an excess of molecules in solution. We tried to perform Raman spectroscopy directly on
the dispersions, but the Raman signal was completely hidden by fluorescence background. It is
also reported that these pyrene-based molecules act as nanographene molecules to heal the
plausible defects in graphene sheets during annealing. Remarkably, they appear to act as
electrical “glue” soldering adjacent to graphene sheets such that electrical contacts between
graphene sheets can be dramatically improved across the film.
The absorption spectral features of Pluronic® P-123 based graphene dispersion is
confirmed by the presence of only 270 nm absorption peak (not shown). The most significant
trend is that non-ionic surfactant (with hyrdrophobic tail and a long hydrophilic head causing
steric repulsion), appears to be more effective in the stabilization of the exfoliated material
compared to ionic surfactants (since they adsorb onto graphene and impart an effective charge,
providing electrostatic repulsion to prevent graphene from aggregation). The concentration
typically achieved lie in 0.2-0.6 mg mL-1 in just five hours of sonication. In comparison with
other surfactants, non-ionic Pluronic® P-123 is significant for higher exfoliation degree (higher
concentrations or yield), structural quality and lateral size. In fact we filtered the graphene
dispersion through micro-filter paper overnight and constructed graphene laminate as shown in
Fig. S1c. An overview of the most recent works is reported in Table SI.
Graphene flakes on the substrates
For a definitive identification of graphene layers, we have used transmission electron
microscopy and Raman spectroscopy techniques as discussed below, an example of which is
shown in Figs. S1d and S3, respectively. From Fig. S1d showing transmission electron
microscopy images at two magnifications, it appears to be graphene monolayer and bilayer and
at times composed of more than one graphene monolayer on top of each other forming
multilayers. It shows large number of flakes with lateral sizes of ~300 nm. Raman spectroscopy
(RS) is a potential analytical technique for characterizing graphene-related materials. We used
RS to characterize the obtained suspensions after deposition onto a Si substrate with 285 nm
thick SiO2 layer. Figure S3a and S3b shows Raman spectra (first- and second- order) for the
solvents and surfactants used in this study besides natural expanded graphite flake used to make
graphene dispersions. A typical Raman spectrum of monolayer graphene shows two main
features namely, the G peak, corresponding to the E2g phonon mode at the Brillouin zone center,
at ~1580 cm-1 and 2D peak, which is activated by two-phonons intervalley assisted Raman
scattering, at ~ 2700 cm-1. The 2D peak can be used to identify graphene, the 2D peak is a single
and sharp peak in the case of monolayer graphene, while in AB-stacked bilayer the 2D peak is
composed of four bands. Graphite shows a broad and up-shifted 2D peak, which in first
approximation can be fitted with two peaks. The 2D peak shape quickly evolves with the number
of layers, so that the 2D band of a sample containing more than 8–10 layers is hardly
distinguishable from that of bulk graphite.8 In case of disorder, defect-activated features appear
in the Raman spectrum: the D peak, first order of the 2D peak, which lies at ~1350 cm-1, and the
D’ peak, which appears at ~1620 cm-1. Pristine graphene does not have enough structural defects
for the D and D’ peaks to be Raman active and they are seen at the edges instead. In order to
have a good statistics of the composition of the material obtained, RS was performed on a large
amount of flakes deposited.
The spectra from our LPE synthesized graphene show common features, such as the G
and 2D peaks. However, D, D’ and 1245 cm-1 peaks are also visible occasionally in the Raman
spectra. The latter is attributed to pyrene aggregates since this peak has been observed in pyrene
crystals9 and re-aggregation is driven by amphiphilic nature of the molecules. Furthermore, a
small fluorescence background in the Raman spectrum indicates that the flakes may be covered
by a layer of solvent molecules. While graphene is able to quench the fluorescence from the
molecules due to charge transfer, it happens only if the molecules are close enough to the surface
of graphene.10
Based on the shape of the 2D peak (Fig. S3), we made an attempt to derive the amount of
graphene in solution. We found that ~ 14% of the flakes are single-layers, ~ 66% are few-layer
graphene flakes (< 7-8 layers) and 20% are thicker flakes. However, they also offer an
approximate idea on the exfoliation efficiency. A few large clusters are also visible and the
thickness of the flake is approximately 5 nm and the distance between the two layers is again 5
nm. Since the flake is smaller than the spot size, we may expect edges to contribute to the D
peak. This has been further confirmed by the intensity ratio between the D and G peak, I(D)/I(G)
on the flakes. This correlation has been used to determine the size of sp2 C domains in various
carbon-based materials including graphene.11,12 We report the ordered-crystalline graphitic
domain was ~ 6-8 nm in as-produced dispersion flakes deposited on Si substrates at the final step
following Tunistra-Koening relation.11 While it is possible that Fig. 4 shows the maximum
I(D)/I(G) is observed at the edges, however, far from the edges, I(D)/I(G) is not null and it is not
uniform over the flake. This confirms that the D peak cannot be entirely assigned to edges and
disorder, but needs to be related to the amount and distribution of the molecules. This can be
confirmed by easily desorbing the molecules by local laser heating. We have tried to remove this
molecules film by washing the sample several times using 100 mL of DI water for 12 h and
refilled the container with fresh water every 5 h. We found that the excess molecules can get
removed further with each wash but a limit is reached at the 3rd or 4th wash, in agreement with
Ref. 13.
We found that by using this protocol presented in this work based on ultra-sonication and
centrifugation with Py–1SO3, Py-Me-NH2 and Pluronic® P-123, it is possible to achieve stable
graphene suspensions directly in water. It is now interesting to compare our results with previous
works in order to achieve a further understanding of the exfoliation mechanism. In particular, it
is interesting to compare exfoliation obtained with Py– 1SO3 and Py-Me-NH2. The difference
between these two molecules is given only by the number of sulphonic versus amine groups.
There is a subtle difference between the two molecules owing to their efficient solubility in
water. Moreover, it is found while the yield of graphene mono- and bi-layers using pyrene-based
molecules is relatively small (as observed from the color of the suspension, almost transparent)
but with better structural quality, the Pluronic® P-123 yielded few-layer graphene with a
reasonable structural quality.
(c)
(a)
Py-MeNH
(b)
(d)
Py-1SO3
Figure S1 (Color online). (a) Schematic of the liquid-phase exfoliation (LPE) of graphite flakes
using 1-pyrenesulfonic acid sodium salt (Py–1SO3), 1-pyrenemethylamine salt (Py–Me-NH2) and
nonionic surfactant Pluronic® P-123 (PEG-PPG-PEG polymer). The process includes different
steps (mixing, exfoliation, ultrasonication, and washing). The washing step is repeated for three
times in order to remove the excess liquid exfoliating agents used in this study. (b) Digital picture of
vials containing the resulting graphene-based dispersion at every step for (top panel) Py–1SO3 and
(bottom panel) Py–Me-NH2. (c) Pluronic® P-123 based graphene laminate (or paper) prepared by
vacuum filtering of dispersion. (d) Typical TEM micrographs in two scales containing several
graphene flakes as an example of mono- and bi-layer graphene layers.
Energy (eV)
4.11
4.94
Absorbance (Arb. Units)
2.5
3.09
2.47
2.06
1.76
Py-Me-NH2
-*
Py-1SO3
2.0
1.5
660 nm used
to determine
LPE graphene
yield
1.0
0.5
0.0
300
400
500
600
700
Wavelength (nm)
Figure S2 (Color online). UV-Vis absorption spectra of the Py–1SO3 and Py–Me-NH2 graphene
dispersions at final step. The absorbance at 660 nm was used to determine graphene yield.
Figure S3 (Color online). Raman spectra excited at EL = 1.92 eV of (a) Py–1SO3 and Py–MeNH2 and (b) Pluronic® P-123 along with graphene flake (at EL = 2.32 eV) placed on SiO2/Si
substrate exhibiting prominent first-order (D and G) and second-order (2D) Raman bands.
(a)
(b)
Figure S4 (Color online). Cyclic voltammograms (CV) for (a) Mono-graphene; Gr_LPE; rGO
and GO modified with Mb in 25 mM, pH of 7.4 PBS (phosphate buffer solution) as aqueous
electrolyte at scan rate of 10 mV/s, (b) CV curves for Mono-graphene; Gr_LPE; rGO and GO
modified with Mb at scan rate of 10 mV/s in the presence of 10 M H2O2 and 40 M H2O2 (Mb
modified Gr_LPE , rGO GC electrodes), both at scan rate of 10 mV/s.
Table S1. List of the works reporting direct liquid-phase exfoliation of graphite producing
graphene with pyrene-based solvents and Pluronic surfactant, as compared with our work.
Details of the exfoliation process, the yield obtained and the Raman spectrum as an assessment
and identifying the graphene quality and layers have been included (e.g., presence or absence of
the D peak and the shape of the 2D peak).
Table S1
Gupta et al.
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