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Chiral Graphene Quantum Dots

Nozomu Suzuki,†,‡,§ Yichun Wang,‡,⊥ Paolo Elvati,⊥,∥ Zhi-Bei Qu,† Kyoungwon Kim,†,‡ Shuang Jiang,†
Elizabeth Baumeister,∥ Jaewook Lee,‡,¶,○ Bongjun Yeom,†,‡,□ Joong Hwan Bahng,‡,⊥ Jaebeom Lee,¶
Angela Violi,*,⊥,∥,△ and Nicholas A. Kotov*,‡,⊥,∥,#
†
Department of Chemical Engineering, ‡Biointerfaces Institute, ⊥Department of Biomedical Engineering, ∥Department of Mechanical
Engineering, #Department of Materials Science and Engineering, and △Department of Macromolecular Science and Engineering,
Biophysics Program, University of Michigan, Ann Arbor, Michigan 48109, United States
§
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 8916-5, Japan
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¶
Department of Cogno-Mechatronics Engineering, Pusan National University, Miryang 627-706, Republic of Korea
*
S Supporting Information

ABSTRACT: Chiral nanostructures from metals and semiconductors attract

wide interest as components for polarization-enabled optoelectronic devices.
Similarly to other fields of nanotechnology, graphene-based materials can
greatly enrich physical and chemical phenomena associated with optical and
electronic properties of chiral nanostructures and facilitate their applications
in biology as well as other areas. Here, we report that covalent attachment of
L/D-cysteine moieties to the edges of graphene quantum dots (GQDs) leads to
their helical buckling due to chiral interactions at the “crowded” edges.
Circular dichroism (CD) spectra of the GQDs revealed bands at ca. 210−220
and 250−265 nm that changed their signs for different chirality of the cysteine
edge ligands. The high-energy chiroptical peaks at 210−220 nm correspond to
the hybridized molecular orbitals involving the chiral center of amino acids
and atoms of graphene edges. Diverse experimental and modeling data,
including density functional theory calculations of CD spectra with probabilistic distribution of GQD isomers, indicate that
the band at 250−265 nm originates from the three-dimensional twisting of the graphene sheet and can be attributed to the
chiral excitonic transitions. The positive and negative low-energy CD bands correspond to the left and right helicity of
GQDs, respectively. Exposure of liver HepG2 cells to L/D-GQDs reveals their general biocompatibility and a noticeable
difference in the toxicity of the stereoisomers. Molecular dynamics simulations demonstrated that D-GQDs have a stronger
tendency to accumulate within the cellular membrane than L-GQDs. Emergence of nanoscale chirality in GQDs decorated
with biomolecules is expected to be a general stereochemical phenomenon for flexible sheets of nanomaterials.
KEYWORDS: chirality, graphene quantum dots, circular dichroism, chiral excitons, biological activity

C hirality of inorganic nanostructures found in individual

inorganic nanoparticles and their assemblies have
attracted increasing attention over the past decade.1−7
Unusually high intensity of chiroptical phenomena due to
but in return, they reveal distinctive chiroptical effects due to
higher chemical activity.8,18,21
Research on different forms of nanoscale carbon22−27 is
abundant and, by some measures, exceeds in volume the
research on nanoscale forms of gold. The synthesis of chiral
plasmonic and excitonic effects characteristic of nanoscale
materials fuels this interest.3−6,8,9 Necessity of further develop- nanocarbons is mostly focused on chirality at the atomic scale
and represents a well-known chemical problem and is difficult
ment of this field is incentivized by technological prospects for
to translate from other areas of asymmetric synthesis. For
chiral nanostructures associated with negative refractive instance, the methods suitable for the synthesis of chiral
materials,10 hyperbolic metasurfaces,11 high-sensitivity bioanal- fullerenes28 cannot be extended to the preparation of
ysis,3,9,12 and chiral catalysis.13 The palette of chiral geometries enantiopure carbon nanotubes (CNTs) because the growth
obtained for inorganic nanostructures is broad, but the variety of the graphene network requires a temperature in the range of
of inorganic materials and organic components used for their several hundred degrees on the Celsius scale, but traditional
construction is not. The chiral nanostructures are being made asymmetric catalysts cannot survive it.25,26,29,30 Synthesis31,32
predominantly from gold due to spectral convenience of its
plasmonic bands, chemical stability, and exciting Received: October 9, 2015
physics.6,9,14−17 The studies of chiral semiconductor nanostruc- Accepted: January 8, 2016
tures18−20 are less frequent due to weaker polarization rotation, Published: January 8, 2016

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Figure 1. (a) Molecular schematics of chiral GQD synthesis. Only a portion of the graphene sheet of GQD is shown. (b,c) TEM images of (b)
L-GQD and (c) D-GQD dispersions. (d−f) Photoluminescence spectra of (d) pristine, (e) L-GQD, and (f) D-GQD dispersions under ambient
conditions. Insets of (d−f): photographs of the corresponding dispersions being illuminated by UV light with λmax = 365 nm.

and separation33,34 of CNTs with chiral conformation of from aqueous dispersions. Most of the work reported below
graphene sheets is possible and symmetry-matched nanotubes used GQD dispersions made according to Peng et al.49 unless
with opposite rotatory optical activity have recently become otherwise stated. Successful formation of a GQD dispersion
available.35,36 Particularly interesting would be to obtain chiral with characteristic bright yellow photoluminescence (PL,
forms of graphene, but chiral nanocarbons based on graphene Figure 1) and a size of 2−7 nm was observed. L-Cysteine or
are virtually unknown. Chirality of graphene nanoribbons was D-cysteine moieties were covalently bound to the edges of the
found only for singular pieces with the help of atom probe carbon sheets using carbodiimide/N-hydroxysuccinimide
microscopy.37 (EDC/NHS) cross-linking protocol;50 the corresponding
The need for comparative evaluation of carbon nanostruc- products are denoted here as L- and D-GQDs (Figure 1a).
tures in symmetrically paired (i.e., mirror image) forms and Note that L- and D-GQD notations refer here to the synthetic
understanding different manifestation of their chirality would route and molecular chirality of the amino acid incorporated in
be essential for many fields of science: quasiparticle physics,38 the product. The reason for this choice of the notations rather
optics,39 electronics,37 drug delivery,40 and biomedical imag- than others exemplified by R/S, M/P, or Δ/Λ notations often
ing.41 Graphene quantum dots (GQDs) represent the basic and used in case of chiral macromolecules and supramolecular
most recent forms of nanocarbon relevant for all of these assemblies, is that prior to the use of these notations we have to
areas.42−45 Additionally, exciton confinement in GQDs44 and identify the geometry of GQDs and the origin of chiroptical
plasmonic effects in graphene sheets46,47 make this nanoscale properties, which is the goal of this study. The chiral symmetry
material similar to both noble metal and semiconductor and handedness of the GQDs will be discussed later based on
nanostructures. Studies of chiral GQDs will be essential for the established 3D geometry of the graphene sheets.
understanding chiroptical phenomena in delocalized states and TEM images show that GQDs before and after modification
their utilization in biology and medicine, which motivated us to have similar sizes in the 2−7 nm range, with fairly broad size
synthesize symmetrically paired GQDs in dispersions. Based on distribution expected of GQDs (Figure 1b,c).49 Atomic force
the previous studies,21 we hypothesized that chiral GQDs and microscopy images confirm the size range of GQDs and their
potentially other forms of graphene sheets with nanoscale polydispersity (Figure S2). Due to the nanoscale dimensions of
chirality can be obtained due to intermolecular interactions of this form of nanoscale carbon, the XRD peak at 2θ = 25° is
chiral surface ligands48 leading to out-of-plane buckling of the broad; it can be attributed to (002) carbon-to-carbon spacing of
graphene sheets (Figure S1). 3.7 Å. The XRD peak is similar for all forms of GQDs and
remains unchanged before and after modification (Figure S3).
RESULTS AND DISCUSSION Small graphene sheets with a few nanometers in diameter are
Synthesis and Structural Characterization. The hy- known to exhibit quantum confinement.42,44,51,52 The UV−
pothesis of asymmetric synthesis of GQDs was tested starting visible absorption spectrum of pristine GQDs reveals a peak at
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Figure 2. (a,b) Circular dichroism (a) and g-factor (b) spectra for L-GQD (red) and D-GQD (black) dispersions. (c,d) Optimized geometry of
a graphene sheet in model GQDs viewed from the direction of the largest dihedral angle, θ. Note that the handedness (i.e., rotatory direction)
of helices is opposite to the handedness of the edge ligands. The chirality of the ligands is noted on the top of the image; they were removed
for clarity.

228 nm, which is strongly blue-shifted compared to that of graphene sheets, which can be further affirmed by both Fourier
graphene or carbon nanotubes.53 The UV−vis absorption peak transform infrared (FT-IR) and Raman spectroscopy. FT-IR
of L/D-GQDs is observed at 265 nm (Figure 2) and indicates spectra of pristine, L-, and D-GQDs show the presence of the
partial relaxation of exciton confinement due to the hybrid- carbonyl, carboxyl, hydroxyl, and epoxy groups (Figure S5a).
ization of the aromatic system of the graphene dot with the The vibrational signatures of O−H bonds can be found at 3400
atomic orbitals on cysteine moieties. Concomitant spectral cm−1, C−H bonds (aliphatic stretching mode) at 2960 cm−1,
changes also develop in PL spectra. Being excited by photons CC bonds (skeletal vibrations of non-oxidized graphitic
with λex = 330 nm, pristine, L-, and D-forms of GQDs all display domains aromatic group) at 1620 cm−1, C−H at 1420 cm−1,
strong emission at 520−550 nm (Figure 1d−f). The red shift and alkoxy C−O bonds at 1170 cm−1.57 After conjugation with
after the modification with the amino acid in the PL peaks is, cysteine, new peaks appeared at 2390 and 930 cm−1 from S−H
nonetheless, subtle (Figure 1d−f). Comparison with the and C−N bonds, respectively. The former one originates from
absorption spectra of both pristine and cysteine-modified attached cysteine ligands, while the latter one indicates covalent
graphene dots in Figure 2 reveals strong Stokes shift of the bonding between the carbon of GQD and the amino group of
synthesized GQDs. The reason for the large energy difference cysteine (Figure 1a). Assignment of the FT-IR peaks was
between the first absorption band with lowest energy at 260− further substantiated by the calculations of vibrational spectra
270 nm and emission band at 520−550 nm can be formation of using Merck molecular force field (MMFF) developed for
small agglomerates in which GQDs of different sizes are likely evaluation of conformations of drugs: all the peaks in the
to be present. In this case, emission should occur from the fingerprint region of 800−1800 cm−1 match those observed
particles with lowest energy, as was observed for semiconductor experimentally (Figure S5b).
quantum dots.54,55 Formation of exciplex states known for the Raman spectroscopy was used to elucidate the chemical state
red shift of the emission of anthracene, coronene, and other of the graphene network of the GQDs (Figure S6a). The
aromatic hydrocarbons is also possible.56 original and modified GQDs display A1g D band at 1355 cm−1
The electrokinetic zeta-potential (ζ) of pristine GQDs is and E2g G band at 1590 cm−1.58,59 The weak ∼2700 cm−1 peak
−26.7 mV (Figure S4a). After attachment of cysteine moieties, corresponding to a 2D harmonic band can be found in all
ζ-potentials of L- and D-form chiral GQDs become −12.7 mV samples. The relative intensities ID/IG are close to 1.0, which is
(Figure S3b) and −10.3 mV (Figure S3c), respectively, which is higher than that for GQDs prepared by electrochemical etching
consistent with amidation of negatively charged −COOH (ID/IG = 0.5).60 Most importantly, the positions and relative
groups at GQD edges while retaining a substantial degree of intensities in Raman scattering peaks do not change after the
ionization. modification with cysteine, indicating intactness of the central
The entirety of spectroscopy and microscopy data points to graphene sheets. Importantly, GQDs also display distinct
the conjugation of cysteine moieties to the edges of the enhancement of Raman bands of crystal violet molecules.61
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Figure 3. (a−d) Molecular models of L-GQDs of different sizes and placements of L-cysteine edge ligands used in the computational evaluation
of their geometry and optical properties.

Figure 4. Molecular orbitals for HZ4 GQDs exemplified by (a) HZ4−A1 and (b) HZ4−A2 regioisomers. Additional MO calculations for HZ3
and HZ2 are given in the Supporting Information.

This typical surface-enhanced Raman scattering (SERS) label be observed in CD spectra (Figure 2). The high-energy peak at
was incorporated between the graphene dots following the 210−220 nm matches that in the CD spectra of free cysteine at
standard process using aqueous solution.62 The sharp intense 209 nm (Figure S7).68 Both L- and D-GQDs concomitantly
SERS lines of crystal violet matching those obtained for gold gave rise to a new peak at 250−265 nm (Figure 2a).
particles61−64 indicate that the synthesized GQDs exhibit some Importantly, this new low-energy chiroptical band has opposite
properties characteristic of plasmonic particles. signs depending on the chirality of the amino acid used for
Chiroptical Properties of L/D-GQDs. The ring-like conjugation to the GQDs, whereas the graphene sheets (Figure
arrangement of chiral ligands in GQDs is conducive to the S8a), unmodified GQDs (Figure S8b), and rac-GQDs (i.e.,
asymmetric preference in the conformation of the entire made with racemic mixture of L- and D-cysteine, Figure S8c)
“molecule” due to collective chiral interactions of the chiral display no chiroptical activity in CD spectra at 250−265 nm.
ligands known for many supramolecular assemblies and The asymmetry of the CD peaks in respect to the abscissa (i.e.
macromolecules.65−67 Chiroptical activity of L- and D-GQDs imperfect mirror-image spectra) was reproducible in several
indicative of the preference toward a specific stereoisomer can independent synthetic series carried out by two experimental
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materials chemists (K.K. and Z.-B.Q.), who performed these hybridization of the chiral electronic states of the edge ligand is
experiments 2 years apart. The low-energy peak for D-GQDs more likely to occur for smaller GQDs. As GQD becomes
was always red-shifted compared with that for L-GQDs. Such larger, the difference in energies between the electronic states of
asymmetries are not uncommon in the studies of chiral L / D -cysteine and the graphene sheet becomes wider.
supramolecules and in case of GQDs are likely to reflect Importantly, HZ4-type GQDs are smaller than the lower size
variable contributions of different isomer sizes of sheets and threshold in 2−7 nm GQD dispersions. As an illustration, 5 nm
GQD agglomerates present in L- and D-dispersions. At the GQDs contain about 10 circles of aromatic rings. Thus, one
present stage of research in this area, it is difficult to speculate would not expect the hybridized orbitals to form for our GQDs
about its origin and should be the subject of a dedicated study. at the MO energies corresponding to the 250−265 nm CD
The chiroptical asymmetry parameter also known as the g- band.
factor of chiral GQDs at the 250−265 nm chiroptical band is We also considered the possibility that chiroptical bands
equal to 1 × 10−4 and comparable to that of many chiral originate not from the chirality induction by the ligands in the
organic compounds (Figure 2b) but lower than for many entire GQD but only in a small segment of the graphene sheet.
chiroplasmonic assemblies.3,4,9,12,69 No circularly polarized For instance, the cysteine ligand can be attached to a benzene
luminescence was observed at room temperature for both ring or a double −CC− bond that does not have strong
types of GQDs despite multiple conditions tested. electron delocalization with the rest of the graphene sheet. It
The low-energy CD band at 250−265 nm is of particular can occur in a variety of chemical structures at the GQD edges
interest because it overlaps with the UV−vis absorption peak at that also sustained partial oxidation/reduction (Figure S12).
260 nm (Figure 2a,c), corresponding to the band gap of GQDs. Such a mechanism can be described as local chirality induction.
Its origin can be associated with a variety of molecular and We calculated UV−vis and CD spectra for HZ2 versions where
nanoscale geometries,14,70−72 which is difficult to establish L-cysteine is covalently bonded to localized benzene, ethane, or
using only experimental methods. Computational tools can be carbonyl groups (Figure S12). Indeed, for several of them, the
particularly helpful in this task, complementing the spectro- in silico spectra show similarities with the experimental ones,
scopic and microscopy data. Moreover, when carrying out such as the presence of the two UV−vis absorption peaks from
quantum mechanical and other computations of GQDs, one cysteine and from graphene segments; they also display a
will benefit from well-established interaction potentials for negative chiroptical band at 250 nm, attributed to MOs
atoms frequently encountered in organic chemistry and biology localized at the edges of GQDs. With the exception of the low
that make calculations of equilibrium geometry of GQDs easier intensity of CD and UV absorption peaks at 210 nm
than for semiconductor or metal NPs. corresponding to cysteine, the combination of two types of
Emergence of chiroptical activity at wavelengths longer than GQDs with cysteine ligands bonded to a localized benzene ring
those of cysteine ligands can be attributed, among other gives spectra that resemble experimental ones (Figure S14).
possibilities, to perturbation of electronic states of the graphene Diverse chemistry of the GQD edges makes it possible that
segment by the ligands and change of the conformational the localized chiral MOs obtained by hybridization of AOs of
structure of the chromophore itself. It is conceivable that the cysteine and graphene segments with partial breakage of sheet
molecular orbitals (MOs) of chiral edge ligands exert the aromaticity may indeed be responsible for chiroptical activity of
symmetry-breaking perturbation to electronic states of L/D-GQDs. If this is true, the relatively large GQDs with a
graphene. We evaluated the hypothesis of symmetry diameter of 10−15 nm produced commercially must also
perturbation (also known as chirality induction) by calculating display the same band. However, no CD or UV−vis bands
MOs of GQD’s using the semiempirical ZINDO algorithm for could be identified above 220 nm for a variety of L/D-GQDs
three sizes of L-GQDs with two, three, and four rings of carbon concentrations (Figure S15). Although localized MOs may be
atoms denoted as HZ2, HZ3, and HZ4 (Figures 3, 4, and S9− responsible for some effects, they are unlikely to play the
S11), respectively. These GQD sizes are realistic for quantum central role in the genesis of the 250−265 nm CD band as well
mechanical methods, while large sizes are possible for less as other chiroptical bands at longer wavelengths.
computationally intense techniques exemplified by MMFF. In Emergence of chiroptical properties of L/D-GQDs can also be
accord with the previously discussed experimental data in associated with the overall shape of this molecule. As the first
Figures S3−S8, L-cysteine ligands were added to the edges of level for in silico evaluation of molecular geometries of GQD,
the graphene sheets via amide bonds. Their number took into we used the same MMFF algorithm used for calculating the IR
account the data from XPS spectroscopy using a sulfur−carbon spectrum (Figure S5). MMFF assumes nearly elastic
atomic ratio (Table S1). deformation of covalent bonds and can be a powerful tool
HZ2 GQDs reveal distinct hybridization between atomic for understanding complex mechanics of large molecules. It is
orbitals (AOs) of the graphene sheet and the edge ligands, for also computationally much less demanding than density
example, for LUMO+2 and LUMO+4 (Figure S9). Similar functional theory (DFT) and semiempirical ZINDO algo-
electronic levels produced by combining AOs of graphene and rithms, and thus, many large atomic systems matching the
cysteine can be found for LUMO+3 in HZ3_A1 GQD, but the median size of our basic 2−7 nm GQD could be calculated with
extent of mixing is much lower; in other regioisomers the MMFF algorithm.
represented by HZ3_A3, one cannot find hybridized MOs As expected, the equilibrium geometries of small unmodified
even for high-level LUMOs and HOMOs (Figures S10 and GQDs display nearly perfect molecular flatness (Figure S16).
S11). HZ4-type GQDs do not show the presence of MO made Angstrom-scale “ripples” appear at the edges of the graphene
from AOs of cysteine and graphene up to LUMO+4 and sheets as the diameter of the sheets becomes bigger, indicating
HOMO−4. the tendency of these structures to relieve some strain from
Heuristic expectations for the hybridization of AOs from atomic repulsion by buckling deformations. When, L-cysteine
graphene sheets and covalently attached functional groups ligands were added around the circumference of GQD, an
based on general MO theory match these calculations: the increase of buckling deformation was observed. In fact, the
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equilibrium geometry of these GQDs displayed a strong twist; venue provided detailed understanding of the influence of
GQDs carrying L-cysteine and D-cysteine revealed right- and dihedral angle on chiroptical properties.
left-handed twists of the GQD, respectively (Figure 2c). The The primary question that we wanted to answer here is
enhancement of buckling is attributed to noncovalent whether the presence of dihedral angle θ may account for the
intermolecular interactions of the ligands with each other and emergence of the (low-energy) 250−265 nm band and resolve
other edge groups. Intermolecular chiral interactions at the the discrepancies discussed above. We largely ignored the high-
periphery of the large molecules are known to cause molecular energy states associated with cysteine and related molecular
deformation and translate the nanoscale chirality in liquid edge segments considered above because the origin of 210−220
crystals, polymers, proteins, and dendrons.66,73−75 nm CD bands in L/D-GQDs caused little doubts. We also need
Their mirror images of twisted graphene sheets are not to point out the limitation of computational methods we
superimposable and therefore should lead to the appearance of applied in this study. ZINDO is known to deviate the energy of
the characteristic chiroptical band(s). The closest symmetry electronic transitions.80 So, it is difficult to expect the perfect
point group describing their geometry would be D2 as they tend match in the spectral location of all the experimental and
to have two perpendicular C2 rotational axes. The twisted calculated CD bands, and we were not able to obtain it except
graphene sheets can also be characterized as axial chiral for those associated with hybridized MO at the edges of GQDs.
structures, and thus, L- and D-GQDs can also be denoted as P- However, the relative energies of transitions for different GQD
and M-GQDs or alternatively as Δ- and Λ-GQDs. The R/S sizes can be properly compared. The sign of the calculated CD
notation is not applicable here because of ambiguity of the bands and the Cotton effect are the parameters that we can use
Cahn−Ingold−Prelog ranking for “substituents” in the GQD as the first level of “yes/no” discrimination when comparing
case, unlike the case of tetrahedral NP assemblies76 or NPs with experimental results.
tetrahedral chiral centers in the apexes70 reported before. The DFT calculations confirmed that the twisted conformation of
L-GQDs (Figure 2c,d) represents the equilibrium geometry.
electronic transition responsible for the 250−265 nm band can
be attributed to promotion of the electron and hole to one of Spontaneous buckling results in complex shapes with multiple
the excitonic states of GQDs and can be referred to as chiral nonzero dihedral angles (Figure S1), and the chiroptical
excitonic transition. properties of the particle will be determined by their
An important point needs to be made that many of the superposition. To obtain a representative set of in silico data,
twisted molecular geometries are dynamic and can flex into the we considered the possibility of incomplete and/or inhomoge-
symmetrically opposite conformation if the energy barrier neous modification of the graphene edge, and multiple
regioisomers of GQDs with different placements of cysteine
between these conformational states is lower than kT, which is
moieties were considered and calculated (A1−3 and B1−3
the essential difference with large-scale supramolecular chiral
isomers, Figure 3b−d) in recognition of the polydispersity of
systems. This energy barrier between different twisted
the molecular structure of GQDs. Therefore, we calculated the
conformations is dependent, among other factors, on the
chiroptical activity of all of the variants and regioisomers of L-
bending modulus of the graphene sheet of the size of starting
GQDs. Some of them have positive and some of them have
GQDs. Since linear mechanics was found to be generally
negative Cotton effects (Figure S12a,b). The calculations of a
applicable to graphene sheets,77 one can conclude that the collective rotatory activity of the GQD dispersion representing
larger the sheets, the smaller their buckling modulus is.78,79 A many conformations must, therefore, take into account the
lower-energy threshold for transition of one buckled state to statistical probability of ligand placement and intensity of the
another leads to the more dynamic twisted state. Eventually, rotatory activity. Hence the cumulative CD spectra were
GQDs of large diameter must exist as a racemic mixture, calculated as a superposition of statistically weighted con-
combining both left- and right-handed twisted sheets in equal tributions from different isomers in Figure S16. The sign of the
amounts regardless of the chirality of the edge ligands because Cotton effects and the first low-energy “wave” in the cumulative
the threshold of racemization becomes smaller than kT. calculated CD spectra in Figure S18 matched the experimental
Therefore, for 10−15 nm L/D-GQDs in Figure S15, the CD ones in Figure 2. The blue shift of the UV−vis and CD
band at 250−265 nm disappears. Instead, the CD bands in the signatures with reduction of the GQD size (Figure 3e)
180−230 nm window characteristic of cysteine showed distinct coincided with the expectations about the excitonic state of
broadening that should be attributed to chiral induction based GQDs being responsible for their rotatory activity. One could
on mixing MOs from edge ligands and graphene discussed also see that in the computational spectra for HZ2-type GQDs,
above. the complexity of CD bands increases due to increasing
MMFF can be versatile, fast, and accessible to many contribution of hybridized MOs. The overall intensity of the
researchers, but it cannot be applied to calculation of optical chiroptical bands decreases in the order HZ4 > HZ3 > HZ2
properties. Taking into account the twist of GQDs, we due to reduced optical cross section of the GQD molecules.
calculated the CD and UV−vis absorption spectra using DFT In the second venue of calculations, we constructed five
+ZINDO algorithms, although these computational tools atomistic models of HZ4 with preset dihedral angles θ = 0, +5,
restricted us to GQDs of smaller sizes presented in Figure 3 +10, +15 and +20° while allowing the GQDs under this
rather than those in Figure 2c,d. boundary condition to optimize its atomic structure. CD
In the first venue of these calculations, we optimized GQD spectra were calculated using the ZINDO algorithm and
conformation for a variety of GQD sizes and regioisomers. The IEFPCM. We found that UV−vis spectra of GQDs were not
computational results obtained in the first venue tested the affected by different values of θ, but the CD spectra were
possibility of spontaneous twisting of GQDs. In the second affected to a large extent (Figure 5). The rotatory activity
venue, we preset the dihedral angle of the base graphene sheets consistently increases, and the CD spectrum becomes ever
of GQD, θ, to have certain values and then calculated CD more complex as θ increases. A parallel can be made with the
spectra of twisted GQDs. The data obtained in the second significance of the twist angle in other chiral molecular and
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nanoscale chemical structures exemplified by a family of axial

compounds based on 1,1′-binaphthyl81 or DNA-bridged
plasmonic assemblies from gold nanorods/nanoparticles.82,83
Similarly to proteins and some drugs, chiral nanostructures
may exhibit different biological activity depending on their
handedness.84 Unlike CdTe and other heavy-metal-based NPs,
the cytotoxicity originating from NP components will not
interfere in this case with the biological activity of the
stereoisomer. So, we tested how L/D-GQDs affect the viability
of human liver hepatocellular carcinoma cells HepG2 that
represent a common model for cytotoxicity studies. Extensive
investigation for different times of exposure, concentrations,
and cell numbers confirmed that biocompatibility of GQDs is
high (Figures S19−S21). The negative effect on cells compared
to the control group is insignificant after 1 h of exposure
Figure 5. CD spectra of HZ4 L-GQDs calculated using the second
(Figure S19). It becomes discernible only after 24 h of
DFT venue (see Methods). All the spectra were calculated as angle- incubation in the presence of GQDs (Figure S20) and results in
averaged functions to reflect random orientation of L-GQDs with a decrease of HepG2 cell viability by ca. 20% for all
respect to the incident light beam in dispersions. In all spectra, concentrations of GQDs used: 0.015, 0.03, and 0.045 mg/
mostly the electronic states corresponding to the graphene sheet, mL. The difference in toxicity between L- and D-GQDs is small.
i.e., the first 20 states, were calculated. For the two highest concentrations of GQDs (i.e., 0.045 or

Figure 6. (a,c) Snapshots from the atomistic MD simulations of a variant of HZ3_A3 D-GQD (a) and HZ4_A1 D-GQD (c) in the vicinity of
the cellular membranes. The chemical groups on the membrane surface are extended toward the GQD. (b,d) Time dependences of the
distances from GQD to the center of the bilayer (projection on the axis normal to the bilayer plane) for HZ3_A3 D-GQD (b) and HZ4_A1
(d). Background colors show the regions of the water (white) and the membrane (light purple). (e−h) Snapshots of GQD/membrane
interactions near the completion point of the simulations. Blue and purple spheres represent the position of phosphoric and nitrogen atoms of
POPC, respectively, while the gray thin lines show the rest of the lipids’ structure.

1750 DOI: 10.1021/acsnano.5b06369

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ACS Nano Article

Figure 7. Time dependence of the (a) mean and (b) minimum distances between any of the chiral carbons of the HZ4_A1 GQD and any of
the chiral carbons of the POPCs.

0.030 mg/mL of GQDs), it becomes statistically significant and During all the MD runs, all GQDs remained less than 1 nm
can be observed for 25 000−100 000−25 000 cells per well away from the membrane surface with the cysteine ligands bent
(Figures S19 and S20). Notably, L-GQDs show slightly higher toward the membrane. This observation is indicative of strong
overall biocompatibility than D-GQDs (Figures S19 and 20). intermolecular interactions between GQD and membrane
The similar relationship between L- and D-enantiomers can be lipids via hydrogen bonding and van der Waals forces. When
seen for free cysteine in our experiments (Figure S21) as well as near the surface, the single D-GQD both for HZ3_A3 and
other studies on chirality effects for the toxicity of drugs and HZ4_A1 cases markedly disturbed the structure of the bilayer,
drug candidates.85−87 It is perhaps instructive to point out that as some lipids extended in the water toward the GQD (Figure
no free amino acid was present in the dialysis-purified 6a,c). After about 15 ns, the D-GQD with HZ3_A3 structure
dispersions of L- and D-GQDs used in experimental series entered the head group region of the membrane, while the
described in Figures S19−S21. surface structure was slowly restored (Figure 6b). Incorporation
There can be multiple biological pathways for how the into the membrane was also observed for D-GQD with
chirality of GQDs and cysteine can affect cellular functions. To HZ4_A1 structure, although this process was about 3 times
our surprise, the mechanism of toxicity differentiation between slower due to the higher molecular mass of the larger GQD
enantiomers is poorly understood even for free L/D-cysteine. In (Figure 6d). In all cases, the graphene (aromatic) part of the
the case of L- and D-GQDs, the difference in biological effects GQDs lay perpendicularly to the bilayer plane, with the two
can be even more complicated than for free amino acids amino acids surrounded by the phosphate and choline groups
because it can be related to both atomic chirality of the edge of the POPC lipid head groups in the membrane.
ligands and the nanoscale chirality of the twisted graphene After different orientational states were explored in half-
sheets. Previous studies of adhesion of neuronal88 and inserted position, D-GQD slowly moved in the glycerol region
mesenchymal bone marrow89 cells on L/D-cysteine-modified of the lipids (Figure 6a−d) and eventually penetrated deep into
surfaces indicate that interactions with cellular membranes are the lipid bilayer (Figure S22). Conversely, L-GQDs never
likely to play the central role in differentiation between the two entered the bilayer. Such lack of affinity between the L-GQDs
isomers. The intermolecular forces causing the difference in and the external part of the cellular membrane was observed for
association with biological membranes and, thus, in toxicity both HZ3 and HZ4 GQDs (Figure 6b,d). To rule out simple
between L- and D-GQDs are hydrogen bonds by van der Waals coincidence, we repeated this simulation five times, placing the
interactions and interactions with hydrophobic forces. It is GQD at different distances and angles with respect to the
essential to note that at the scale of a few nanometers these membrane. In all cases, the L-GQD stayed parallel to the bilayer
interactions become nonadditive,90 and thus, the traditional at less than 1 nm from the surface (Figure 6b,d). We can
distinction between the forces becomes ambiguous. Atomistic speculate that the difference in association with the membranes
molecular dynamics (MD) studies become one of the best ways between L- and D-GQDs is determined in the case of small
to elucidate them, although limitations of the current atomistic HZ3-type and HZ4-type quantum dots by the edge groups
potentials should also be taken into account. because the nanoscale twist for small graphene sheets is not yet
Interactions of the GQD stereoisomer with cellular pronounced as it is for larger GQDs (see above). The
membranes were evaluated using advanced MD simulations contribution of the nanoscale twist is, at the moment, uncertain
where both GQD and a bilayer lipid membrane composed of 1- and will require further MD study evaluating the behavior of
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the system at much longer times, which presents multiple
cholesterol was described at an atomistic level using the fundamental and hardware limitations.
CHARMM force field91 with particle mesh Ewald method92 to Considering stronger toxicity differentiation between the
evaluate long-range forces. The simulations were carried out in stereoisomer at higher concentrations of L- and D-GQDs
six different systems: two systems with a single D/L-GQD of (Supporting Information, Figures S13 and S14), we analyzed
HZ3_A3-type (Figure 6a,b,e,f), two systems with a single D/L- the behavior of a pair of stacked GQDs having the same
GQD of HZ4_A1-type (Figure 6c,d), and two with a stack of chirality. The GQDs were prepared in a “closed stack”
identical GQDs of HZ3_A3-type and to reflect the possibility configuration that is likely to be formed in dispersions as
of agglomeration (Figure 6g,h). The evidence of possible their concentration increases.93 L-GQD and D-GQD stacks
agglomeration can be seen in the large Stokes shift between the showed that they remained intact for the duration of the whole
first excitonic transition of GQDs at 260 nm and the maximum simulation. Overall, the behavior of agglomerates is similar to
of photoluminescence at ca. 530 nm (Figure 2). that of single GQDs. D-GQD stacks quickly entered in the
1751 DOI: 10.1021/acsnano.5b06369
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bilayer and remained halfway inserted, with the aromatic plane g/mol) was purchased from Thermo Scientific. The GQDs were
laying normal to the membrane for the rest of the simulation synthesized by a modified protocol from a previous report.49 Through
(Figure 6g). On the other hand, L-GQD stacks stayed near the a top-down process, 0.5 g of carbon fibers was dispersed into a 40 mL
surface but remained parallel to it, never leaving the water mixture of sulfuric acid and nitric acid (3:1 v/v). The black solution
was sonicated for 2 h and mechanically stirred for one additional day at
region above the bilayer (Figure 6h). Considering the 80 °C. After the reaction, the mixture solution was cooled and diluted
intermolecular forces that could lead to such a difference of with DI water (0.15 mg/mL). To adjust to pH 8, sodium hydroxide
behavior between aggregates of L- and D-GQDs, it is essential to was added into the solution. For purification of GQDs, the mixture
point out that the carbon dots explored the full range of angles was dialyzed for 3 days. Large GQDs with diameters exceeding 15 nm
even when they were 3 nm away from the bilayer center were obtained from Sigma-Aldrich.
(Figure S22). The mean and minimal distances between the Synthesis of Chiral GQD. Synthesis of chiral GQDs was carried
chiral centers of GQDs and those of POPC in the membrane out at room temperature using L/D-cysteine. In order to impart
model indicate that L-GQDs tend to stay closer to the contact chirality to the GQDs, the carboxylic group of GQDs was connected
with the chiral center of the lipid than do D-GQDs (Figure 7). with the amine group of L-(or D-)cysteine by the EDC/NHS method.
A solution of EDC (30 μL, 6 mM) was added into 0.5 mL of GQD
This may suggest that intermolecular interactions between L-
solution. After 10 min stirring, the same amount of NHS (6 mM) was
GQDs and the lipid layer are favorable to the parallel added to the solution and it was mixed for 30 min. Finally, 30 μL of L-
orientation of the graphene sheet and the membrane. Such a (or D-)cysteine (6 mM) was added into the graphene GQD-NHS, and
configuration makes entering the bilayer more difficult for L- the mixture was stirred for 30 min. The surplus L/D-form of cysteine
GQDs than for D-GQDs. was removed by a dialysis membrane (Viskase, Membra-cell MD 34).
Surface-Enhanced Raman Scattering Measurements. Crystal
CONCLUSIONS violet (Fisher) was used without further purification; its 1 × 10−6 M
solution in water was mixed with isovolumetric L /D-GQDs.
In this study, we described the asymmetric synthesis of chiral Approximately, 20 μL of this solution was deposited on clean silicon
GQDs by covalent edge modification with L- and D-cysteine, wafers and dried. Raman spectra were recorded using a Witec
leading to the nanoscale twist of the flexible graphene sheets. microscope with a 100× Zeiss objective. A 532 nm continuous-wave
Quantum mechanical chiral induction occurs at the edges of the laser light was used for excitation of the Raman scattering with a
GQDs due to hybridization of MOs of the the edge-ligands and spectral collection time of 1 s for all of the measurements.
those of peripheral carbon atoms. Resulting L- and D-GQDs Instrumentation. The chiroptical activity of the dispersions was
represent an example of symmetry-related carbon nanostruc- measured by CD spectroscopy (JASCO, J-815), and the chemical
tures with nearly mirror image optical activity. L-GQDs and D- reaction progress was monitored by FT-IR spectroscopy (Nicolet
6700) and Raman spectroscopy (Witec, Alpha 300 D, USA). The
GQDs reveal a 210−220 nm CD peak corresponding to the absorbance of chiral GQD was analyzed by UV/vis spectroscopy
amino acid broadened by hybridization with edge groups of the (Agilent, 89090A). The fluorescence property of chiral GQDs was
graphene sheets. GQDs also display a new chiroptical band at characterized by PL spectroscopy (Horiba, Fluoromax-3). The
250−265 nm that is associated with the twisted excitonic state morphology of chiral GQDs was observed by HR-TEM (JEOL,
of the graphene sheet. The twist in the graphene sheet JEM-3011). Their surface potential was analyzed by a Zetasizer
originates from collective interactions of amino acid moieties (Malvern Instruments, Nano ZS).
bound to the graphene base via amide bonds. While multiple DFT Quantum Mechanical Studies of the GQD Conformers.
regioisomers and conformations are possible for cysteine- Conformation search was carried out using the CONFLEX method
modified GQDs, atomistic simulations using multiple methods (searching limit = 3.0 kcal/mol) with MMFF94S as a force field. After
the search, the most stable conformation of each structure was chosen
indicate that L-GQDs preferentially display right helicity in their
for each configuration (first venue) or each structure with preset θ = 0,
twisted state whereas D-GQDs have predominantly left-handed +5, +10, +15, and +20° (second venue). DFT calculation at the
conformation. Therefore, L- and D-GQDs can also be identified B3LYP/6-31G(d) level with IEFPCM (solvent = water) level was
as P- and M-GQDs, respectively, following the notations for carried out followed by ZINDO calculation to obtain the CD
helical structures with clockwise and counterclockwise spectrum. Generalized Born/solvent-accessible surface (GB/SA)
rotations. Strong luminescence and SERS activity point to the model (solvent = water) was used for GQDs with L-cysteine (first
possibility of circular polarization effects in both photo- venue). For GQDs with preset θ (second venue), the twisted forms
luminescence94 and Raman scattering95 that need to be were maintained during the CONFLEX search as well as DFT
investigated further. calculation (see Supporting Information for further description).
In vitro evaluation of GQD biocompatibility with liver cells These calculations can be carried out only for L-GQD because
geometry optimization by CONFLEX used a deterministic rather than
demonstrated their low cytotoxicity and differentiation of a stochastic algorithm; it has to yield mirror inversion of equilibrium
cytotoxicity between GQD stereoisomers. Atomistic simula- geometry.
tions of L- and D-GQDs interacting with cellular membranes Cell Cultures. Hepatocellular carcinoma human cells (HepG2)
demonstrated stronger binding of D-GQDs to the lipid bilayer (ATCC, VA) were maintained with Eagle’s minimum essential
of the cells. medium (EMEM) supplemented with 10% fetal bovine serum
Biocompatibility of chiral GQDs opens new routes for (FBS) and 1% penicillin−streptomycin (ATCC) in a humidified
development of drug delivery vehicles and more selective incubator (MCO-15AC, Sanyo) at 37 °C in which the CO2 level was
phototherapies, while the twisted electronic states can lead to maintained at 5% before seeding. All of the medium was filtered using
polarization-based optoelectronic devices and (photo)catalysts. 0.22 μ SteriCup filter assembly (Millipore, USA) and stored at 4 °C
for no longer than 2 weeks. For cells incubated with the L-form, D-
form, and control GQDs, the cells were cultured overnight to allow
METHODS attachment in a 96-well plate, washed with FBS-free EMEM, and then
Materials and GQD Synthesis. The carbon fiber was obtained incubated with L-GQDs, D-GQDs, and control GQDs with a
from Fiber Glast Development Co. (Brookville, OH). L/D-Cysteine, concentration of 0.015 mg/mL at 37 °C for 1 h in FBS-free medium.
sulfuric acid, nitric acid, and N-ethyl-N′-(dimethylaminopropyl)- After incubation with GQD, the cells were washed repeatedly with
carbodiimide (EDC, 191.7 g/mol) were purchased from Sigma- sterilized PBS and maintained in culture medium before further
Aldrich and Thermo Scientific. N-Hydroxysuccinimide (NHS, 115.09 analysis.

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Cell Viability Assays. PrestoBlue cell viability reagent (Life University of Michigan’s EMAL for its assistance with electron
Technologies) was used to measure cell viability. In brief, HepG2 cells microscopy, and for the NSF Grant No. DMR-9871177 for
were seeded into a 96-well flat culture plate (Corning). After being funding of the JEOL 2010F analytical electron microscope used
cultured overnight, the cells were washed with FBS-free EMEM and
incubated with a specific concentration of L-GQD and D-GQD in FBS-
in this work.
free medium at 37 °C for 1 h. The cells were then washed three times
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