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Physical Chemistry Chemical Physics View Article Online
Size-dependent penetration of carbon dots inside the ferritin
Published on 17 April 2015. Downloaded by University of Birmingham on 21/04/2015 16:41:56.
nanocages: evidence for quantum confinement effect in carbon dots†
Arpan Bhattacharya, Surajit Chatterjee, Roopali Prajapati, and Tushar Kanti Mukherjee∗
Discipline of Chemistry, Indian Institute of Technology Indore, M-Block, IET-DAVV Campus, Khandwa Road, Indore-452017, M.P., India
† ∗
Electronic supplementary information (ESI) available. Corresponding author. E-mail: [email protected]; Tel: +91-7312438738. 1
Physical Chemistry Chemical Physics Accepted Manuscript
DOI: 10.1039/C5CP00543D
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1
Abstract
2
The origin of the excitation wavelength (λex)-dependent photoluminescence (PL) of carbon dots (CDs) is poorly understood and
3
still remains obscured. This phenomenon is often explained on the basis of surface trap/defect states, while the effect of quantum
4
confinement is highly neglected in the literature. Here, we have shown that the λex-dependent PL of CDs is mainly due to the
5
inhomogeneous size distribution. We have demonstrated the λex -dependent PL quenching of CDs inside the ferritin nanocages
6
through selective optical excitation of differently sized CDs. It has been observed that Fe3+ ions of ferritin effectively quench the
7
PL of CDs due to static electron transfer, which is driven by favorable electrostatic interactions. However, control experiment
8
with aqueous Fe3+ ions in bulk medium revealed λex-independent PL quenching of CDs. The λex-dependent PL quenching of CDs
9
by Fe3+ ions of ferritin has been rationalized on the basis of different extent of accessibility of Fe3+ ions by differently sized CDs
10
through the funnel-shaped ferritin channels. PL microscopy of individual CD has been performed to get further information about
11
their inherent PL properties at single dot resolution. Our results have shown that these hydrophilic CDs can be use as potential
12
iron sensors in biological macromolecules.
13 14 15 16 17 18 19
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1. Introduction
21
In recent years, carbon-based fluorescent nanomaterials have gained vast attention due to their superior physicochemical
22
properties compared to the semiconductor based nanomaterials.1–5 Among these nanomaterials, CDs have emerged as highly
23
promising candidates for biomedical imaging application due to their low cytotoxicity and smaller size with comparable PL
24
characteristics as inorganic core-shell QDs.6,7
25
CDs show optoelectronic properties like excitation wavelength (λex) dependent PL,8,9 high PL quantum yield,10 low
26
photobleaching,11 and no PL blinking (fluorescence on–off).12 However, the origins of their intense PL are a matter of debate and
27
not yet entirely understood.8 It is believed that the PL occurs from the radiative recombination of excitons at the surface trap
28
states. Earlier, It has been reported that CDs containing carboxylic (-COOH) and hydroxyl (-OH) groups are highly luminescent
29
as surface oxidation introduce surface defects to CDs which are responsible for their PL.13,14 Another unresolved unifying feature
30
of the PL of CDs is the λex dependence of the emission wavelength and intensity. Whether this arises because of differently sized
31
CDs and/or different emissive surface traps on each CD surface are still unresolved. Most of the earlier literatures successfully
32
demonstrated the role of surface trap states on the origin and λex dependence PL of CDs.12,15,16 It has been speculated that the PL
33
of CDs could be due to the distribution of emissive defect sites on the CD surface. However, the role of quantum confinement
34
effect due to inhomogeneous size distribution of CDs on the λex dependence of PL is poorly understood and neglected in the
35
literature except few reports on size-dependent PL of size separated CDs.11,17–20 Earlier, Zhao et al. have synthesized CDs via
36
electrochemical oxidation of graphite and shown that size separated CDs exhibit size-dependent but λex-independent PL.11
37
Similarly, Dong et al. have observed that both excitation and PL spectra of size separated CDs red-shift with increase in their
38
size.17 Recently, by using single particle PL microscopy, Ghosh et al. have shown that the λex-dependent ensemble PL of CDs
39
arises due to excitation of different subsets of CDs in heterogeneous sample.18 Here, it is important to stress that, our aim in the
40
present study is to address the origin behind the λex dependence PL of CDs without their size separation, as size separation will
41
lead to λex-independent PL. In the present study, our approach is based on a unique strategy that, in the presence of a
42
heterogeneous system, differently sized CDs are expected to behave differently, and hence, by selective optical excitation of
43
differently sized CDs, we can monitor the PL characteristics of various sized CDs present in our system. We have shown that the
44
λex dependence PL of CDs arises due to the inhomogeneous size distribution rather than distribution of different surface trap
45
states in each CD by selective optical excitation of differently sized CDs in the wavelength range 320-450 nm. Earlier, using the
46
same approach we have demonstrated the size-dependent partitioning of Si-QDs inside the micellar aggregates.21 However, in the
47
present study we have chosen a protein nanocage namely, ferritin to probe the dynamics of differently sized CDs.
48
Ferritin is a spherical cage-like major intracellular iron storage protein in eukaryotes with a molecular weight of 450
49
000 Da (Scheme 1).22 Iron is stored in a soluble non-toxic ferrihydrite form inside the ferritin nanocage which can accommodate
50
upto ~ 4500 Fe3+ ions.23 Interstingly, iron enters and leaves the ferritin cavity as Fe2+ ions.22 Hence, migration of Fe2+ ions to the
3
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ferroxidase center inside the ferritin cavity occurs before its oxidation. On the other hand, reduction of iron from Fe3+ to Fe2+
52
within the ferritin cavity is essential to its release. Apoferritin is composed of 24 subunits, which self-assemble into a hollow
53
cage-like nanosphere with external and internal diameters of ~12-13 nm and ~7-8 nm, respectively.24 The outer surface is
54
connected with the inner cavity through funnel-shaped channels on the protein surface. It contains eight 3-fold hydrophilic
55
channels which are involve in Fe2+ transportation and six 4-fold hydrophobic channels which facilitate the transfer of protons
56
across the protein shell.25,26 The main objective of the present study is to shed light on the origin of λex dependence of the
57
emission wavelength and intensity. To the best of our knowledge, this is the first direct report (without size separation) to show
58
that the λex-dependent PL of CDs is due to the quantum confinement effect of the surface trap states.
59 60
2. Experimental Section
61
2.1. Materials. Ethylenediamine (EDA, ≥ 99.5%), ferritin from equine spleen (Type I, Saline solution), apoferritin from
62
equine spleen and Pur-A-Lyzer™ Dialysis Kit (MWCO 3.5 kDa) were purchased from Sigma-Aldrich. Citric acid monohydrate
63
(99.5%) and Fe (III) nitrate nonahydrate were purchased from Merck (Germany). Milli-Q water was obtained from a Millipore
64
water purifier system (Milli-Q integral).
65
2.2. Synthesis of CDs. CDs were synthesized according to the previously reported method (Scheme 1).27 In brief, citric acid
66
(1.015 g) and ethylenediamine (335 µL) were dissolved in Milli-Q water (10 mL) and sonicated for 5 min. Then the solution was
67
transferred to a Teflon-coated stainless steel autoclave (25 mL) and heated at 200˚C for 5 h. Subsequently the reactor was cooled
68
to room temperature and the solution was dialyzed (MWCO 3.5 kDa) for 48 hrs. The water for dialysis was changed after every 6
69
h. Finally the black brown transparent CD solution was obtained.
70
2.3. Sample Preparation. All solutions were prepared in pH 7.4 phosphate buffer (25 mM). Ferritin solution was prepared
71
by dilution from 125 mg/mL ferritin (MW 450 000 Da) stock solution. For microscopy experiment samples were spin coated
72
with a spin-coater (Apex Instruments Spin NXG-P1) on a clean glass cover slide at 2000 rpm for 3 min. The cover slides were
73
first cleaned with 2% Hellmanex III (Sigma-Aldrich) followed by piranha solution (3:1 concentrated sulfuric acid and 30%
74
hydrogen peroxide) and then with chromic acid. Each of these cleaning steps was followed by repeated washing with Milli-Q
75
water. Finally, these washed slides were rinsed with methanol (Sigma-Aldrich) and dried in vacuum oven at 100˚C.
76
2.3. Instrumentation. Absorption spectra were recorded in a quartz cuvette (10 × 10 mm) using a Varian UV−Vis
77
spectrophotometer (Carry 100 Bio). The PL spectra were recorded in a quartz cuvette (10 × 10 mm) using Fluoromax-4
78
Spectrofluorimeter (HORIBA Jobin Yvon, model FM-100) with excitation and emission slit width at 2 nm. The PL spectra of
79
CDs in the presence of ferritin and aqueous Fe3+ ions were corrected for the absorbance,28 as both of them strongly absorbs light
80
in the wavelength range 200-450 nm. The PL spectrum of CDs was deconvoluted using Origin software. The binding constants
81
(Kb) and the number of binding sites (n) were determined using the Scatchard equation, which can be expressed as follows:
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82 83
Where Kb is the binding constant of the CDs-ferritin complex and n is the number of binding sites. The plot of log[(F0 - F)/F]
84
against log[Q] should yield a straight line with an intercept equal to log Kb and a slope equal to n. Circular Dichroism (CD)
85
spectra were measured on a JASCO J-815 CD spectropolarimeter using a quartz cell of 1 mm path length. Scans were made with
86
a slit width of 1 mm and speed of 20 nm/min. Dynamic light scattering (DLS) experiments were performed on a Brookhaven
87
particle size analyzer (model 90 Plus). All the samples for DLS measurements were filtered through 0.22 µm syringe filter
88
(Whatman). PL imaging of individual CD samples were performed on a home-built epi-fluorescence microscope. An air-cooled
89
argon ion laser (Melles Griot, model 400-A03) with excitation wavelength at 457 nm was used to excite the CDs sample placed
90
on an inverted microscope (Nikon, model Eclipse Ti-U). The laser beam was expanded and subsequently focused on the back-
91
focal plane of an oil immersion objective (100× 1.49 NA Nikon) to illuminate 60 × 60 µm2 area of the sample. The PL from the
92
sample was collected through a B2A filter cube (Nikon) with a 505 nm dichroic mirror and a 520 nm long-pass filter and finally
93
imaged with a back-illuminated EMCCD camera (Andor, model iXon X3 897). The exposure time was 200 ms. The images were
94
analyzed with ImageJ (Version 1.46r) NIH. AFM images were recorded on a cleaned glass coverslip using an AIST-NT
95
microscope (model SmartSPM-1000). Powder X-ray diffraction spectra (XRD) were recorded on a Rigaku SmartLab, Automated
96
Multipurpose X-ray Diffractometer with Cu Kα source (wavelength of X- rays was 0.154 nm). FTIR spectra were recorded in a
97
KBr pellet using Bruker spectrometer (Tensor-27). PL decays were recorded on a HORIBA Jobin Yvon picosecond time
98
correlated single photon counting (TCSPC) spectrometer (model Fluorocube-01-NL). The samples were excited at 376 nm by a
99
picoseconds diode laser (model Pico Brite-375L). The decays were collected with the emission polarizer at a magic angle of
100
54.7° by a photomultiplier tube (TBX-07C). The instrument response function (IRF, fwhm ~140 ps) was recorded using a dilute
101
scattering solution. The fluorescence decays were analyzed using IBH DAS 6.0 software by the iterative reconvolution method,
102
and the goodness of the fit was judged by reduced χ-square (χ2) value. The decays were fitted with three-exponential function.
Ft = exp 1⁄!
103 104
Where F (t) denotes normalized PL decay and a1, a2 and a3 were the normalized amplitude of decay component τ1, τ2 and τ3 respectively. The average lifetime was obtained from the equation:
105
< ! >= !
106
5
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3. Results and Discussion
108
3.1. Characterization of CDs. These CDs show PL quantum yield of ~ 60.2% and content 51.13% carbon, 5.81% hydrogen,
109
26.80% oxygen, and 16.25% nitrogen.27 The structure and morphology of synthesized CDs are confirmed from FTIR
110
spectroscopy, powder XRD and AFM. Fig. 1A shows the FTIR spectrum of CDs. The broad peak at 3420 cm-1 is assigned to the
111
stretching vibration of O-H and N-H moieties. The peak at 2925 cm-1 is due to C-H stretching vibration. Two prominent peaks at
112
1690 and 1566 cm-1 arise due to stretching and bending vibration of C=O and N-H moieties respectively. Other noticeable peak
113
at 1394 cm-1 is assigned to the bending vibrations of C-H moieties. The estimated zeta potential of these synthesized CDs is -
114
25.45 mV at pH 7.4.
115
Fig. 1B shows the XRD patterns of the synthesized CDs. A broad peak centered at 250 (0.34 nm) has been observed,
116
which exactly matches with the earlier reported value,27 and signify poorly crystalline nature of these CDs. Fig. 1C displays a
117
typical AFM image of CDs. The CDs are spherical in nature. The size distribution histogram is generated from AFM height
118
profiles of 276 CDs. Fig. 1D shows the size distribution histogram of CDs. Two clear peaks; one centered at 1.73 ± 0.08 nm and
119
the other at 4.86 ± 0.08 nm have been observed. The diameter of the first peak varies from 0.50 to 3.50 nm while the diameter of
120
the second peak varies from 3.60 to 6.60 nm. These results clearly indicate that the synthesized CDs have inhomogeneous and
121
broad size distribution.
122
The absorption spectrum of CDs exhibits a weak shoulder at 250 nm and a prominent peak at 342 nm (Fig. 2A), which
123
is similar to that reported earlier. The shoulder at 250 nm is assigned due to the π–π* transition of C=C bonds from the CDs core,
124
while the prominent peak at 342 nm originates due to n–π* transition of C=O bonds from the surface of CDs.27,29 Excitation at
125
and above 320 nm results in strong PL in the visible region. Moreover, the PL maxima strongly depend on the excitation
126
wavelength (λex). Fig. 2A shows the effect of λex on the PL spectra of CDs. With gradual increase in λex from 320 to 450 nm, the
127
PL maxima progressively red shift from 433 to 512 nm (Fig. 2A). Interestingly, excitation below 300 nm results in a broad PL
128
spectrum. Fig. 2A and B shows the PL spectrum of CDs at λex = 280 nm. Deconvolution of this broad spectrum (fwhm ~144 nm)
129
with multiple Gaussian functions reveals very good fit with four Gaussian peaks in the range 417-524 nm (Fig. 2B). On the basis
130
of these results and earlier reports it is clear that excitation below 300 nm results in excitation of the carbogenic core of CDs,
131
while excitation at and above 320 nm results in excitation and subsequent PL from surface trap states which progressively red-
132
shift with increase in λex. Moreover, excitation of the carbogenic core results in PL from different surface trap states. The origin
133
of these different surface trap states have been discussed later. Here it is important to mention that the importance of surface
134
donor/acceptor states have also been realized earlier in other nanostructure materials including GaN/AlGaN core/shell
135
nanowires.30,31 Fig. 2C exhibits the variation of PL intensities of CDs with λex from 300–510 nm. Interestingly, the histogram
136
reveals a clear peak at 336 nm with a broad and weak shoulder centered at 420 nm. Here, it is important to note the similarity
137
between size distribution histogram estimated from AFM height profile (Fig. 1D) with the PL intensity histogram against λex.
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This is expected as PL intensity is directly proportional to the number of particles of a given size present in the solution.
139
Moreover, this similarity strongly indicates that the bimodal size distribution obtained from AFM measurement is not an artifact.
140
Fig. 2D shows the variation of band gap energy with λex in the range of 320–450 nm. These results clearly establish the idea that
141
by proper selection of λex, it is possible to excite differently sized CDs present in our system. For the present study, we have
142
selected three well-separated λex namely, 320, 350, and 450 nm to excite differently sized CDs present in the system.
143
3.2. PL quenching of CDs in the presence of ferritin. Excitation at 350 nm results in blue PL centered at 437 nm. The PL of
144
CDs progressively quenches upon gradual addition of ferritin (Fig. 3A). The PL spectrum of CDs in presence of 2.02 µM ferritin
145
quenches by 2.7 times with a red-shift of 10 nm. In contrast, apoferritin in the same concentration range does not quench the PL
146
of CDs (Fig. 3B). Fig. 3C shows the steady-state Stern-Volmer (SV) plots of CDs in presence of ferritin and apoferritin. The SV
147
constant in presence of ferritin has been estimated from the slope of the fitted straight line, which is 9.13 × 105 M-1 at 298 K. The
148
only difference between ferritin and apoferritin is that the latter lacks Fe3+ ions in its core. Hence, it is clear that Fe3+ ions of
149
ferritin are involved in the observed PL quenching of CDs. To gain further insight into the mechanistic aspect of this PL
150
quenching process, we measured the PL decay traces of CDs in absence and presence of different concentrations of ferritin. Fig.
151
S1 of the Supporting Information shows the PL decay traces of CDs in absence and presence of 2.02 µM ferritin. The decay trace
152
of CDs in absence of ferritin exhibits three exponential decay kinetics with lifetime components of 3.90 ns (31%), 10.62 ns
153
(29%) and 0.93 ns (40%). It is evident from Table S1 of the Supporting Information that the lifetime components of CDs remain
154
unaltered in presence of 2.02 µM ferritin. Fig. 3D shows the time-resolved SV plot of CDs with average lifetimes. The plot is
155
parallel to the X-axis with a slope of zero. These results clearly signify the formation of static ground state complex between CDs
156
and ferritin.
157
Earlier, Zhu et al. have reported similar kind of PL quenching of CDs by aqueous Fe3+ ions and assigned it due to
158
nonradiative electron transfer from CDs to the vacant d-orbital of Fe3+ ions.27 It has also been proposed that the specificity of Fe3+
159
ions to quench the PL of CDs arises due to special coordination interaction between Fe3+ ions and the phenolic hydroxyl groups
160
on the CDs surface. Moreover, it has been shown that the quenching process proceeds through dynamic mechanism rather than
161
static mechanism. In contrast, the static quenching process observed in the present study with ferritin indicates altered
162
interactions of CDs with Fe3+ ions of ferritin.
163
3.3. Effect of temperature and estimation of thermodynamic parameters. To estimate the binding constant (Kb) and various
164
thermodynamic parameters, we have performed temperature variation study. Fig. 3D shows the effect of temperature in the range
165
298–318K on the steady-state SV plot of CDs. As expected for static ground state quenching process, the SV constant decreases
166
with increase in temperature. The binding constant was determined using the Scatchard equation. Fig. 4A shows the Scatchard
167
plot at three different temperatures. All the plots are linear and the estimated binding constant (λex = 350 nm) for CDs-ferritin
7
Physical Chemistry Chemical Physics Accepted Manuscript
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168
complex is 7.35 (± 0.02) × 105 M-1 at 298 K. The binding constant gradually decreases with increase in temperature from 298–
169
318 K, indicating the static nature of the complex.
170
To gain further insights into the thermodynamics of their association reactions, we used van’t Hoff equation to extract
171
various thermodynamic parameters such as enthalpy change (∆H), entropy change (∆S), and Gibbs free energy change (∆G). Fig.
172
4B shows the linear plot of lnKb against 1/T. The estimated thermodynamic parameters for the CDs-ferritin complex at λex = 350
173
nm are listed in Table 1. The ground state association reaction is found to have a favorable negative enthalpy change (∆H < 0)
174
with a positive entropy change (∆S > 0), giving rise to an overall negative free energy change. In general, processes triggered by
175
hydrophobic interactions between proteins and ligands proceed with a large positive entropy change with a positive enthalpy
176
change while processes triggered by electrostatic interactions proceed with a positive entropy change with a small negative
177
enthalpy change.32,33 Hence, on the basis of our present results we propose that electrostatic interactions between positively
178
charged ferritin cores with the negatively charged CDs surface trigger their association process.
179
Further evidence of their ground state association comes from DLS measurement (Fig. 5). The hydrodynamic diameter
180
of ferritin is estimated to be 14.65 ± 0.10 nm (Fig. 5), which is close to the earlier reported diameter of 12 nm from TEM
181
measurement.34 However, the mean diameter of ferritin in the presence of 0.01 mg/ml CDs increases to 17.37 ± 0.05 nm (Fig. 5).
182
This increase in mean diameter clearly justifies the proposed mechanism of static ground state association of CDs with ferritin
183
through electrostatic interactions. Here it is important to note that the ferritin iron is not exposed to surrounding environment,
184
rather it is buried inside its core. The only way CDs can access the iron core is through the hydrophilic 3-fold channels. In order
185
to know whether the presence of CDs disrupt the secondary structure of ferritin or not, we have performed CD measurements.
186
Fig. S2 of the Supporting Information shows the CD spectra of ferritin in absence and presence of 0.01 mg/mL of CDs.
187
Although, the overall secondary structures of ferritin remain unperturbed in presence of CDs, we cannot rule out local structural
188
alterations near the channels.26,35
189
3.4. Excitation wavelength-dependent PL quenching of CDs by ferritin. Next, we extend our study to explore the size-
190
dependent partitioning of CDs through the funnel-shaped channels of ferritin. Here, our aim is to selectively excite differently
191
sized CDs through proper selection of λex and study their dynamics in presence of ferritin. In the present study, we have measured
192
the PL quenching efficiencies at three different λex namely, 320, 350, and 450 nm. Here, it should be noted that the λex-dependent
193
PL quenching of CDs may arise either due to the inherent λex-dependent PL properties of CDs or due to the presence of
194
differently sized CDs present in the solution. To distinguish between these two possibilities, we have performed control
195
experiment with aqueous Fe3+ ions in bulk solution. Fig. 6A shows the steady-state SV plots of CDs in presence of different
196
concentrations of ferritin at three different excitation wavelengths. It is evident that the slope of the SV plots decreases
197
significantly upon increasing the λex from 320 nm to 450 nm. The SV constant decreases from 13.98 × 105 M-1 at λex of 320 nm to
8
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198
3.66 × 105 M-1 at λex of 450 nm. The calculated SV constants for all the three wavelengths are listed in Table 2. In marked
199
contrast to the above results, no such λex-dependent PL quenching of CDs have been observed with aqueous Fe3+ ions in pH 7.4
200
buffer (Fig. 6B,C). The SV constants for all the three wavelengths are similar in presence of aqueous Fe3+ ions (Table 2).
201
Moreover, the PL lifetime (λex = 376 nm) of CDs gradually decreases with increase in aqueous Fe3+-ions concentrations (Fig.
202
7A). The lifetime components are listed in Table S2 of the Supporting Information. The time-resolved SV plot was constructed
203
using average lifetimes in presence of different concentrations of aqueous Fe3+ ions (Fig. 7B). These results clearly indicate
204
dynamic collisional quenching of CDs by aqueous Fe3+ ions.27
205
The λex-independent PL quenching of CDs in presence of aqueous Fe3+ ions in combination with λex-dependent PL
206
quenching by Fe3+ ions of ferritin clearly signify the presence of differently sized CDs in the solution as in bulk aqueous medium
207
all the differently sized CDs are equally accessible by aqueous Fe3+ ions. The λex-dependent PL quenching of CDs by Fe3+ ions of
208
ferritin can be explained by considering different extent of accessibility of the core Fe3+ ions of ferritin by various sized CDs
209
through the funnel-shaped channels.
210
These channels are highly dynamic and flexible in nature. It is well established that various reducing, chelating and
211
chaotropic molecules larger than the size of the ferritin channels can diffuse and migrate into ferritin core through the hydrophilic
212
3-fold channels and the maximum diameter for the permeation is smaller than 2.0 nm.26,36–38 Earlier, Hoare et al. have reported 6
213
Å resolution electron density map of cubic horse-spleen apoferritin and shown that the outer and inner diameter of these channels
214
are 0.9-1.2 nm and 1.7-2.0 nm, respectively.39 It has also been speculated that molecules with a diameter of 1.5 nm can penetrate
215
the protein shell. Later, Clegg et al. have reported the crystal structure of ferritin and shown that molecules (0.7-1.0 nm) larger
216
than the channel diameter (~0.5 nm) can penetrate into the protein cavity.40 On the basis of these literatures and our present
217
results we propose a mechanism where selective optical excitation by varying the λex results in PL from differently sized CDs and
218
in the presence of ferritin, these differently sized CDs exhibit size-dependent penetration into the ferritin shell (Scheme 2). Size-
219
distribution histogram from AFM measurements strongly justify this proposed mechanism where smaller sized CDs (diameter
PCCP Accepted Manuscript
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Size-dependent penetration of carbon dots inside the ferritin
Published on 17 April 2015. Downloaded by University of Birmingham on 21/04/2015 16:41:56.
nanocages: evidence for quantum confinement effect in carbon dots†
Arpan Bhattacharya, Surajit Chatterjee, Roopali Prajapati, and Tushar Kanti Mukherjee∗
Discipline of Chemistry, Indian Institute of Technology Indore, M-Block, IET-DAVV Campus, Khandwa Road, Indore-452017, M.P., India
† ∗
Electronic supplementary information (ESI) available. Corresponding author. E-mail: [email protected]; Tel: +91-7312438738. 1
Physical Chemistry Chemical Physics Accepted Manuscript
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1
Abstract
2
The origin of the excitation wavelength (λex)-dependent photoluminescence (PL) of carbon dots (CDs) is poorly understood and
3
still remains obscured. This phenomenon is often explained on the basis of surface trap/defect states, while the effect of quantum
4
confinement is highly neglected in the literature. Here, we have shown that the λex-dependent PL of CDs is mainly due to the
5
inhomogeneous size distribution. We have demonstrated the λex -dependent PL quenching of CDs inside the ferritin nanocages
6
through selective optical excitation of differently sized CDs. It has been observed that Fe3+ ions of ferritin effectively quench the
7
PL of CDs due to static electron transfer, which is driven by favorable electrostatic interactions. However, control experiment
8
with aqueous Fe3+ ions in bulk medium revealed λex-independent PL quenching of CDs. The λex-dependent PL quenching of CDs
9
by Fe3+ ions of ferritin has been rationalized on the basis of different extent of accessibility of Fe3+ ions by differently sized CDs
10
through the funnel-shaped ferritin channels. PL microscopy of individual CD has been performed to get further information about
11
their inherent PL properties at single dot resolution. Our results have shown that these hydrophilic CDs can be use as potential
12
iron sensors in biological macromolecules.
13 14 15 16 17 18 19
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1. Introduction
21
In recent years, carbon-based fluorescent nanomaterials have gained vast attention due to their superior physicochemical
22
properties compared to the semiconductor based nanomaterials.1–5 Among these nanomaterials, CDs have emerged as highly
23
promising candidates for biomedical imaging application due to their low cytotoxicity and smaller size with comparable PL
24
characteristics as inorganic core-shell QDs.6,7
25
CDs show optoelectronic properties like excitation wavelength (λex) dependent PL,8,9 high PL quantum yield,10 low
26
photobleaching,11 and no PL blinking (fluorescence on–off).12 However, the origins of their intense PL are a matter of debate and
27
not yet entirely understood.8 It is believed that the PL occurs from the radiative recombination of excitons at the surface trap
28
states. Earlier, It has been reported that CDs containing carboxylic (-COOH) and hydroxyl (-OH) groups are highly luminescent
29
as surface oxidation introduce surface defects to CDs which are responsible for their PL.13,14 Another unresolved unifying feature
30
of the PL of CDs is the λex dependence of the emission wavelength and intensity. Whether this arises because of differently sized
31
CDs and/or different emissive surface traps on each CD surface are still unresolved. Most of the earlier literatures successfully
32
demonstrated the role of surface trap states on the origin and λex dependence PL of CDs.12,15,16 It has been speculated that the PL
33
of CDs could be due to the distribution of emissive defect sites on the CD surface. However, the role of quantum confinement
34
effect due to inhomogeneous size distribution of CDs on the λex dependence of PL is poorly understood and neglected in the
35
literature except few reports on size-dependent PL of size separated CDs.11,17–20 Earlier, Zhao et al. have synthesized CDs via
36
electrochemical oxidation of graphite and shown that size separated CDs exhibit size-dependent but λex-independent PL.11
37
Similarly, Dong et al. have observed that both excitation and PL spectra of size separated CDs red-shift with increase in their
38
size.17 Recently, by using single particle PL microscopy, Ghosh et al. have shown that the λex-dependent ensemble PL of CDs
39
arises due to excitation of different subsets of CDs in heterogeneous sample.18 Here, it is important to stress that, our aim in the
40
present study is to address the origin behind the λex dependence PL of CDs without their size separation, as size separation will
41
lead to λex-independent PL. In the present study, our approach is based on a unique strategy that, in the presence of a
42
heterogeneous system, differently sized CDs are expected to behave differently, and hence, by selective optical excitation of
43
differently sized CDs, we can monitor the PL characteristics of various sized CDs present in our system. We have shown that the
44
λex dependence PL of CDs arises due to the inhomogeneous size distribution rather than distribution of different surface trap
45
states in each CD by selective optical excitation of differently sized CDs in the wavelength range 320-450 nm. Earlier, using the
46
same approach we have demonstrated the size-dependent partitioning of Si-QDs inside the micellar aggregates.21 However, in the
47
present study we have chosen a protein nanocage namely, ferritin to probe the dynamics of differently sized CDs.
48
Ferritin is a spherical cage-like major intracellular iron storage protein in eukaryotes with a molecular weight of 450
49
000 Da (Scheme 1).22 Iron is stored in a soluble non-toxic ferrihydrite form inside the ferritin nanocage which can accommodate
50
upto ~ 4500 Fe3+ ions.23 Interstingly, iron enters and leaves the ferritin cavity as Fe2+ ions.22 Hence, migration of Fe2+ ions to the
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ferroxidase center inside the ferritin cavity occurs before its oxidation. On the other hand, reduction of iron from Fe3+ to Fe2+
52
within the ferritin cavity is essential to its release. Apoferritin is composed of 24 subunits, which self-assemble into a hollow
53
cage-like nanosphere with external and internal diameters of ~12-13 nm and ~7-8 nm, respectively.24 The outer surface is
54
connected with the inner cavity through funnel-shaped channels on the protein surface. It contains eight 3-fold hydrophilic
55
channels which are involve in Fe2+ transportation and six 4-fold hydrophobic channels which facilitate the transfer of protons
56
across the protein shell.25,26 The main objective of the present study is to shed light on the origin of λex dependence of the
57
emission wavelength and intensity. To the best of our knowledge, this is the first direct report (without size separation) to show
58
that the λex-dependent PL of CDs is due to the quantum confinement effect of the surface trap states.
59 60
2. Experimental Section
61
2.1. Materials. Ethylenediamine (EDA, ≥ 99.5%), ferritin from equine spleen (Type I, Saline solution), apoferritin from
62
equine spleen and Pur-A-Lyzer™ Dialysis Kit (MWCO 3.5 kDa) were purchased from Sigma-Aldrich. Citric acid monohydrate
63
(99.5%) and Fe (III) nitrate nonahydrate were purchased from Merck (Germany). Milli-Q water was obtained from a Millipore
64
water purifier system (Milli-Q integral).
65
2.2. Synthesis of CDs. CDs were synthesized according to the previously reported method (Scheme 1).27 In brief, citric acid
66
(1.015 g) and ethylenediamine (335 µL) were dissolved in Milli-Q water (10 mL) and sonicated for 5 min. Then the solution was
67
transferred to a Teflon-coated stainless steel autoclave (25 mL) and heated at 200˚C for 5 h. Subsequently the reactor was cooled
68
to room temperature and the solution was dialyzed (MWCO 3.5 kDa) for 48 hrs. The water for dialysis was changed after every 6
69
h. Finally the black brown transparent CD solution was obtained.
70
2.3. Sample Preparation. All solutions were prepared in pH 7.4 phosphate buffer (25 mM). Ferritin solution was prepared
71
by dilution from 125 mg/mL ferritin (MW 450 000 Da) stock solution. For microscopy experiment samples were spin coated
72
with a spin-coater (Apex Instruments Spin NXG-P1) on a clean glass cover slide at 2000 rpm for 3 min. The cover slides were
73
first cleaned with 2% Hellmanex III (Sigma-Aldrich) followed by piranha solution (3:1 concentrated sulfuric acid and 30%
74
hydrogen peroxide) and then with chromic acid. Each of these cleaning steps was followed by repeated washing with Milli-Q
75
water. Finally, these washed slides were rinsed with methanol (Sigma-Aldrich) and dried in vacuum oven at 100˚C.
76
2.3. Instrumentation. Absorption spectra were recorded in a quartz cuvette (10 × 10 mm) using a Varian UV−Vis
77
spectrophotometer (Carry 100 Bio). The PL spectra were recorded in a quartz cuvette (10 × 10 mm) using Fluoromax-4
78
Spectrofluorimeter (HORIBA Jobin Yvon, model FM-100) with excitation and emission slit width at 2 nm. The PL spectra of
79
CDs in the presence of ferritin and aqueous Fe3+ ions were corrected for the absorbance,28 as both of them strongly absorbs light
80
in the wavelength range 200-450 nm. The PL spectrum of CDs was deconvoluted using Origin software. The binding constants
81
(Kb) and the number of binding sites (n) were determined using the Scatchard equation, which can be expressed as follows:
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Where Kb is the binding constant of the CDs-ferritin complex and n is the number of binding sites. The plot of log[(F0 - F)/F]
84
against log[Q] should yield a straight line with an intercept equal to log Kb and a slope equal to n. Circular Dichroism (CD)
85
spectra were measured on a JASCO J-815 CD spectropolarimeter using a quartz cell of 1 mm path length. Scans were made with
86
a slit width of 1 mm and speed of 20 nm/min. Dynamic light scattering (DLS) experiments were performed on a Brookhaven
87
particle size analyzer (model 90 Plus). All the samples for DLS measurements were filtered through 0.22 µm syringe filter
88
(Whatman). PL imaging of individual CD samples were performed on a home-built epi-fluorescence microscope. An air-cooled
89
argon ion laser (Melles Griot, model 400-A03) with excitation wavelength at 457 nm was used to excite the CDs sample placed
90
on an inverted microscope (Nikon, model Eclipse Ti-U). The laser beam was expanded and subsequently focused on the back-
91
focal plane of an oil immersion objective (100× 1.49 NA Nikon) to illuminate 60 × 60 µm2 area of the sample. The PL from the
92
sample was collected through a B2A filter cube (Nikon) with a 505 nm dichroic mirror and a 520 nm long-pass filter and finally
93
imaged with a back-illuminated EMCCD camera (Andor, model iXon X3 897). The exposure time was 200 ms. The images were
94
analyzed with ImageJ (Version 1.46r) NIH. AFM images were recorded on a cleaned glass coverslip using an AIST-NT
95
microscope (model SmartSPM-1000). Powder X-ray diffraction spectra (XRD) were recorded on a Rigaku SmartLab, Automated
96
Multipurpose X-ray Diffractometer with Cu Kα source (wavelength of X- rays was 0.154 nm). FTIR spectra were recorded in a
97
KBr pellet using Bruker spectrometer (Tensor-27). PL decays were recorded on a HORIBA Jobin Yvon picosecond time
98
correlated single photon counting (TCSPC) spectrometer (model Fluorocube-01-NL). The samples were excited at 376 nm by a
99
picoseconds diode laser (model Pico Brite-375L). The decays were collected with the emission polarizer at a magic angle of
100
54.7° by a photomultiplier tube (TBX-07C). The instrument response function (IRF, fwhm ~140 ps) was recorded using a dilute
101
scattering solution. The fluorescence decays were analyzed using IBH DAS 6.0 software by the iterative reconvolution method,
102
and the goodness of the fit was judged by reduced χ-square (χ2) value. The decays were fitted with three-exponential function.
Ft = exp 1⁄!
103 104
Where F (t) denotes normalized PL decay and a1, a2 and a3 were the normalized amplitude of decay component τ1, τ2 and τ3 respectively. The average lifetime was obtained from the equation:
105
< ! >= !
106
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107
3. Results and Discussion
108
3.1. Characterization of CDs. These CDs show PL quantum yield of ~ 60.2% and content 51.13% carbon, 5.81% hydrogen,
109
26.80% oxygen, and 16.25% nitrogen.27 The structure and morphology of synthesized CDs are confirmed from FTIR
110
spectroscopy, powder XRD and AFM. Fig. 1A shows the FTIR spectrum of CDs. The broad peak at 3420 cm-1 is assigned to the
111
stretching vibration of O-H and N-H moieties. The peak at 2925 cm-1 is due to C-H stretching vibration. Two prominent peaks at
112
1690 and 1566 cm-1 arise due to stretching and bending vibration of C=O and N-H moieties respectively. Other noticeable peak
113
at 1394 cm-1 is assigned to the bending vibrations of C-H moieties. The estimated zeta potential of these synthesized CDs is -
114
25.45 mV at pH 7.4.
115
Fig. 1B shows the XRD patterns of the synthesized CDs. A broad peak centered at 250 (0.34 nm) has been observed,
116
which exactly matches with the earlier reported value,27 and signify poorly crystalline nature of these CDs. Fig. 1C displays a
117
typical AFM image of CDs. The CDs are spherical in nature. The size distribution histogram is generated from AFM height
118
profiles of 276 CDs. Fig. 1D shows the size distribution histogram of CDs. Two clear peaks; one centered at 1.73 ± 0.08 nm and
119
the other at 4.86 ± 0.08 nm have been observed. The diameter of the first peak varies from 0.50 to 3.50 nm while the diameter of
120
the second peak varies from 3.60 to 6.60 nm. These results clearly indicate that the synthesized CDs have inhomogeneous and
121
broad size distribution.
122
The absorption spectrum of CDs exhibits a weak shoulder at 250 nm and a prominent peak at 342 nm (Fig. 2A), which
123
is similar to that reported earlier. The shoulder at 250 nm is assigned due to the π–π* transition of C=C bonds from the CDs core,
124
while the prominent peak at 342 nm originates due to n–π* transition of C=O bonds from the surface of CDs.27,29 Excitation at
125
and above 320 nm results in strong PL in the visible region. Moreover, the PL maxima strongly depend on the excitation
126
wavelength (λex). Fig. 2A shows the effect of λex on the PL spectra of CDs. With gradual increase in λex from 320 to 450 nm, the
127
PL maxima progressively red shift from 433 to 512 nm (Fig. 2A). Interestingly, excitation below 300 nm results in a broad PL
128
spectrum. Fig. 2A and B shows the PL spectrum of CDs at λex = 280 nm. Deconvolution of this broad spectrum (fwhm ~144 nm)
129
with multiple Gaussian functions reveals very good fit with four Gaussian peaks in the range 417-524 nm (Fig. 2B). On the basis
130
of these results and earlier reports it is clear that excitation below 300 nm results in excitation of the carbogenic core of CDs,
131
while excitation at and above 320 nm results in excitation and subsequent PL from surface trap states which progressively red-
132
shift with increase in λex. Moreover, excitation of the carbogenic core results in PL from different surface trap states. The origin
133
of these different surface trap states have been discussed later. Here it is important to mention that the importance of surface
134
donor/acceptor states have also been realized earlier in other nanostructure materials including GaN/AlGaN core/shell
135
nanowires.30,31 Fig. 2C exhibits the variation of PL intensities of CDs with λex from 300–510 nm. Interestingly, the histogram
136
reveals a clear peak at 336 nm with a broad and weak shoulder centered at 420 nm. Here, it is important to note the similarity
137
between size distribution histogram estimated from AFM height profile (Fig. 1D) with the PL intensity histogram against λex.
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138
This is expected as PL intensity is directly proportional to the number of particles of a given size present in the solution.
139
Moreover, this similarity strongly indicates that the bimodal size distribution obtained from AFM measurement is not an artifact.
140
Fig. 2D shows the variation of band gap energy with λex in the range of 320–450 nm. These results clearly establish the idea that
141
by proper selection of λex, it is possible to excite differently sized CDs present in our system. For the present study, we have
142
selected three well-separated λex namely, 320, 350, and 450 nm to excite differently sized CDs present in the system.
143
3.2. PL quenching of CDs in the presence of ferritin. Excitation at 350 nm results in blue PL centered at 437 nm. The PL of
144
CDs progressively quenches upon gradual addition of ferritin (Fig. 3A). The PL spectrum of CDs in presence of 2.02 µM ferritin
145
quenches by 2.7 times with a red-shift of 10 nm. In contrast, apoferritin in the same concentration range does not quench the PL
146
of CDs (Fig. 3B). Fig. 3C shows the steady-state Stern-Volmer (SV) plots of CDs in presence of ferritin and apoferritin. The SV
147
constant in presence of ferritin has been estimated from the slope of the fitted straight line, which is 9.13 × 105 M-1 at 298 K. The
148
only difference between ferritin and apoferritin is that the latter lacks Fe3+ ions in its core. Hence, it is clear that Fe3+ ions of
149
ferritin are involved in the observed PL quenching of CDs. To gain further insight into the mechanistic aspect of this PL
150
quenching process, we measured the PL decay traces of CDs in absence and presence of different concentrations of ferritin. Fig.
151
S1 of the Supporting Information shows the PL decay traces of CDs in absence and presence of 2.02 µM ferritin. The decay trace
152
of CDs in absence of ferritin exhibits three exponential decay kinetics with lifetime components of 3.90 ns (31%), 10.62 ns
153
(29%) and 0.93 ns (40%). It is evident from Table S1 of the Supporting Information that the lifetime components of CDs remain
154
unaltered in presence of 2.02 µM ferritin. Fig. 3D shows the time-resolved SV plot of CDs with average lifetimes. The plot is
155
parallel to the X-axis with a slope of zero. These results clearly signify the formation of static ground state complex between CDs
156
and ferritin.
157
Earlier, Zhu et al. have reported similar kind of PL quenching of CDs by aqueous Fe3+ ions and assigned it due to
158
nonradiative electron transfer from CDs to the vacant d-orbital of Fe3+ ions.27 It has also been proposed that the specificity of Fe3+
159
ions to quench the PL of CDs arises due to special coordination interaction between Fe3+ ions and the phenolic hydroxyl groups
160
on the CDs surface. Moreover, it has been shown that the quenching process proceeds through dynamic mechanism rather than
161
static mechanism. In contrast, the static quenching process observed in the present study with ferritin indicates altered
162
interactions of CDs with Fe3+ ions of ferritin.
163
3.3. Effect of temperature and estimation of thermodynamic parameters. To estimate the binding constant (Kb) and various
164
thermodynamic parameters, we have performed temperature variation study. Fig. 3D shows the effect of temperature in the range
165
298–318K on the steady-state SV plot of CDs. As expected for static ground state quenching process, the SV constant decreases
166
with increase in temperature. The binding constant was determined using the Scatchard equation. Fig. 4A shows the Scatchard
167
plot at three different temperatures. All the plots are linear and the estimated binding constant (λex = 350 nm) for CDs-ferritin
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168
complex is 7.35 (± 0.02) × 105 M-1 at 298 K. The binding constant gradually decreases with increase in temperature from 298–
169
318 K, indicating the static nature of the complex.
170
To gain further insights into the thermodynamics of their association reactions, we used van’t Hoff equation to extract
171
various thermodynamic parameters such as enthalpy change (∆H), entropy change (∆S), and Gibbs free energy change (∆G). Fig.
172
4B shows the linear plot of lnKb against 1/T. The estimated thermodynamic parameters for the CDs-ferritin complex at λex = 350
173
nm are listed in Table 1. The ground state association reaction is found to have a favorable negative enthalpy change (∆H < 0)
174
with a positive entropy change (∆S > 0), giving rise to an overall negative free energy change. In general, processes triggered by
175
hydrophobic interactions between proteins and ligands proceed with a large positive entropy change with a positive enthalpy
176
change while processes triggered by electrostatic interactions proceed with a positive entropy change with a small negative
177
enthalpy change.32,33 Hence, on the basis of our present results we propose that electrostatic interactions between positively
178
charged ferritin cores with the negatively charged CDs surface trigger their association process.
179
Further evidence of their ground state association comes from DLS measurement (Fig. 5). The hydrodynamic diameter
180
of ferritin is estimated to be 14.65 ± 0.10 nm (Fig. 5), which is close to the earlier reported diameter of 12 nm from TEM
181
measurement.34 However, the mean diameter of ferritin in the presence of 0.01 mg/ml CDs increases to 17.37 ± 0.05 nm (Fig. 5).
182
This increase in mean diameter clearly justifies the proposed mechanism of static ground state association of CDs with ferritin
183
through electrostatic interactions. Here it is important to note that the ferritin iron is not exposed to surrounding environment,
184
rather it is buried inside its core. The only way CDs can access the iron core is through the hydrophilic 3-fold channels. In order
185
to know whether the presence of CDs disrupt the secondary structure of ferritin or not, we have performed CD measurements.
186
Fig. S2 of the Supporting Information shows the CD spectra of ferritin in absence and presence of 0.01 mg/mL of CDs.
187
Although, the overall secondary structures of ferritin remain unperturbed in presence of CDs, we cannot rule out local structural
188
alterations near the channels.26,35
189
3.4. Excitation wavelength-dependent PL quenching of CDs by ferritin. Next, we extend our study to explore the size-
190
dependent partitioning of CDs through the funnel-shaped channels of ferritin. Here, our aim is to selectively excite differently
191
sized CDs through proper selection of λex and study their dynamics in presence of ferritin. In the present study, we have measured
192
the PL quenching efficiencies at three different λex namely, 320, 350, and 450 nm. Here, it should be noted that the λex-dependent
193
PL quenching of CDs may arise either due to the inherent λex-dependent PL properties of CDs or due to the presence of
194
differently sized CDs present in the solution. To distinguish between these two possibilities, we have performed control
195
experiment with aqueous Fe3+ ions in bulk solution. Fig. 6A shows the steady-state SV plots of CDs in presence of different
196
concentrations of ferritin at three different excitation wavelengths. It is evident that the slope of the SV plots decreases
197
significantly upon increasing the λex from 320 nm to 450 nm. The SV constant decreases from 13.98 × 105 M-1 at λex of 320 nm to
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198
3.66 × 105 M-1 at λex of 450 nm. The calculated SV constants for all the three wavelengths are listed in Table 2. In marked
199
contrast to the above results, no such λex-dependent PL quenching of CDs have been observed with aqueous Fe3+ ions in pH 7.4
200
buffer (Fig. 6B,C). The SV constants for all the three wavelengths are similar in presence of aqueous Fe3+ ions (Table 2).
201
Moreover, the PL lifetime (λex = 376 nm) of CDs gradually decreases with increase in aqueous Fe3+-ions concentrations (Fig.
202
7A). The lifetime components are listed in Table S2 of the Supporting Information. The time-resolved SV plot was constructed
203
using average lifetimes in presence of different concentrations of aqueous Fe3+ ions (Fig. 7B). These results clearly indicate
204
dynamic collisional quenching of CDs by aqueous Fe3+ ions.27
205
The λex-independent PL quenching of CDs in presence of aqueous Fe3+ ions in combination with λex-dependent PL
206
quenching by Fe3+ ions of ferritin clearly signify the presence of differently sized CDs in the solution as in bulk aqueous medium
207
all the differently sized CDs are equally accessible by aqueous Fe3+ ions. The λex-dependent PL quenching of CDs by Fe3+ ions of
208
ferritin can be explained by considering different extent of accessibility of the core Fe3+ ions of ferritin by various sized CDs
209
through the funnel-shaped channels.
210
These channels are highly dynamic and flexible in nature. It is well established that various reducing, chelating and
211
chaotropic molecules larger than the size of the ferritin channels can diffuse and migrate into ferritin core through the hydrophilic
212
3-fold channels and the maximum diameter for the permeation is smaller than 2.0 nm.26,36–38 Earlier, Hoare et al. have reported 6
213
Å resolution electron density map of cubic horse-spleen apoferritin and shown that the outer and inner diameter of these channels
214
are 0.9-1.2 nm and 1.7-2.0 nm, respectively.39 It has also been speculated that molecules with a diameter of 1.5 nm can penetrate
215
the protein shell. Later, Clegg et al. have reported the crystal structure of ferritin and shown that molecules (0.7-1.0 nm) larger
216
than the channel diameter (~0.5 nm) can penetrate into the protein cavity.40 On the basis of these literatures and our present
217
results we propose a mechanism where selective optical excitation by varying the λex results in PL from differently sized CDs and
218
in the presence of ferritin, these differently sized CDs exhibit size-dependent penetration into the ferritin shell (Scheme 2). Size-
219
distribution histogram from AFM measurements strongly justify this proposed mechanism where smaller sized CDs (diameter
