Uncovering the actual inner-filter effect between highly efficient carbon dots and nitroaromatics

Article abstract of DOI:10.1016/j.saa.2020.118342

High performance sensors can be produced by adequately designing the chemical structure and uncovering the actual detection mechanism. In this study, a fluorescent probe was synthesized for various nitroaromatic molecules, including stereochemically varied nitrophenols and nitroaniline. A systematic investigation of the influence of various analytes on the luminescence behavior of the as-synthesized carbon dot (CDs) revealed the inner-filter effect to be the major detection mechanism. The extinction coefficient and spectral overlap were found to be the critical parameters for high sensitivity and good selectivity rather than the functional groups of the CDs and analytes.

Full text of DOI:10.1016/j.saa.2020.118342

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Uncovering the actual inner-filter effect between highly efficient
carbon dots and nitroaromatics
Hye Jin Lee, Jayasmita Jana, Jin Suk Chung, Seung Hyun Hur
PII:
S1386-1425(20)30320-6
https://doi.org/10.1016/j.saa.2020.118342
SAA 118342
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 6 March 2020
Revised date:
31 March 2020
3 April 2020
Accepted
date:
Please cite this article as: H.J. Lee, J. Jana, J.S. Chung, et al., Uncovering the actual
inner-filter effect between highly efficient carbon dots and nitroaromatics, Spectrochimica
Acta Part A: Molecular and Biomolecular Spectroscopy (2020), https://doi.org/10.1016/
j.saa.2020.118342
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Uncovering the actual Inner-filter Effect between highly efficient carbon
dots and nitroaromatics
Authors name: Hye Jin Lee, Jayasmita Jana, Jin Suk Chung and Seung Hyun Hur*
School of Chemical Engineering and Bioengineering, University of Ulsan, Daehak-ro 93,
Nam-gu, Ulsan 44610, South Korea
Email: shhur@mail.ulsan.ac.kr
Abstract
High performance sensors can be produced by adequately designing the chemical
structure and uncovering the actual detection mechanism. In this study, a fluorescent probe
was synthesized for various nitroaromatic molecules, including stereochemically varied
nitrophenols and nitroaniline. A systematic investigation of the influence of various analytes
on the luminescence behavior of the as-synthesized carbon dot (CDs) revealed the inner-filter
effect to be the major detection mechanism. The extinction coefficient and spectral overlap
were found to be the critical parameters for high sensitivity and good selectivity rather than
the functional groups of the CDs and analytes.
Keyword: carbon dots; nitrophenol; nitroaniline; inner-filter effect (IFE); fluorescence;
quenching
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Introduction
Nitroaromatics are used widely as intermediates in the dye, pharmaceutical, pesticide,
and explosives industries. On the other hand, these compounds are hazardous to humans and
wildlife because of their toxicity, carcinogenicity, and mutagenicity [1]. In addition, their
widespread use has resulted in serious environmental contamination in air, soil, and water.
Nitrophenols have negative effects on health, such as skin irritation, blood disorders, and
even death [2]. For example, 2,4-dinitrophenol (DNP) causes increased basal metabolic rates,
a feeling of warmth, sweating, weight loss, and increased heart rate, breathing rate, and body
temperature [3]. Nitroaniline also induces hypoactivity, convulsions, prostration, salivation,
piloerection, shallow respiration, and a loss of muscle coordination [4].
Recently, fluorescence-based sensing of nitroaromatics has been attempted widely
because of its high sensitivity, excellent accuracy, rapid response, and simple operation
process. Soni et al. reported a fluorescent sensor for mono-, di-, and tri-nitrophenols operated
in the micromolar range using graphene quantum dots (GQDs) [5]. Tian et al. fabricated a
paper-based sensor for trinitrotoluene in the nanomolecular range using carbon dots (CDs)
and CDs-polyvinyl alcohol composites [6]. Yuan et al. demonstrated a fluorescent probe for
p-nitroaniline sensing in both aqueous and ethanol solutions using CDs [7].
Fluorescent CDs are promising candidates for fluorescence probes because of their
non-toxicity, photostability, and excellent luminescent properties [8-11]. Their properties can
be controlled easily by precursor design [12], surface passivation [13], and optimization of
the reaction conditions [14]. To obtain the desirable sensing properties, understanding the
detection mechanism and designing their chemical structures is crucial. Many researchers
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have suggested photo-induced electron transfer (PET), Förster resonance energy transfer
(FRET), and inner-filter effect (IFE) [15-17], as a major detection mechanism. In particular,
Inner filter effect (IFE) was widely applied on the fields of chemical sensing [18-21]. Cayuela
et al. fabricated Inner-filter effect based sensor for detection of silver nanoparticles, whose
aggregation shows high absorption in visible range [19]. Sai et al. used CDs for determining
acetone via inner filter effect because of spectral overlap in range of 240-320 nm [20]. Fan et
al. fabricated inner-filter effect based fluorescent probe for for detecting 2,4,6-trinitrophenol
[21]. Although there are a lot of interest of IFE based probe, but it is not clearly explained or
misunderstood between other mechanisms.
This study investigated the detection mechanism of CDs towards various
nitroaromatics, including 2-nitroaniline (2-NA), 3-nitrophenol (3-NP), 2,4-dinitrophenol
(DNP), and 4-nitrophenol (4-NP). The IFE and FRET mechanisms in sensing can be
confused because both are based on spectral overlap between the emission band of the energy
donor and the absorption of the acceptor. The IFE and FRET models were compared and
discussed through mathematical models, including Lakowicz [22] and the Parkers and Barnes
correction [23]. The CDs used in this study were synthesized from citric acid and uric acid as
novel precursors after optimizing the reaction conditions, which exhibited the excellent
linearity and low detection limits towards nitroaromatics. As shown in Table S2, previous
studies used the functional group-induced specific interaction between CDs and each analyte,
which resulted in limited sensing of nitroaromatics. In contrast, the CDs fabricated in this
study can detect a range of nitroaromatics by the IFE.
Experiments
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The detailed information on the chemicals and instruments used in this study is
provided in the supporting information.
Synthesis of CDs
As illustrated in scheme 1, CDs were synthesized from citric acid (105 mg) and uric
acid (168 mg) via a hydrothermal process. First, a pre-determined NaOH solution (0, 0.2, 0.4,
0.6, 0.8, and 1.0 M) was prepared. Uric acid and citric acid were then dissolved in 10 mL of
the above solutions with stirring for 10 min using a magnetic bar. To achieve a homogeneous
dispersion, the solution was sonicated for 1 h. After the solution was transferred to a Teflon-
lined stainless-steel autoclave, it was placed into a muffle furnace, heated to 160 ℃, and
maintained at that temperature for 6 h for CD formation. After cooling to room temperature,
the resulting solution was mixed with 20 mL of ethanol and centrifuged at 5000 rpm for 10
min to remove the residues and organic byproducts. The sediment was collected and re-
dispersed in 10 mL of D.I. water. To obtain the solid CDs, 2 mL of CD solution was freeze-
dried for one day at -46 ℃. CDs synthesized by the 0.8 M NaOH solution was selected to
examine the physicochemical properties and sensing performance after optimizing the optical
properties and yields.
Detection of nitroaromatics
Two milliliters of a CD solution (34 mg/mL) was diluted in D.I. water, while the total
volume of solution was kept to 40 mL. Subsequently, 3.6 mL of a CD solution was mixed
with 0.4 ml of the target analyte solutions, which were prepared beforehand with a pre-
determined amount of various analytes. The concentration of organic molecules, including
aniline o-PD, m-PD, p-PD, 2-NA, 3-NP, DNP, and 4-NP was kept to 1 mM for the sensitivity
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test. To determine the limit of detection (LOD), fluorescence and UV-Vis absorption spectra
were collected for a specific range (0-100 μM , and 0-1000 μM , respectively). All
measurements were taken with a 2.5nm excitation slit.
Determination of Quantum Yield
The quantum yields (QYs) of the CDs fabricated in this study were determined by
comparing the fluorescence spectra at four absorbance values with those of a standard sample,
quinine bisulfate (0.1M H2SO4, ϕ=0.54). The fluorescence spectra were collected with a 2.5
nm excitation slit bandwidth and 5 nm emission slit bandwidth. The following equation was
used to determine the quantum yields.
퐴푠 퐹 푛푥
푥
2
휙푥 = 휙
(
)
푠 퐴푥 퐹 푛푠
푠
The subscript, “χ”, means the sample and “s” means the reference material. 휙, A, F,
and n are the quantum yield, absorbance at the excitation wavelength, integrated emission
area across the band, and refractive index of the solvents, respectively. The absorbance was
kept at approximately 0.2-0.6, and the measurements were taken at room temperature.
Results and discussion
Effect of basicity in the CD formation
The effects of basicity on the yield and optical properties of CDs were investigated to
optimize the reaction conditions. As shown in Fig S1a and Table S1, both the quantum yield
and yield increased remarkably under high basic conditions (NaOH 0.8-1.0 M). No CDs
formed under low basic conditions (NaOH 0-0.2 M), indicating that a base catalyst is
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necessary for the carbonization process [14]. A red shift of the maximum absorbance
wavelength was also observed (Fig. S2 and Table S1) under high basic conditions, which can
be due to the increased sp2 cluster of the carbon core and reduced band gaps [24,25]. The
constant maximum excitation and emission wavelengths, and the excitation-wavelength
independent behavior shown in Figs. S1b and S3b suggest that the CDs possess uniform
edge/surface states [26,27]. The CDs exhibited deep-blue luminescence near UV (Fig. S3a),
which was attributed to the small amounts of doped nitrogen atoms that can cause long-
wavelength emission [28,29].
Characterization of as-synthesized CDs
The morphology and the size distribution of CDs were investigated by transmission
electron microscopy (TEM). As shown in Fig 1, the CDs exhibited a spherical morphology
with an average size of 2.32 nm. The broad size distribution resulted in a broad fluorescence
emission band due to the quantum size effect [24]. The high resolution-TEM images in the
inset in Fig. 1a suggest that CDs possess the (100) plane of a graphitic structure,
corresponding a lattice spacing of 0.213nm [28,30]. XRD spectra (Fig S4) show a broad peak
near 20°, corresponded with (002) plane [31]. And, the sharp peaks near 30-40° are due to
presence of sodium [32].
XPS was performed to investigate the elemental composition and chemical structure
of CDs. As shown in Fig S5, strong peaks were observed at 1071 (Na1s), 531 (O1s), and 289
eV (C1s). The strong Na1s peak indicates the formation of a salt by an acid-base reaction
between the carboxylic acid groups of CDs and NaOH under high basic conditions [33]. The
deconvoluted O1s spectrum revealed quinone C=O at 530.7 eV, which indicates the presence
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of oxygen-related functional groups on the CDs [34]. The C1s spectra could be deconvoluted
into three peaks centered at 284.8, 287.5, and 289.0 eV, which were assigned to carbon-
carbon (C-C/C=C), carbonyl (C=O), and carboxyl/carboxylate groups (C=O-O) [34,35].
Surface functional groups were analyzed by Fourier transform infrared (FT-IR)
spectroscopy, as shown in Fig S6. A broad absorption band centered at 3390 cm-1 was
ascribed to O-H stretching vibrations. Two broad absorbances at 1593 and 1455 cm-1 were
assigned to C=C aromatic stretching vibrations and carboxylate symmetric and asymmetric
vibrations, respectively [35,36].
The measured zeta potential was -23.0 mV, which also indicates that large amounts
of oxygen-related functional groups are present in the as-synthesized CDs [37].
Detection of nitroaromatics
The sensing performances of the nitroaromatics, including 4-NP, DNP, 3-NP, and 2-
NA were investigated using a fluorometric method, as shown in Figs. 2 and S7-S9. Table S3
lists all the sensing parameters. CDs have shown good linearity in a range of 0-20 and 0-
200 μM for 4-NP and DNP, 3-NP and 2-NA, respectively. They possess high sensitivity
(0.02967, 0.03414 μM−1) for 4-NP and DNP compared to previous study as shown in Table
S2. And, this is the first study to detect 3-NP and 2-NA and they also shown good Ksv value
(0.00495, 0.00647 μM−1), respectively. The fluorescence intensity of the CDs decreased
sharply with increasing concentration of analytes. A Stern-Volmer plot was used to
investigate the quenching behaviors (Fig. 2b). There was a significant positive deviation from
linearity as the concentration of the analytes increased, suggesting that both dynamic and
static quenching co-exist [22]. The ground-state and sphere action model can explain this
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type of quenching behavior [22,38], but the Stern-Volmer quenching constant is not
recovered fully in both models, as shown in Fig. 2c and Table S4. This indicates that the
dynamic and static quenching model cannot explain the quenching behavior sufficiently. The
recovery values were calculated using the following equation:
K푆푉,ꢀꢁ표푢푛푑(표푟 K푆푉,푠푝ℎ푒ꢁ푒
)
푅푒푐표푣푒푟푦 =
× 100 (%)
K푆푉 (푓표푟 푑푦푚푎푚푖푐 푙푖푛푒푎푟 푟푎푛푔푒)
Proving Inner-filter Effect (IFE)
To examine the rational quenching mechanism, the absorbance spectra of specific
molecules were collected, and then their spectral overlap with the excitation and emission
spectra of the CDs were investigated. As shown in Fig. 3a, a large amount of spectral overlap
was observed between the PL and PLE of CDs and the absorbance of 4-NP, DNP, 3-NP, and
2-NA, which shows that both FRET (Förster resonance energy transfer) and secondary IFE
(Inner-filter effect) can exist in this sensing system. FRET can occur in the presence of
spectral overlap between the emission spectrum of the fluorescent probe and the absorption
spectrum of the acceptor. IFE is involved with the absorption of the quencher overlapped
with the excitation (primary inner-filter effect) or emission of the CDs (secondary inner-filter
effect) [16-17]. For these reasons, the quenching mechanism can sometimes be confusing.
Nevertheless, the obvious difference between them is that energy or electron transfer
accompanies the decrease in lifetime. As shown in Fig. 3b and Table S5, the lifetime of the
photon remains the same even after adding the analyte, which can exclude the FRET
mechanism. In addition, the lack of a relationship between the energy transfer efficiency and
spectral overlap in CDs-nitroaromatics system (Table S6) indicates non-FRET quenching.
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To verify the IFE-induced quenching mechanism, the observed fluorescence
intensity was corrected using the following mathematical models. The Parker and Barnes
model is the first model containing a correction of IFE-induced deviation of the fluorescence
parameter [23], which considers the geometry and fluorescence observation field [39].
2.303퐴푒푥(푥2 − 푥1)
2.303퐴푒푚(푦2 − 푦1)
퐹
푐표ꢁꢁ
= 퐹표푏푠 × 푓 × 푓 = 퐹표푏푠
×
×
−퐴 ×푥
2
푝
푠
10−퐴푒푥×푥 − 10
10−퐴푒푚×푦 − 10
−퐴 ×푦
푒푚 2
1
푒푥
1
2.303퐴푒푥(푥2 − 푥1)
2.303퐴푒푚(푦2 − 푦1)
푓 =
푝
푓 =
푠
10−퐴푒푥×푥 − 10
10−퐴푒푚×푦 − 10
−퐴 ×푥
−퐴 ×푦
푒푚 2
1
푒푥
2
1
where 푓 is the factor for primary IFE involved with excitation and 푓 is the factor for
푝
푠
secondary IFE from the absorption effect of emission light. The correction factor was
calculated using the suggested geometry parameter in the case of a Cary Eclipse
spectrophotometer. 푥1, 푥2, 푦1, and 푦2 are 0.207, 1.085, 0.258 and 0.727, respectively [40].
Lakowicz suggested a correction of the IFE-induced deviation of the fluorescence
intensity using the following equation [22]:
퐴
푒푥
+퐴
푒푚
2
퐹
푐표ꢁꢁ
= 퐹표푏푠10
퐹
푐표ꢁꢁ
is the corrected fluorescence intensity. 퐹표푏푠 is the experimental fluorescence intensity.
퐴푒푥 is the absorbance value at the excitation wavelength. 퐴푒푚 is the absorbance value at the
emission wavelength. This can be used generally because it avoids considering the instrument
geometry, which is not provided by the manufacturers.
As shown in Figs. 3c and S10 and summarized in Tables 1, 2, and S7-S9, the
deviations from linearity converge almost to zero for all nitroaromatic analytes, regardless of
the correction equations, which confirms that the quenching of CDs by the nitroaromatic
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analytes that occurred in this study was due mainly to the inner-filter effect.
Table 1. Parker and Barnes correction parameter
퐹표푏푠,0
/퐹표푏푠
1
퐹
푐표ꢁꢁ,0
4-NP concentration
Correction
Factor
푓
푓
푠
퐹표푏푠
퐹
푐표ꢁꢁ
푝
(μM)
/퐹
푐표ꢁꢁ
0
2
1.172645 0.999
1.185513 1.03
1.190655 1.07
1.207658 1.10
1.219949 1.14
1.228485 1.18
1.291465 1.40
1.409696 1.89
1.544952 2.42
1.691417 3.42
1.845509 4.44
1.17
1.23
1.27
1.33
1.39
1.45
1.81
2.67
3.74
5.79
8.19
273.989
257.128
242.48
321
315
308
310
308
309
309
296
272
289
279
1
1.066
1.13
1.18
1.24
1.29
1.60
2.47
3.76
5.49
8.03
1.02
1.04
1.04
1.04
1.04
1.04
1.08
1.18
1.11
1.15
4
6
232.216
220.989
212.559
171.214
111.135
72.877
8
10
20
40
60
80
100
49.902
34.119
Table 2. Lakowicz correction parameter
퐹표푏푠,0
/퐹표푏푠
1
퐹
푐표ꢁꢁ,0
4-NP concentration
Correction
Factor
퐴푒푥
퐴푒푚
퐹표푏푠
퐹
푐표ꢁꢁ
(μM)
/퐹
푐표ꢁꢁ
0
2
0.10829 -0.00006
0.11582 0.02988
0.11881 0.05661
0.12862 0.08712
0.13564 0.11643
0.14048 0.14826
0.17535 0.29908
0.23715 0.57637
1.13
1.18
1.22
1.28
1.34
1.39
1.73
2.55
273.989
257.128
242.48
310
304
297
298
295
296
296
284
1
1.066
1.13
1.18
1.24
1.29
1.60
2.47
1.02
1.05
1.04
1.05
1.05
1.05
1.09
4
6
232.216
220.989
212.559
171.214
111.135
8
10
20
40
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60
80
0.30278 0.80653
3.59
5.68
8.21
72.877
261
283
280
3.76
5.49
8.03
1.19
1.09
1.11
0.36870 1.13965
0.43317 1.39576
49.902
34.119
100
The selelectivity of nitroaromatics sensing can be achieved using this inner-filter
effect. The spectral overlap between the CDs and the analytes is the essential condition for
the inner-filter effect. As shown in Table 3 and Fig. 4a, aniline and m-, o- and p-
phenylenediamine exhibit minimal spectral overlap with CDs, despite their similar chemical
structures as nitroaromatics, which results in a very low quenching property (Fig. 4b). The
selectivity depends on their spectral properties of the nitroaromatics compound. For precise
recognition, the peaks at 400, 353, 390, 412nm, for 4-NP, DNP, 3-NP and 2-NA, respectively,
can be observed in the absorbance spectra as shown in Fig S11. Again, isobestic point
methods proposed by El-Ghobashy et al. help to distinguish the nitroaromatic compound
when present at the mixture and avoid interference from other absorbed compounds [41].
Table 3. Spectral overlap calculation between the excitation and emission spectra of the CDs
and absorbance spectra of the nitroaromatics
Parameter* 4-NP
DNP
7780
9057
16837
3-NP
1560
643
2-NA
619
p-PD
290
o-PD
98
m-PD
68
aniline
53
푱풆풙
푱풆풎
퐉′
5750
12766
18516
45
0.17
97.8
-2.60
65.4
-1.94
51.4
1727
2346
335
2203
* 푱풆풙 (spectral overlap from excitation spectra) and 푱풆풎 (spectral overlap from
excitation spectra) are defined as:
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∞ 퐹 (휆)휀(휆)푑 휆
∞ 퐹 (휆)휀(휆)푑 휆
푒푚
∫
∫
0
푒푥
0
퐉′ = 푱풆풙 + 푱풆풎
=
+
∞ 퐹 (휆)푑 휆
∞ 퐹 (휆)푑 휆
∫
∫
0
푒푥
푒푚
0
∞ 퐹 (휆)휀(휆)푑 휆
∞ 퐹 (휆)휀(휆)푑 휆
푒푚
∫
∫
0
푒푥
0
푱풆풙
=
푱풆풎 =
∞ 퐹 (휆)푑 휆
∞ 퐹 (휆)푑 휆
∫
∫
0
푒푥
푒푚
0
퐹 (휆) is the intensity of the fluorescence excitation spectra and 퐹 (휆) is the intensity of
푒푥
푒푚
the emission spectra. 휀(휆) is the absorbance at the excitation wavelength. ∞ 퐹 (휆)푑 휆
∫
푒푥
0
and ∞ 퐹 (휆)푑 휆 are used for normalization.
∫
푒푚
0
Summary and Conclusions
Highly luminescent CDs were fabricated using novel precursors of citric acid and
uric acid by facile hydrothermal synthesis. The as-synthesized CDs could detect a range of
nitroaromatics by the IFE mechanism. Mathematical correction and lifetime measurements
showed that PL quenching occurred by the IFE instead of the FRET mechanism. In addition,
spectral overlap between the CDs and analytes was the critical parameter rather than the
specific interaction between the CDs and analyte during the quenching process, which
resulted in excellent selectivity towards interfering molecules and a wide range of
nitroaromatic detections.
Acknowledgements
This study was supported by the Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by The Ministry of Science, ICT and
Future Planning (NRF-2019R1A2B5B02069683).
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Figures Captions
Scheme 1. Schematic diagram of carbon dot synthesis.
Fig 1. (a) TEM images of CDs (inset shows HR-TEM images of CDs). (b) Size distribution
of CDs.
Fig 2. (a) PL spectra (inset shows photographs of CDs under UV illumination). (b) Stern-
Volmer Plot of the PL intensity change at various 4-NP concentrations of 0 - 100μM. (c)
Parameters obtained from the quenching equations.
Fig 3. (a) Spectra overlap between PL, PLE of CDs, and the absorbance of various
nitroaromatics (1000 μM). (b) Time-correlated single-photon counting (TCSPC) of CDs. (c)
Inner filter effect (IFE)-corrected fluorescence intensity versus analyte concentration.
Fig 4. (a) Spectra overlap between PL, PLE of CDs, and the absorbance of phenylenediamine
isomers (1000 μM). (b) Relative PL intensity change of CDs for various analytes (1000 μM).
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Scheme 1
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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CRediT author statement
Seung Hyun Hur: Writing- Reviewing and Editing, Supervision. Hye Jin Lee:
Methodology, Investigation, Visualization, Data curation, Writing- Original draft preparation.
Jayasmita Jana: Resources, Investigation. Jin Suk Chung : Project Administration
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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Graphical Abstract
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Highlights
 Highly luminescent carbon dots (CDs) were fabricated using citric acid and uric acid
as novel precursors.
 The as-synthesized CDs can detect a range of nitroaromatics by the inner filter effect
(IFE)
 The quenching mechanisms were systematically studied and mathematically
calculated.
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