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Carbon dots prepared from citric acid and urea

by microwave-assisted irradiation as a turn-on
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Cite this: New J. Chem., 2021,

45, 22424 fluorescent probe for allantoin determination†
Ratchadaporn Seedad, Sasimaporn khuthinakhun, Nuanlaor Ratanawimarnwong,
Piyada Jittangprasert, Thitirat Mantim and Kriangsak Songsrirote *

Received 7th July 2021,
Accepted 4th November 2021
DOI: 10.1039/d1nj03284d
rsc.li/njc
This research aimed to develop a simple and selective method for allantoin determination to overcome
the limitations of existing colorimetric and alternative methods. A fluorometric detection-based method
was developed and used carbon dots (CDs) synthesized from citric acid (CA) and urea as a carbon
source. CDs selective for allantoin were synthesized from a mixture of 0.5 g CA and 2.5 g urea exposed
to 800 watts of microwave-assisted pyrolysis for 9 minutes. A fluorescence emission spectrum of
438.0 nm was obtained when CDs were excited with a 370.0 nm wavelength. Several detection
parameters were investigated including pH, sample: reagent ratio, and reaction time. Under optimum
conditions, the proposed method resulted in a detection limit of 0.30 mg L1, a quantitation limit of
0.98 mg L1, and a detection range of 1.0–100.0 mg L1. In summary, the application of this method for
measuring the allantoin content of skincare products was demonstrated and accurate and selective
detection of allantoin was achieved. Furthermore, a mechanism of fluorescent enhancement of CDs by
allantoin was also proposed in this research.

1. Introduction these technologies suffer from disadvantages such as poor selec-

Allantoin is the end product of purine catabolism in most organ-
isms including mammals, plants, and bacteria.1–3 It contains high
levels of urea to form a heterocyclic organic molecule. Allantoin is a
pharmacologically effective compound that has been shown to
have protective, keratolytic, and anti-inflammatory properties that
help to soothe the skin and promote cell proliferation.2 As a result,
allantoin has been widely used in topical treatments for ulcers,
acne, hypertrophic scars, atopic dermatitis, and psoriasis and is a
common ingredient in various cosmetic and skincare products.4–10
Recommended allantoin concentrations in cosmetics are between
0.1% and 0.5% (w/w) and the U.S. Food and Drug Administration
(FAD) lists allantoin as a Category 1 (safe and effective) active
ingredient for skin protection at concentrations of 0.5% to 2.0%.11
Therefore, effective quality control methods are needed to deter-
mine the content of allantoin in skincare products. Several meth-
ods have previously been used for allantoin determination
including colorimetric methods,9,12,13 UV derivative spectropho-
tometry,2 IR analysis,14 alkaline titration,9 high performance liquid
chromatography,15–17 and enzymatic assay.18 However, most of
Department of Chemistry, Faculty of Science, Srinakharinwirot University,
Sukhumvit 23, Wattana, Bangkok 10110, Thailand. E-mail: kriangsaks@g.swu.ac.th
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1nj03284d
tivity and sensitivity, high operation costs, and long reaction times.
Therefore, the development of a simple approach with a high
analytical performance for allantoin determination is challenging.
Since the discovery of the fluorescent properties of carbon
nanotube fragments in 2004,19 carbon dots (CDs), carbon-based
fluorescent nanomaterials, have attracted extensive attention
because of their intriguing optical and electronic properties.20 In
addition, the use of CDs has several advantages such as high and
tunable fluorescence, low toxicity, good water solubility, and facile
synthesis.21 As a result, CDs have been exploited in a wide range
of applications such as sensing,22,23 bioimaging,24 mimic enzy-
me catalyst,25,26 cancer treatment,27 photo-catalysis,28 superca-
pacitor,29 and lighting and displays.30 Furthermore, the use of
CDs as fluorescent probes has dramatically increased and has
demonstrated potential for sensing cations and anions,31–34 small
molecules and macromolecules,35–37 and cells.38–40 Currently,
numerous synthetic methods have been developed to produce
fluorescent CDs.21,41,42 Microwave-assisted synthesis is one of the
most widely used techniques for CD formation because it is a
simple, rapid, green, and low-cost method that produces a relatively
high quantum yield of fluorescence.43,44 Furthermore, a domestic
microwave oven can be used to synthesise CDs with strong
fluorescent characteristics.45,46
CDs can be prepared from a variety of precursors ranging from
waste materials, natural molecules,47 and complex, expensive

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compounds.48 However, citric acid (CA) is one of the most
popular carbon sources because it has broad photoluminescent
regions and high quantum yields but is relatively cheap and
biocompatible.20,23,49 As a building block of allantoin, urea can
be coupled with CA for CD preparation.46,50 As a result, the
synthesized CDs might have the ability to interact with allantoin,
which would cause a change in their photoluminescent proper-
ties. Therefore, this work aimed to develop a simple method for
the determination of allantoin content in skincare products using
CDs prepared from CA and urea by microwave irradiation. The
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blue-fluorescent CDs were obtained and showed a potential to be
applied as a selective and sensitive probe for allantoin detection.
The conditions for detection and mechanism of interaction
between allantoin and CDs were elucidated and presented herein.
2. Experimental section
2.1. Materials
CA and urea were purchased from Fisher Chemicals. Standard
allantoin, melamine, and cyanuric acid were purchased from
Sigma-Aldrich. Dialysis bag (cut of molecular weight 500 Da)
was purchased from Thermo Invitrogen, USA. Samples of facial
creams and body lotions were obtained from beauty shops in
Bangkok, Thailand.
2.2. Methods
2.2.1. Synthesis of the CDs. Different ratios of CA : urea in
grams (2.5 : 0.5, 2.0 : 1.0, 1.5 : 1.5, 1.0 : 2.0, and 0.5 : 2.5 g) were
dissolved in 20 mL of deionized (DI) water. Beakers containing
the mixture solutions were placed in a domestic microwave
oven at 800 watts for 9 minutes. Black solids obtained from
microwave irradiation were re-dissolved in 20 mL of DI water
and then centrifuged at 7000 rpm for 15 minutes to remove
large residues. The supernatant was transferred to a dialysis
bag and placed in a beaker containing 50 mL of DI water. The
solution outside the dialysis bags (containing low molecular
fractions) was exchanged with distilled water every 24 hours for
5 days. The solutions containing low molecular fractions were
combined and dried under a vacuum using a lyophilizer. The
dried CDs were re-suspended with 10 mL of DI water under
sonication and kept at 4 1C until use.
2.2.2. Characterization of synthesized CDs and their activ-
ity with allantoin. Absorption spectra of the aqueous solutions
of CDs prepared from different mass ratios of CA:urea were
acquired using a spectrophotometer (Agilent 8453 UV-visible
spectrophotometer with micro-volume cuvette) and the excita-
tion and fluorescence emission spectra were measured using a
fluorometer (Shimadza RF-5301PC spectrofluorophotometer).
A 250 mL volume of 10 ppm allantoin solution was mixed with
250 mL of 100 ppm CDs prepared from each CA:urea ratio and
left for 20 minutes prior to measuring fluorescence. The
fluorescence spectra of the mixtures were recorded to deduce
the effect of precursor ratio on allantoin determination.
The particle sizes of CDs that provided the best activity for
allantoin detection were characterized using high-resolution
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transmission electron microscopy (HR-TEM) (FEI Tecnai G2 20,
200 kV TEM/STEM/EDX/Tomography).
The functional groups of the CDs were analyzed by FTIR
(Spectrum GX FTIR System, PerkinElmer, USA) using attenu-
ated reflection mode. FTIR spectra were obtained in the range
of wavenumbers from 800 to 4000 cm1 from 64 scans with a
2 cm1 resolution.
X-Ray photoelectron spectroscopy (XPS) was used to eluci-
date the mechanism of interaction between allantoin molecules
and functional groups at the surface of CDs. XPS spectra were
recorded by the AXIS Ultra DLD spectrometer with a mono-
chromatic Al Ka X-ray source (1486.6 eV). Then, C 1s peak was
used as an inner standard calibration peak at 285.0 eV.
2.2.3. Conditions and stability of CDs for allantoin deter-
mination. Optimum conditions were identified by examining
the effect of pH (3–9), volume ratios of allantoin and CDs (1 : 4,
2 : 3, 1 : 1, 3 : 2, and 4 : 1), and reaction time (1–30 minutes) on
allantoin determination by CDs.
The stability of CDs on allantoin detection was investigated
by determining the relative fluorescent intensity of the CDs in
the presence of 5.0 and 50.0 ppm of allantoin under optimum
conditions. The signals obtained from the same batch of the
synthesized CDs were monitored and compared for three
months.
2.2.4. Study of effect of interfering compounds on allan-
toin detection. The effects of compounds analogous to allan-
toin including cyanuric acid and melamine on the fluorescent
intensity of CDs were observed under optimum conditions to
investigate the selectivity of the CDs for allantoin.
2.2.5. Extraction of allantoin from skincare products. The
sample preparation procedure was modified from Sherma and
Cortelyou.50 In brief, 1.0 g samples of facial creams and body
lotions were weighed, dissolved in 20 mL of water, and heated
to 55 1C with stirring for 10 minutes. After cooling to room
temperature, each sample was passed through a reverse-phase
VertiPak SPE cartridge (500 mg) that was conditioned with 5 mL
of methanol followed by 5 mL of DI water. The sample solution
was loaded through the cartridge at a flow rate of 1 mL min1
and the loading fraction was collected before washing the
cartridge with 5 mL of DI water. The wash fraction was
combined with the loading fraction, and then adjusted the
volume to 100.0 mL with DI water. The optimum conditions
identified for allantoin determination were used for further
analysis.
2.2.6. Method validation. The method of allantoin deter-
mination was validated by measuring the recovery of the
standard allantoin spiked to the sample before the extraction
process. In addition, the linearity, limit of detection (LOD),
limit of quantification (LOQ), precision, and accuracy of the
method was determined. Microsoft Excel 1997–2003 was used
to calculate the repeatability of the measurement. All analyses
were performed in triplicate (three replicates of measurement
were performed for each of the samples and their spiked
standards) and compared to those obtained by the conven-
tional RP-HPLC method using an Inertsils ODS-3 C18
column (4.6 mm 205  150 mm, 5 mm) coupled with a
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Shimadzu LC-20AD HPLC pump. The mobile phase was
80% (v/v) ethanol in water at a flow rate of 0.8 mL min1 using
isocratic elution. Allantoin was detected at the wavelength of
210 nm using an SPD-M20A diode array detector. Significant
differences between the two groups were identified using a
paired sample t-test.
3. Results and discussion
3.1. Characterization of CA:urea CDs
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CDs synthesized from CA and urea had strong optical absorp-
tion in the UV region with a tail extending to the visible range
(Fig. 1). Furthermore, different ratios of CD:urea resulted in
CDs that showed altered absorption characteristics. For exam-
ple, CDs synthesized from 2.5 : 0.5 g CA:urea had a maximum
absorption at 230 nm and 332 nm that could be attributed to
the p– p* transition of the CQC bonds as the n–p* transition of
CQO bonds of the carbon core has been previously observed.51
In agreement with Wang et al., (2019), the CDs of 0.5 : 2.5 g CA:
urea exhibited additional strong absorption peaks at 275 nm
and 413 nm, which could result from the molecules at the
surface of the CDs.22 Finally, the CDs of 1.5 : 1.5 g CA:urea
shared characteristic peaks of both of the ratios described. This
suggested that a mixture of different CDs species was present in
the solution. In summary, these results showed that different
molar ratios of starting reagents yielded different CD products
that had a range of optical properties.
The fluorescent spectra of CDs synthesized from different
ratios of CA:urea were investigated (Fig. 2). Blue fluorescence
was obtained from the CDs with CA:urea ratios of 2.5 : 0.5 and
2.0 : 1.0 g with the former ratio showing the highest emission
intensity (438.0 nm) with excitation at 370.0 nm. In contrast,
CDs formed from a ratio of 0.5 : 2.5 g produced green fluores-
cence with emission at 540.0 nm and excitation at 408.5 nm.
The effect of excitation on the emission wavelength was also
investigated. It was found that the emission wavelength was not
affected by using different excitation wavelengths as long as
Fig. 1 Absorption spectra and appearance of CDs synthesized from CA :
urea at the ratios of 2.5 : 0.5, 1.5 : 1.5, and 0.5 : 2.5 g.
22426 | New J. Chem., 2021, 45, 22424–22431 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
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Fig. 2 Fluorescent emission spectra of the CDs from different ratios of
CA : urea and appearance of samples produced from ratios of 2.5 : 0.5 and
0.5 : 2.5 g under UV light.
less than 438.0 nm was applied (Fig S1, ESI†). Excitation only
affected the intensity of fluorescence, and the excitation of
370 nm gave the strongest fluorescent emission.
CDs from CA : urea ratios of 2.5 : 0.5 g and 0.5 : 2.5 g were
chosen for further investigation owing to the high fluorescence
and emission spectra characteristics produced by these samples.
Subsequently, the potential use of these CDs as detection probes
for allantoin determination was assessed. A significant increase in
the fluorescence intensity of blue CDs was shown in response to
changes in the concentration of allantoin. However, there were no
significant differences in the fluorescence intensity of green CDs
in response to allantoin (data not shown).
HR-TEM was used to examine the morphology of blue CDs
(Fig. 3). Image analysis showed that CDs were less than 5.0 nm
in diameter and had an almost spherical shape. Furthermore,
the lattice spacing of CDs (0.228 nm) corresponded to the (100)
plane of graphite. These results are in agreement with previous
findings.22
FTIR was used to characterize the functional groups of the CDs
in comparison to the starting reagents (Fig. 4). In general, the
spectrum of CDs resembled the signals of citric acid and urea with
slight shifts due to bond formations, as their peaks occurred in
similar wavenumbers. The broad band at 3207–3442 cm1 can be
assigned to stretching vibration of hydrophilic amine (N–H) and
hydroxyl (O–H) groups.22 The signal at 3075 cm1 represents C–H
stretching vibration of aromatic ring.52 The strong signal at 1700
and 1591 cm1 corresponded to CQO and CQN stretching modes,
respectively.53 In addition, the vibration peak ca. 1620–1610 cm1
can be assigned to CQC stretching which is consistent with the
presence of benzene ring structure and also core structure of the
CDs.52 The three peaks at 1405, 1354, and 1286 cm1 corresponded
to the stretching mode of aromatic C–N vibration.54 The signals at
1182 and 1079 cm1 were attributed to the vibration mode of C–N
and C–O, respectively.22 Based on the FTIR results, the functional
groups of CDs were hypothesized to contain citrazinic acid as
proposed by Kasprzyk, et al.49

Paper
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Fig. 3 High resolution TEM image of blue CDs from CA : urea (2.5 : 0.5 g)
with lattice spacing of 0.228 nm (inset).
3.2. Optimum conditions for allantoin determination
3.2.1. Effect of pH. The effect of pH on allantoin detection
was investigated by comparing blue CDs redissolved in buffer
solutions (0.1 M) that ranged in pH (pH 3–9) with those
dissolved in DI water (pH 6.5). The relative fluorescent intensity
in terms of the enhancement factor (eqn (1)) of blue CDs in the
Fig. 4 FTIR spectra of blue CDs and the starting reagents.
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buffer solutions and DI water in the presence of 10 ppm
allantoin at a volume ratio of 1 : 1 was determined (Fig. S2, ESI†)
Enhancement factor = (F  F0)/F0 (1)
where F refers to the fluorescence intensity of each sample with
allantoin, and F0 is the intensity of the reagent blank.
The results showed that CDs in buffer solutions exhibited
less fluorescent enhancement compared to those in DI water.
This suggested that ions contained in the buffer interrupted the
fluorescent emission efficiency of CDs or inhibited the reaction
between CDs and allantoin. Therefore, DI water was selected as
the solvent for CD dissolution.
3.2.2. Effect of the volume ratio. The volume ratio of CDs
redissolved in DI water and the allantoin solution was opti-
mized to achieve the highest sensitivity. Total volume mixtures
of 500 mL were prepared that contained 1 : 4, 2 : 3, 1 : 1, 3 : 2, or
4 : 1 ratios of CD:allantoin and the relative fluorescent inten-
sities of these mixtures were compared to their blank solutions.
The results showed that solutions with a higher volume ratio of
CD to allantoin produced the highest fluorescent intensities
(Fig. S3, ESI†). In particular, the highest fluorescence emission
was obtained with a ratio of 4 : 1 CD:allantoin.
3.2.3. Effect of reaction time. The fluorescence intensity
was monitored over time to determine the reaction time needed
for completion of the reaction. The fluorescence was recorded
immediately after mixing CDs with standard allantoin
(0 minutes) and subsequently measured in 3 minute intervals.
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molecules (cyanuric acid and melamine) and ascorbic acid,

which is often present in various skincare products. These
organic molecules were mixed with blue CDs under the optimal
conditions for allantoin detection and the fluorescence inten-
sities of the mixtures were measured. The results showed that
the fluorescent intensity of CDs was not significantly different
in the presence of cyanuric acid and melamine up to 500 ppm
and 350 ppm, respectively. Conversely, the tolerance limit for
ascorbic acid was relatively low (75 ppm). However, it was found
that the presence of these compounds at levels above their
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tolerance limits quenched fluorescence while allantoin

Fig. 5 Spectra of blue CDs in the presence of different concentrations of
allantoin and a calibration curve (inset) for allantoin detection using
CA:urea CDs as a detection probe.
The fluorescence intensity of the mixture increased over time
and plateaued after 18 minutes (Fig. S4, ESI†). Therefore, an
optimal reaction time of 18 minutes was chosen for allantoin
determination.
3.2.4. Stability of CDs on allantoin detection. The relative
fluorescent intensities of the CDs in the presence of 5.0 and
50.0 ppm allantoin were determined and monitored for three
months by using the same batch of CDs. In comparison to the
signals obtained from freshly prepared CDs, the intensities of
the CDs after 1, 2, 4, 8, and 12 weeks of synthesis did not
change significantly (Fig. S5, ESI†). This indicates that the CDs
had a long shelf life, which can potentially be applied in the
field for real-time application.
3.3. Selectivity of the CDs on allantoin determination
Our initial results suggested that the fluorescence emission of
blue CDs in allantoin determination was affected by the buffer
composition. Therefore, the activity of CDs in the presence of
organic molecules with allantoin-like structures was further
investigated. In particular, the effects of nitrogen-rich
enhanced the emission intensity.
3.4. Performance of CD fluorescence on allantoin
determination
A calibration curve was constructed following the correlation
between the enhancement factor, (F  F0)/F0, versus different
concentrations of allantoin using blue CDs prepared from CA
and urea under optimum conditions. Fig. 5 shows the fluores-
cence signal increasing with increasing concentrations of
allantoin (1.0 to 100.0 ppm) relative to the blank solution. This
was used to obtain a calibration curve for allantoin detection
with a linearity range between 1.0 – 100.0 ppm (inset of Fig. 5).
LOD and LOQ were calculated based on the standard devia-
tion (SD) at very low concentrations of allantoin and the slope
of the calibration curve and they were found to be 0.30 ppm and
0.98 ppm, respectively, which corresponded to the experi-
mental results. Furthermore, the precision of the method was
determined from the relative standard deviation (% RSD) of the
standard allantoin solution at the concentrations of 1.0 ppm
(2.83%) and 100.0 ppm (1.26%). The performance of the
fluorescence-based method using CDs synthesized from CA
and urea as a detection probe for allantoin was compared to
alternative approaches shown in Table 1. Overall, fluorescence-
based CD probes had high simplicity, sensitivity, precision, and
selectivity with a short analysis time and low cost. In addition,

Table 1 Comparison of the performance of CD fluorescent probes and alternative methods for allantoin detection

Method Reagents Sensitivitya Reaction time Sample

12 1
Colorimetry H3PW12O40, Sat. Pb(CH3OO)2, 5% H2SO4, 200 mg L 10 min at 100 1C Urine
folin ammoniacal copper reagent, acid molyb-
date reagent
Colorimetry13 Phenylhydrazine, K4[Fe(CN)6], KH2PO4, NaOH 1.0 mg L1 2 min at 100 1C Urine
and HCl
UV derivative 0.1 M NaOH 50 mg L1 — Liposomes and pharmaceutical
spectrophotometry2 formulations
HPLC-MS/MS15 Polymer based amino column with acetonitrile/ 14.6 mg L1 Retention time of Urine
water as mobile phase 9 min
RP-HPLC with UV detector16 C18 column with KH2PO4 (aq) as mobile phase 0.94 mg L1 Retention time of Urine
(LOD) 3.3 min
Ion pairing RP-HPLC with UV C18 column with 5.0 mM H3PO4, pH 2.4 as 0.04 mg L1 Retention time of Brain dialysis
detector17 mobile phase with 1.5 mM TBAHS as ion pairing (LOD) 3.6 min
reagent
Enzymatic assay with fluor- Enzyme allantoinase, resorcinol, HCl 0.08 mg L1 420 min with Serum, urine, saliva
18
escent detection heat
CDs as fluorescent probe [This Citric acid and urea 0.30 mg L1 18 min Facial creams and body lotions
work] (LOD)
a
Sensitivity = Lowest concentration of standard used by author.

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Table 2 Concentration and recovery of allantoin determined in skincare

samples using CA:urea CDs and RP-HPLC (n = 3)

Detection (mg L1)

Std. allantoin added
Sample (mg L1) RP-HPLC CA:urea CDs % Recovery
No. 1 0.0 0.0 0.0 —
1.0 0.96  0.88 1.18  1.52 118.0
50.0 51.21  1.16 48.57  1.78 97.1
No. 2 0.0 28.17  1.24 27.40  1.65 —
1.0 28.96  1.20 28.23  1.84 83.0
50.0 79.64  1.22 75.87  1.92 96.9
No. 3 0.0 48.07  1.37 46.67  2.16 —
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1.0 50.15  1.35 47.84  2.01 117.0

50.0 96.72  1.27 97.91  1.86 102.5

very small amounts of reagents and CDs were needed for this
detection method.

3.5. Allantoin determination in real samples and method

validation
Optimal conditions for the detection approach were used for
allantoin determination with fluorescent CDs in 3 samples of
commercial facial creams and body lotions. Measurement
results for the different samples and the percent recovery of
detection after spiking samples with 1.0 and 50.0 mg L1 of a
standard allantoin solution were compared with those obtained
by RP-HPLC (Table 2). Two out of three samples were
found to contain allantoin at concentrations of 27.40 mg L1
(0.27% (w/w)) and 46.67 mg L1 (0.47% (w/w)) and the recovery
of allantoin spiked into the samples ranged from 83.0–118.0%.
In addition, there were no significant differences between the
results obtained using CA:urea CDs and RP-HPLC (95% CI,
p o 0.05). Therefore, these findings show that CA:urea CDs are
an effective alternative method for the determination of allan-
toin in skincare products. Fig. 6 XPS spectra of CDs before allantoin addation (a), deconvoluted
peaks of O 1s before (b) and after allantoin addition (c), and those of N 1s
3.6. Mechanism of interaction between CDs and allantoin before (d) and after allantoin addition (e).

Kasprzyk, et al.49 reported that the chemical structure of the

presence of citrazinic acid functionalized on the CDs, while
functional group responsible for blue CDs was citrazinic acid,
pyrrolic N and graphitic N possibly existed in core structure of
which resulted from microwave-assisted pyrolysis of citric acid
the CDs. The peak shift of pyridinic N to lower B.E. at 398.280 eV
and urea. An XPS analysis was conducted on CDs before and
was monitored (Fig. 6e). The decrease in B.E. resulted from an
after the addition of allantoin standard solution to elucidate
the interaction mechanism. The photoelectron spectra of O 1s,
N 1s, and C 1s were recorded (Fig. 6a). In CDs without allantoin,
the binding energies (B.E.) of 531.936 eV and 533.053 eV were
the first two major signals of O 1s, corresponding to CQO 0 and
C–O 0 –H, respectively (Fig. 6b).55 After adding allantoin, the B.E.
of CQO 0 did not change significantly (532.096 eV), while the
B.E. for C–O 0 –H increased to 533.354 eV possibly due to the
H-bonding interaction (Fig. 6c). The XPS peak shift to higher
binding energy induced by H-bonding resulted from an
enhancement of bond strength between C–O because of the
creation of the relatively ionic O–H bond.56
In addition to the change in B.E. of O 1s, the shifts of N 1s B.E.
were also observed. The signals at 399.505 eV, 400.299 eV, and
401.197 eV corresponded to pyridinic N, pyrrolic N, and graphitic
N, respectively, and are in agreement with the previous report57 Fig. 7 The proposed mechanism for fluorescent enhancement of blue
(Fig. 6d). Moreover, the signal of pyridinic N also confirmed the CDs by allantoin.

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electron donor in H-bonding formation with the allantoin mole-
cule, while the peak of 399.314 eV remained relatively high
because of the presence of amide groups of allantoin.
Based on the results of XPS analysis, and structure of both
allantoin and citrazinic acid, hydrogen bonding could occur
between these molecules and cause an enhancement of the fluores-
cence. The proposed interaction mechanism is as shown in Fig. 7.
The connection of CDs via hydrogen bonding with allantoin results
in increased structural rigidity of their functional groups. As a result,
restriction of structural rotation and vibration causes an increase in
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the radiative rate and decreases the non-radiative rate.23
4. Conclusions
The method developed in this work utilized the fluorescent
detection of blue CDs for allantoin determination. Our
approach offers several advantages to conventional methods
such as increased simplicity, high sensitivity, selectivity, low
cost, and low reagent consumption. This study showed that
different mass ratios of the precursors CA and urea resulted in
changes in the characteristics of fluorescence emission owing
to different moieties at the surface of the CDs synthesized from
microwave-assisted pyrolysis. Furthermore, blue CDs that had a
high selectivity for allantoin were produced from a CA : urea
ratio of 0.5 : 2.5 g. Under optimum conditions, the sensitivity of
the developed method was better or comparable to alternative
methods for allantoin determination. In addition, results for
this method were equivalent to those obtained using RP-HPLC
for the measurement of allantoin in commercial skincare
products. Hydrogen bond formation resulting in increased
rigidity of the citrazinic acid structure attached to CDs core is
likely to increase the rate of radiative decay in photolumines-
cence process. Therefore, this mechanism could account for
the fluorescent enhancement of blue CDs in the presence of
allantoin.
Author contributions
Ratchadaporn Seedad: investigation, writing – original draft.
Sasimaporn khuthinakhun: investigation, writing – original
draft. Nuanlaor Ratanawimarnwong: investigation, methodo-
logy. Piyada Jittangprasert: validation. Thitirat Mantim: formal
analysis. Kriangsak Songsrirote: methodology, project admin-
istration, resources, supervision, writing – review & editing.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors gratefully acknowledge the financial supports from
Srinakharinwirot University (Grant No. 657/2563).
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