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Green Synthesis of Various Heteroatom-doped Carbon

Quantum Dots from Urine, Whey, and Their Mixture: The
Optimization of Synthesis and Potential Applications
Selin Aydin,[a] Asli Yilmaz,*[a] and Mehmet Yilmaz*[b, c]

The employment of biomass waste for the fabrication of carbon with an excitation-dependent emission nature were obtained
quantum dots (CQDs), as a novel fluorescent material with high with a quantum yield (QY) value of 48 %, 28 %, and 39 % for U,
photoluminescence (PL) activity, has gained intense interest for W, and U/W, respectively. For each case, doping of various
the last decade. However, the fabrication of CQD from biomass heteroatoms such as N, P, S, etc., in the structure of CQDs
waste encounters challenges including low fluorescence yield, contributed PL characteristics with high QY, reasonably low
reproducibility, and stability. Therefore, the novel, simple, cytotoxicity, and, robust pH and storage stability which indicate
flexible fabrication routes for CQDs using biomass waste as a their high potential in various biomedical applications such as
precursor are still essential for practical applications. In this bioimaging and pH sensors. This study proves the utilization of
study, we reported the employment of human urine (U), sour human urine and whey as the precursors for the fabrication of
whey (W), and their mixture (U/W) as precursors for the CQDs through a low-cost, flexible, and eco-friendly green
hydrothermal green synthesis approach. For all cases, CQDs synthesis procedure.

Introduction arc discharge, electrochemical processing, and ultrasonic

methods.[19–22] The requirement of high-cost preparation devices
For the last two decades, carbon quantum dots (CQDs) have (for example laser sources and electrochemical workstations)
emerged as an extraordinary nanomaterial and attracted and harsh synthesis conditions with long durations, low product
remarkable attention as a robust alternative to their semi- yield, and the lack of size control are major drawbacks of top-
conductor-based counterparts.[1–3] CQDs have distinct benefits down approaches which limit their employment for practical
compared to inorganic QDs, such as low toxicity, excellent applications.[2,23] Contrary to top-down strategies, through
biocompatibility, good solubility in water, and, high photo- bottom-up approaches, CQDs are synthesized via the treatment
stability. They also exhibit strong and excitation wavelength- of carbon-based small molecules as precursor material by
dependent photoluminescence (PL) activity with a flexible various methods including microwave synthesis, pyrolysis,
structure for surface modification.[2,4–8] With these unique combustion, thermal decomposition, solvothermal, and hydro-
properties, CQDs have been employed in chemical and bio- thermal synthesis.[6–8,24,25] Of these approaches, the hydrothermal
logical sensor systems,[9–12] diagnostics,[13] bio-imaging,[13,14] drug method is the most preferred synthesis procedure due to its
delivery,[15] and photocatalytic and electrocatalytic simplicity, low cost, high flexibility in the selection of the
applications.[16–18] For the preparation of CQDs, both top-down precursor material, and non-toxic and environmentally friendly
and bottom-up approaches have been widely employed. The nature which are essential for green synthesis routes.[6–8]
top-down strategies basically relied on the breaking down of Recently, there has been a lot of interest in the use of
carbon-based bulk materials through various chemical and biomass waste from many sources, including food, agriculture,
physical treatments such as laser ablation, chemical oxidation, and domestic waste, as precursors in the manufacture of
CQDs.[15,26] Biomass waste has a rich content of carbon (C),
[a] S. Aydin, A. Yilmaz oxygen (O), and nitrogen (N) as main components. These
Department of Molecular Biology and Genetics, Ataturk University, 25240 elements create the main skeleton of CQDs. Also, in most cases,
Erzurum, Turkey biomass waste contains various trace elements such as
E-mail: [email protected]
phosphorus (P), sulfur (S), and, potassium (K). These trace
[email protected]
elements take part in the structure of CQDs as doped
[b] M. Yilmaz
Department of Nanoscience and Nanoengineering, Ataturk University, heteroatoms and may provide unique optical characteristics
25240 Erzurum, Turkey including enhanced PL activity and creation of new energy
E-mail: [email protected] levels to form trap states leading to excitation-dependent PL
[c] M. Yilmaz characteristics.[6,27,28] Various biomass wastes including walnut
Department of Chemical Engineering, Ataturk University, 25240 Erzurum,
shell,[29] orange peel,[30] crop straws,[1] watermelon peel,[31]
Turkey
E-mail: [email protected] orange juice,[32] and lemon peel[33] have been employed for the
Supporting information for this article is available on the WWW under fabrication of CQDs. Most of the biomass waste-derived CQDs
https://doi.org/10.1002/slct.202304930 exhibited unique performances in many chemical sensing,

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biological sensing, and bioimaging applications.[2] Despite the cases, doping of various heteroatoms such as N, P, S, etc., in the
significant progress in terms of the abundance of precursors structure of CQDs has a dominant effect on their PL character-
and synthesis methods, novel, simple, flexible, and low-cost istics with high QY, excitation-dependent emission nature,
strategies are still highly demanded to fabricate CQDs with high reasonably low cytotoxicity, and, robust pH and storage
PL activity and excitation-dependent nature from biomass stability. The present report proved the definite potential of
waste sources. human urine and whey as the precursors for the fabrication of
Herein, we propose a simple yet efficient hydrothermal CQDs, the conversion/elimination of these biomass wastes into
procedure for the fabrication of various heteroatom-doped valuable products and unquestionable potential in various
CQDs utilizing human urine (U), sour whey (W), and their biomedical applications.
mixture (U/W) as precursors. Both urine and whey have very
complex and rich content. Human urine is composed of urea,
creatinine, uric acid, and many ions such as Na, K, phosphate, Results and Discussion
sulfate, and, ammonium.[34] Sour whey has a high content of
sugars, fat, proteins, and some ions including Ca and K.[35] Characterization of U-CQDs
Despite their rich content for the fabrication of CQDs, to the
best of our knowledge, there is only one report on human In the first part, we performed some experimental studies to
urine,[36] one report on sour whey,[37] and no report for their evaluate the PL characteristics of U-CQDs and, W-CQDs via
mixture as precursors. In the first part, we systematically various analyses such as PL, UV-vis absorption, FT-IR and XPS
evaluated the optical properties of CQDs from urine or whey spectra, and relevant TEM images. For this, both QDs were
through various methods. Then, the mixture of the precursors fabricated at fixed synthesis time (12 h) and temperature
was employed and various experimental parameters such as (180 °C). For the case of U-CQDs, normalized PL spectra
fabrication time, temperature, and the ratio of urine to whey (Figure 1a) showed the excitation-dependent emission in a
precursors were determined in detail. We observed that for all wide range of excitation wavelengths. The increase in the

Figure 1. Characterization of U-CQDs. Normalized PL spectra under different excitation wavelengths (a), UV-vis absorption spectrum and optical images of
CQD suspension under daylight and a 365 nm UV-lamp (b), FT-IR spectrum (c), XPS survey with elemental composition data (d), high-resolution C1s (e), O1s
(f), N1s (g) and, representative TEM image with relevant particle size distribution graph (h).

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excitation wavelengths from 280 to 600 nm led to a dramatic mostly in spherical morphology in the range of 5–15 nm with
red-shift in PL peak maxima from 394 to 616 nm. The tunable an average size of 7.7 nm.
PL emission through manipulating excitation wavelength
provides unquestionable opportunities in various applications.
So far, many convincing mechanisms such as the quantum Characterization of W-CQDs
confinement effect and surface traps have been proposed to
elucidate the excitation-dependent PL emission.[6–8,38–41] UV-vis Similar to U-CQDs, we performed many characterization
absorption spectra of U-CQDs (Figure 1b) indicated that various methods to evaluate the optical and chemical properties of W-
transitions contributed to the final optical characteristics of the CQDs. W-CQDs were synthesized at the same synthesis time
material. We noticed a weak shoulder at 271 nm assigned to a (12 h) and temperature (180 °C). The normalized PL spectra of
π–π* transition band indicating the emergence of aromatic C C W-CQDs (Figure 2a) showed that as the excitation wavelength
bonds in the structure.[6–8,42,43] Another weak peak at 338 nm increased from 280 to 600 nm, the PL absorption maximum was
showed the presence of n–π* transition of C=O bonds.[6–8,44,45] redshifted from 440 to 620 nm indicating an excitation-depend-
Also, a broad weak peak at 494 nm was attributed to functional ent emission nature. Unlike U-CQDs, only a broader shoulder at
surface states. The presence of various transitions may be due 292 nm was observed in UV-vis absorption spectra of W-CQDs
to the complex content of human-originated urine as the (Figure 2b) confirming a π–π* transition band associated with
precursor. The colloidal suspension of U-CQDs (see insets of the presence of aromatic C C bonds in the composition.[6–8,42,43]
Figure 1b) showed a pale brown color under daylight but The optic image colloidal suspension of W-CQDs was provided
represented a very high and bright blue emission under a in the insets of Figure 2b indicating a pale yellow color under
365 nm UV lamp. This robust emission created high PL activity daylight while it has a very high and bright blue-white emission
with a high quantum yield (QY) of 43 % which was determined under a 365 nm UV lamp. These optical properties led to a
for a 360 nm excitation wavelength by employing quinine strong PL emission with a QY of 28 % for a 360 nm excitation.
sulfate as reference material. The FT-IR spectrum of U-CQDs Various surface functional groups-associated bands were de-
summarized in Figure 1c provided valuable insight to evaluate tected in the FT-IR spectrum of W-CQDs which was provided in
the probable surface functional groups. From this spectrum, Figure 2c. We observed prominent bands at 3181, 3042, 2968,
prominent bands were detected at 3032, 1602, 1406, and 1580, 1410, and 1117 cm 1 which were assigned to υ (–OH), υ
1130 cm 1 were assigned to υ ( NH)/ υ (C H), δ (C=C)/ υ (C=O), ( NH), υ (C H), δ (C=C)/ υ (C=O), υ (C H) and, υ (C N),
υ (C H), and υ (C N), respectively.[46,47] The hydrophilic groups respectively.[46,47,53] While the hydrophilic groups such as υ
such as υ ( NH) and υ (C=O) enable the high stability of CQDs ( OH), υ ( NH) and, υ (C=O) provide excellent solubility in
in the water phase.[6,48,49] However, υ (C H), δ (C=C), and, υ (C N) water,[7,8,48,54–56] the bands for δ (C=C), υ (C H) and, υ (C N)
functional groups indicate the polyaromatic nature of the groups show the presence of polyaromatic characteristic of the
material.[7,8] The XPS survey of U-CQDs (Figure 1d) shows the CQDs’ core.[57,58] The wide-scanning XPS spectra of W-CQDs
presence of mainly C (C1s), N (N1s), and, O (O1s) at 284, 400, (Figure 2d) indicated the presence of mainly C (C1s), N (N1s),
and 530 eV binding energies, respectively. In addition, some and, O (O1s) at 284, 399, and 530 eV binding energies,
elements such as S, Na, and P were detected in trace amounts respectively. The elemental composition of the material for C, N,
showing the complex composition of urine as the precursor. and O was found to be 66 %, 5 %, and 21 %, respectively. Also,
The elemental composition of the material for C, N, and O was we detected the presence of some elements such as K, and Ca
found to be 66 %, 3 %, and 22 %, respectively. This data supports indicating the complex content of whey as a precursor. This
the UV-vis absorption, PL, and FT-IR spectra in terms of the data is highly compatible with the UV-vis absorption, PL, and
abundance of functional-surface states and resultant excitation- FT-IR spectra concerning the presence of functional-surface
dependent PL emission. It is well-known that heteroatoms such states and obtained high PL activity with an excitation-depend-
as N, P, S, etc., in the structure of CQDs may create new energy ent nature. Similar to U-CQDs, heteroatoms such as N, K, Ca,
levels to form trap states leading to excitation-dependent PL etc., in the structure of W-CQDs may lead to the emergence of
characteristics.[6,27,28] The high-resolution XPS spectra for C1s new energy levels to form trap states and resultant excitation-
(Figure 1e) indicated the convolution into three peaks at 283.3, dependent PL characteristics.[6,27,28] The high-resolution XPS
287.1, and 291.3 eV assigned to C C/C=C, C O and C=O bonds, spectrum for core level C1s (Figure 2e) was convoluted to three
respectively.[50–52] However, the O1s region (Figure 1f) was peaks at 283.3, 286.8, and 290.6 eV assigned to C C/C=C, C O
convoluted into two peaks at 528.7 and 530.8 eV showing the and C=O bonds, respectively.[50–52] However, O1s’ high-resolu-
C=O and C O bonds, respectively.[6–8] Similarly, the N1s tion spectrum (Figure 2f) was convoluted into two peaks at
spectrum (Figure 1g) was convoluted to two main peaks at 529.6 and 530.8 assigned to the C=O and C O bonds,
398.5 and 400.2 eV which were attributed to the presence of respectively.[6–8] Similarly, the N1s spectrum (Figure 2g) showed
pyrrolic-N and graphitic-N, respectively.[7,8] The XPS data proves three main peaks at 397.7, 399.2, and 400.9 eV due to the
that U-CQDs have numerous surface functional groups and presence of pyridinic-N, pyrrolic-N and graphitic-N,
abundant heteroatoms in the structure. Lastly, we collected respectively.[7,8] The XPS data for W-CQDs shows the numerous
some TEM images (Figure 1h) to determine the size and surface functional groups and abundant heteroatoms in the
morphology of the CQDs. It was observed the U-CQDs are structure. TEM analysis (Figure 2h) depicted W-CQDs in mostly

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Figure 2. Characterization of W-CQDs. Normalized PL spectra under different excitation wavelengths (a), UV-vis absorption spectra and optical images of CQD
suspension under daylight and 365 nm UV-lamp (b), FT-IR spectra (c), XPS survey with elemental composition data (d), high-resolution C1s (e), O1s (f), N1s (g),
representative TEM image with relevant particle size distribution graph (h).

spherical structure with particle sizes ranging from 5 to 13 nm constant reaction temperature (180 °C) and precursor ratio
with an average size of 7.4 nm. (1 : 1). UV-vis absorption and PL spectra were collected and
summarized in Figure 3. For all synthesis times, we observed a
shoulder ranging from 291 to 327 nm, as the synthesis time
Characterization of U/W-CQDs increased from 3 to 24 h (Figure 3b,d,f). This band was ascribed
to the n–π* transition due to the C=O bonds or other surface
The previous analyses on U-CQDs and W-QDs demonstrated groups in the U/W-CQDs.[59,60] Also, another transition band was
that the type of the precursor material has a dominant effect on observed in the range of 267–269 nm for the cases of 12 and
the chemical content and resultant PL activity of CQDs. In this 24 h of synthesis times (Figure 3d,f). This band is attributed to
part, we furtherly investigated the contribution of experimental the π–π* transition band of aromatic C C bonds. However, at
parameters such as synthesis time, temperature, and volumetric low synthesis time (3 h, Figure 3b), no π–π* transition band was
ratio of precursors (urine and whey) on the PL activity. For the detected indicating the low carbonization of urine and whey as
evaluation, UV-vis and PL spectra were collected and analyzed precursors.[7,8,56] Also, at prolonged times of fabrication, we
in detail. detected the emergence of some weak peaks in the visible
range at around 600 nm demonstrating the presence of the
functional-surface states.[23,61] The PL nature of U/W-CQDs was
The Effect of Synthesis Time on U/W-CQDs investigated for different excitation wavelengths in the range of
280–640 nm (Figure 3a,c,e). For all synthesis times, a strong PL
Firstly, we performed some experiments to determine the effect activity with an excitation-dependent property was observed.
of synthesis time on the optical nature of U/W-CQDs. For this, PL emission peak maxima ranged from 439 to 660 nm, from
we manipulated the synthesis time in the range of 3–24 h at 426 to 662 nm, and from 457 to 640 nm for the cases of 3, 12,

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Figure 3. The effect of synthesis time (3 h (a–b), 12 h (c–d), and 24 h (e–f)) on PL and UV-vis absorption spectra of U/W-CQDs. The PL spectra were collected
at different excitation wavelengths. The synthesis temperature and the volumetric ratio of initiators were 180 °C and, 1 : 1, respectively.

and 24 h of synthesis times, respectively. It seems that surface respectively. As mentioned earlier, surface groups with probable
groups with probable heteroatom content could create trap heteroatom composition of U/W-CQDs could form trap states
states with different energy levels and lead to an excitation- with different energy levels and result in an excitation-depend-
dependent nature at all reaction times.[7,8,56] For the higher ent property for the given temperature range.[7,8,56] For the
synthesis time (24 h, Figure 3e), relatively higher PL activity (2 higher synthesis temperature (180 and 220 °C, Figure 4c,e),
times) was noticed in comparison to lower synthesis times relatively higher PL activity (4 and 6 times, respectively) was
(Figure 3a,c). This issue is in line with the relevant UV-vis spectra observed in comparison to the lower synthesis temperature
(Figure 3b,d,f) and due to the lower carbonization at a lower (140 °C, Figure 4a). This phenomenon is in accordance with the
reaction time. Briefly, the prolonged synthesis times resulted in relevant UV-vis spectra (Figure 4b,d,f) and may be attributed to
higher PL activity with an excitation-dependent nature as a the lower carbonization at a lower reaction temperature. In
consequence of the presence of various transition bands and summary, the higher synthesis temperature led to higher PL
functional surface groups with probable heteroatom content. activity with an excitation-dependent behavior due to the
presence of various transition bands and functional surface
groups with probable heteroatom content.
The Effect of Synthesis Temperature on U/W-CQDs

Secondly, we tuned the synthesis temperature ranging from The Effect of the Ratio of Precursors on U/W-CQDs
140 to 220 °C at constant synthesis time (12 h) and precursor
ratio (1 : 1) and collected UV-vis and PL spectra of U/W-CQDs Lastly, we performed the same strategy to evaluate the effect of
which were provided in Figure 4. From UV-vis absorption the ratio of the precursors on the optical properties of U/W-
spectra (Figure 4b,d,f), we detected similar bands for each CQDs (Figure 5). For this, the ratio of urine to whey (1 : 1, 1 : 3,
reaction temperature case. The shoulder at around 270 nm 3 : 1 in volume) was manipulated at fixed synthesis temperature
indicated the π–π* transition band due to the aromatic C C and time. UV-vis absorption spectra (Figure 5b,d,f) showed
bonds. Also, the shoulder at around 330 nm was attributed to similar bands for each of the ratios of the precursors case. We
the n–π* transition band showing the presence of C=O bonds observed a shoulder at around 270 nm for the π–π* transition
or other surface groups.[59,60] At higher synthesis temperatures band assigned the aromatic C C bonds. In addition, the n–π*
(180 and 220 °C), we detected some weak peaks in the visible transition band indicating the presence of C=O bonds or other
range at around 600 nm (Figure 4d,f) showing the emergence surface groups was noticed at around 325 nm.[59,60] For the case
of the functional-surface states.[23,61] In the PL spectra (Fig- of a 1 : 1 ratio of precursors (Figure 5b), a weak peak in the
ure 4a,c,e), as the excitation wavelength was increased from visible range at around 600 nm (Figure 5b) shows the emer-
280 to 600 nm, PL emission peak maxima ranged from 449 to gence of the functional surface states.[23,61] PL spectra (Fig-
632 nm, from 443 to 641 nm, and from 433 to 621 nm for the ure 5a,c,e) depicted that the increase in the excitation wave-
cases of 140, 180 and 220 °C of synthesis temperature, length from 280 to 600 nm tuned the PL emission peak maxima

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Figure 4. The effect of synthesis temperature (140 °C (a–b), 180 °C (c–d), and 220 °C (e–f)) on PL and UV-vis absorption spectra of U/W-CQDs. The PL spectra
were collected at different excitation wavelengths. The synthesis time and the volumetric ratio of initiators were 12 h and, 1 : 1, respectively.

Figure 5. The effect of the ratio of precursor (1 : 1 (a–b), 1 : 3 (c–d), and 3 : 1 (e–f)) on PL and UV-vis absorption spectra of U/W-CQDs. The PL spectra were
collected at different excitation wavelengths. The synthesis time and temperature were 12 h and, 180 °C, respectively.

in the range of 425–623 nm, 445–625 nm, and 425–623 nm for Further Characterization of U/W-CQDs
the cases of 1 : 1, 1 : 3, and 3 : 1 of the ratio of precursors,
respectively. For all cases, we could attain high PL activity In this part, we utilized various analysis methods (Figure 6) to
indicating the high potential of both precursors for the green evaluate U/W-CQDs in detail. For this, U/W-CQDs were
synthesis of CQDs. fabricated at 18 h, 180 °C, and 1 : 1 of the ratio of precursors.
After determination of synthesis time, temperature, and the The suspension of CQDs showed a dark brown color in daylight
ratio of precursors on optical properties, we performed detailed and a bright blue-white emission under a 365 nm UV lamp (see
analyses for U/W-CQDs fabricated under optimal conditions. insets of Figure 6a). As the excitation wavelength increased

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Figure 6. Further characterization of U/W-CQDs. Normalized PL spectra under different excitation wavelengths and optical images of CQD suspension under
daylight and 365 nm UV-lamp (a), Raman spectrum (b), FT-IR spectrum (c), XPS survey with elemental composition data (d), high-resolution C1s (e), O1s (f),
N1s (g), representative TEM image with relevant particle size distribution graph (h), zeta-potentials (i) the change in PL intensity (ex:360 nm) for storage under
daylight (j) PL spectra at different pH values (k) and normalized PL intensity (l).

from 280 to 600 nm, the PL maximum was redshifted from 421 band (ID/IG) was calculated to be 1.25. This value indicated the
to 620 nm (Figure 6a). The bright and strong PL emission high content of defects or disorders in comparison to
created a robust QY of 39 %. This value is highly acceptable for graphite.[63–65] FT-IR spectrum (Figure 6c) demonstrated the
various applications and compared with some materials in the presence of abundant functional groups in the structure of U/
literature provided in Table S1. We also collected the Raman W-CQDs. We noticed significant bands at 3171, 3042, 2963,
spectrum (Figure 6b) of U/W-CQDs to investigate the carbon 1585, 1411, 1119, and 1055 cm 1 which were assigned to υ
characteristics of the material. Raman spectrum showed two ( OH), υ ( NH), υ (C H), δ (C=C)/ υ (C=O), υ (C H), υ (C N), and
broad peaks at 1307 and 1594 cm 1, indicating the D-band and υ (C O), respectively.[46,47,53] The hydrophilic groups including υ
G-band, respectively.[62] While the D band shows structural ( OH), υ ( NH), υ (C=O) proved excellent solubility and high
defects or disorders due to the sp3 defects, the G band stability in water.[7,8,48,54–56] However, the bands for δ (C=C), υ
represents the vibration of sp2 carbon rings in the structure. By (C H), and υ (C N) groups show the presence of polyaromatic
utilizing this data, the ratio of intensity of the D band to the G characteristics of the CQDs’ core.[57,58] The wide-scanning XPS

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spectra of U/W-CQDs (Figure 6d) indicated the presence of The Storage and pH Stability of U/W-CQDs
mainly C, N, and O in the structure at 70 %, 10 %, and 17 %,
respectively. Also, we noticed the presence of some trace The long-term storage and pH stability of CQDs is a major
elements including Na, and S showing the complex content of concern that governs their employment in practical applica-
both urine and whey as precursors. The C1s high-resolution XPS tions. To evaluate the storage stability of as-prepared U/W-
spectrum (Figure 6e) exhibited three peaks at 283.7, 287.1, and CQDs in daylight, we collected PL (Figure S1) and UV-vis
291.9 eV assigned to C C/C=C, C O and C=O bonds, absorption spectra (Figure S2) at different time intervals. Also,
respectively.[50–52] O1s high-resolution spectrum (Figure 6f) was the change in PL intensity over time is shown in Figure 6j. This
convoluted into two peaks at 528.7 and 530.8 eV associated data indicated that U/W-CQDs preserved 65 % of their original
with the C=O and C O bonds, respectively.[6–8] N1s spectrum PL activity after they were stored in daylight even for 60 days
(Figure 6g) showed three main peaks at 397.2, 398.9, and (see Figure 6j and Figure S1). Considering the storage duration,
400.6 eV due to the presence of pyridinic-N, pyrrolic-N, and this PL activity is still satisfying. Also, no significant change was
graphitic-N, respectively.[7,8] The XPS data for U/W-CQDs shows observed in their excitation-dependent behavior (Figure S1).
the numerous surface functional groups and abundant heter- However, after storage in daylight for 60 days, UV-vis absorption
oatoms in the structure. This data is a confirmation of the UV- spectra (Figure S2) showed a slight decrease in the π-π* and n-
vis absorption, PL, and FT-IR spectra concerning the presence of π* band transition compared with freshly prepared samples.
functional-surface states and obtained high PL activity with an This issue was due to probable structural changes in the
excitation-dependent nature. It seems that heteroatoms such as material during the storage.
N, Na, S, etc., in the structure of U/W-CQDs may create new In addition to storage stability, since many practical
energy levels to form trap states and resultant excitation- applications of CQDs require usage in various pH environments,
dependent PL characteristics.[6,27,28] TEM images (Figure 6h) we tested their PL stability in a wide range of pH (Figure 6k,l). It
showed that the U/W-CQDs are mostly in spherical morphology was observed that PL yield is highly dependent on pH in the
in the range of 5–17 nm with an average size of 8.6 nm. In range of 3–10. The maximum yield was attained at pH 3. The
summary, all presented data proved that U/W-CQDs with increase in pH led to a dramatic decrease in intensity (Figure 6l)
graphite core, high heteroatom content, and a high degree of but almost no change (~5 nm) in the location of emission
defects and disorders were sufficiently fabricated through the maxima (Figure 6k). However, for the wide range of pH, the
green hydrothermal synthesis method employing urine and emission was satisfying to determine the change by the naked
whey as precursors. Lastly, we collected the Zeta potential of U- eye. The decrease in emission intensity with increasing pH can
CQDs, W-CQDs, and U/W-CQDs which were found to be 51.4, be attributed to the deprotonation of surface groups and
4.3, and 21.6 mV, respectively (Figure 6i). The potential values resultant aggregation.[8] This phenomenon shows the potential
for U-CQDs and U/W-CQDs indicate their high colloidal stability of U/W-CQDs in pH sensor applications including pH monitor-
which has an important role in the storage and various ing in colloid systems and inside the cell. However, further
chemical and biological applications. The difference in Zeta structural and spectroscopic analyses are required which will be
potential values can be attributed to different compositions of the topic of our perspective work.
precursor.
Lastly, for the comparison, the main characteristics of U-
CQDs, W-CQDs, and U/W-CQDs were summarized in Table 1. Cytotoxicity Evaluation of CQDs
Considering whole characterization data, various parameters
including the type and content of dopped elements (N, S, P, The cytotoxicity of CQDs governs their employment in various
etc.) determine the structure and resultant optical nature (QY, biomedical applications. Hence, we performed an MTT test to
excitation-dependent, etc.) of the material. These results were evaluate the cytotoxicity of U-CQDs, W-CQDs, and U/W-CQDs
governed by the type of the precursor and synthesis conditions. on MRC5 and A549 cell lines. For this, cell lines were exposed to
CQDs at different concentrations in the range of 31.2–
1,000 ppm for a 24-hour incubation. The cell viability results are
provided in Figure 7. For all materials and cell lines, CQDs

Table 1. The comparison of CQDs fabricated from different precursors.

U-CQDs W-CQDs U/W-CQDs

Precursors Urine Sour Whey Urine and Sour Whey

Quantum Yield 48 % 28 % 39 %
(QY)

Elemental Con- C(66 %), O(22 %), N(3 %), Na(1 %), Si(4 %), P(1 %), C(66 %), O(21 %), N(5 %), Na(2 %), Ca(1 %), C(70 %), O(17 %), N(10 %), Na(2 %),
tent* S(1 %) K(1 %) S(1 %)

*The data was obtained from XPS data.

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Figure 7. Cell viability of U/W-CQDs (a), W-CQDs (b), and U-CQDs (c) on MRC5 and A549 cell lines after 24 h incubation at different concentrations.

Experimental Section
showed similar cytotoxic effects in a dose-dependent manner.
For MRC-5 cell lines, IC50 values were found to be > 1,000, 755, Hydrothermal Production and Characterization of CQDs
and 780 ppm for U/W-CQDs, W-CQDs and U-CQDs, respectively.
For the synthesis of CQDs, we employed a simple yet effective
For A549 cell lines, IC50 values were detected to be 625, 181, green hydrothermal procedure that utilized urine (U-CQDs), whey
and 384 ppm for U/W-CQDs, W-CQDs, and U-CQDs, respectively. (W-CQDs), and their mixture (U/W-CQDs) as initiators. For this, urine
These results were very similar to the other carbon-based was obtained from a healthy female individual (informed consent
fluorescent materials reported in the literature (see Table S2). was obtained from all participants) and whey was purchased from a
We envision that various parameters including the type and local store in Erzurum-Türkiye. To eliminate the suspending
particles, both urine and whey were passed through filter paper
content of dopped elements as well as surface functional
(MF-Millipore® Membrane Filter, 0.22 μm pore size). Then, 10 mL of
groups have a significant effect on the resultant cytotoxicity. urine or whey or their mixture was transferred to a Teflon-lined
The cytotoxicity test shows the low toxicity and satisfying stainless steel reactor (40 mL) and kept in the oven for different
biocompatibility of CQDs in both cell lines in a wide range of reaction times and temperatures. For the synthesis procedure of U/
concentrations. Lastly, the higher sensitivity of the A549 cell line W-CQDs, some major synthesis parameters such as the reaction
(lung cancer) against CQDs in comparison to the MRC-5 cell line time (3–24 h), temperature (140–220 °C), and the ratio of precursors
(1 : 1, 1 : 3, 3 : 1 ratio of urine to whey in volume) were manipulated
(healthy lung) indicates the potential of these materials in
to determine the optimum conditions with the highest PL activity.
cancer treatment. At the end of the period, the reactor was removed from the oven
and kept at ambient conditions to cool to room temperature. For
the purification, the suspension was centrifuged at 13,000 rpm for
Conclusions 1 h to remove aggregates. Then, unreacted impurities were
removed with the dialysis membrane (MW cut-off 1000 Da) for
24 h.
In conclusion, we systematically investigated the fabrication of
CQDs from urine or whey or their mixture via a simple yet The fabricated CQDs were characterized via various techniques
flexible green hydrothermal synthesis. All synthesized nano- such as fluorescence spectroscopy (Agilent Cary Eclipse), UV-vis
absorption spectroscopy (Shimadzu UV-1800), Raman spectroscopy
systems exhibited bright blue-white emission under the UV
(WITech alpha 300R), Zetasizer (Malvern Hydro 2000MU), Fourier
light with an excitation-dependent nature. Various heteroatoms transform infrared spectroscopy (FT-IR, Bruker VERTEX 70v) and X-
such as N, P, S, etc. doped in the structures had a dominant ray photoelectron spectroscopy (XPS, Specs-Flex XPS). The pH
effect on the PL characteristics of the material. For the cases of stability of CQDs was tested through fluorescence and UV-vis
U/W-CQDs, W-CQDs, and U-CQDs, QY values of 39 %, 28 %, and spectra using 1 M of HCl (37 %, Sigma-Aldrich) or 1 M of NaOH
48 % were obtained, respectively. Cytotoxicity tests proved the (98 %, Sigma-Aldrich).
low toxicity and satisfying biocompatibility of CQDs in MRC5
and A549 cell lines in a wide range of concentrations. Also, U/ Cytotoxicity Evaluation of CQDs
W-CQDs showed high storage stability over long durations and
For the cytotoxicity evaluation, healthy lung fibroblast (MRC5) and
pH-dependent PL activity indicating their potential in various
lung cancer (A549) cell lines were employed for the comparison. In
biomedical applications. We strongly believe that this report the first stage, similar to our earlier studies, both cell lines were
confirms the role of heteroatom doping in the CQDs and its grown to obtain satisfying cell confluency. Then, 1 × 105 cells were
effect on PL activity as well as their potential application. seeded in 96-well plates and treated with as-prepared CQDs in
different concentrations in the range of 1,000–31.2 ppm. The
untreated and an H2O2 solution (250 μM) treated cells were
considered as negative and positive control, respectively. After a
24-h of exposure, the cytotoxicity was determined through an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test

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which measures the mitochondrial activity by collecting absorbance [27] H. Wang, P. Sun, S. Cong, J. Wu, L. Gao, Y. Wang, X. Dai, Q. Yi, G. Zou,
value (Biotek Epoch microplate reader) at 570 nm. Nanoscale Res. Lett. 2016, 11, 1–6.
[28] J. Xu, K. Cui, T. Gong, J. Zhang, Z. Zhai, L. Hou, F. u. Zaman, C. Yuan,
Nanomater. 2022, 12, 312.
[29] C. Cheng, Y. Shi, M. Li, M. Xing, Q. Wu, Mater. Sci. Eng. C. 2017, 79, 473–
480.
Acknowledgements [30] A. Prasannan, T. Imae, Ind. Eng. Chem. Res. 2013, 52, 15673–15678.
[31] J. Zhou, Z. Sheng, H. Han, M. Zou, C. Li, Mater. Lett. 2012, 66, 222–224.
The authors also kindly acknowledge the East Anatolia High [32] S. Sahu, B. Behera, T. K. Maiti, S. Mohapatra, Chem. Commun. 2012, 48,
8835–8837.
Technology Application and Research Center (DAYTAM) at
[33] S. Ghosh, K. Ghosal, S. A. Mohammad, K. Sarkar, Chem. Eng. J. 2019, 373,
Ataturk University for providing laboratory spaces and charac- 468–484.
terization devices. [34] N. Sarigul, F. Korkmaz, İ. Kurultak, Sci. Rep. 2019, 9, 20159.
[35] J. B. Królczyk, T. Dawidziuk, E. Janiszewska-Turak, B. Solowiej, Polish J.
Food Nutr. Sci. 2016, 66, 157.
[36] X.-D. Zhang, J. Li, J.-N. Niu, X.-P. Bao, H.-D. Zhao, M. Tan, Methods 2019,
Conflict of Interests 168, 84–93.
[37] V. Lacivita, F. Tarantino, R. Molaei, M. Moradi, A. Conte, M. A. Del Nobile,
Food Res. Int. 2023, 113159.
The authors declare no competing financial interest. [38] Z. Gan, H. Xu, Y. Hao, Nanoscale. 2016, 8, 7794–7807.
[39] Y. Deng, X. Chen, F. Wang, X. Zhang, D. Zhao, D. Shen, Nanoscale. 2014,
6, 10388–10393.
Keywords: Carbon quantum dots · Heteroatom doping · Green [40] Z. Gan, S. Xiong, X. Wu, T. Xu, X. Zhu, X. Gan, J. Guo, J. Shen, L. Sun, P. K.
synthesis · Urine · and Whey Chu, Adv. Opt. Mater. 2013, 1, 926–932.
[41] J. Lee, K. Kim, W. I. Park, B.-H. Kim, J. H. Park, T.-H. Kim, S. Bong, C.-H.
Kim, G. Chae, M. Jun, Nano letters. 2012, 12, 6078–6083.
[42] P. Wu, W. Li, Q. Wu, Y. Liu, S. Liu, RSC Adv. 2017, 7, 44144–44153.
[1] S. Ding, Y. Gao, B. Ni, X. Yang, Inorg. Chem. Commun. 2021, 130, 108636. [43] Y. Jiang, Q. Han, C. Jin, J. Zhang, B. Wang, Mater. Lett. 2015, 141, 366–
[2] L. J. Desmond, A. N. Phan, P. Gentile, Environ. Sci.: Nano 2021, 8, 848– 368.
862. [44] R. Liu, H. Li, W. Kong, J. Liu, Y. Liu, C. Tong, X. Zhang, Z. Kang, Mater. Res.
[3] X. Xu, R. Ray, Y. Gu, H. J. Ploehn, L. Gearheart, K. Raker, W. A. Scrivens J. Bull. 2013, 48, 2529–2534.
Am. Chem. Soc. 2004, 126, 12736–12737. [45] Y. Zhuo, H. Miao, D. Zhong, S. Zhu, X. Yang, Mater. Lett. 2015, 139, 197–
[4] L. Hu, C. Zhang, G. Zeng, G. Chen, J. Wan, Z. Guo, H. Wu, Z. Yu, Y. Zhou, 200.
J. Liu, RSC Adv. 2016, 6, 78595–78610. [46] P. Keerthana, A. R. Cherian, U. Sirimahachai, D. A. Thadathil, A. Varghese,
[5] D. L. Zhao, T.-S. Chung, Water Res. 2018, 147, 43–49. G. Hegde, J. Environ. Chem. Eng. 2022, 10, 107209.
[6] S. Aydin, O. Ustun, A. Ghosigharehaghaji, T. Tavaci, A. Yilmaz, M. Yilmaz, [47] L. Yue, H. Li, Q. Sun, J. Zhang, X. Luo, F. Wu, X. Zhu, ACS applied nano
Coatings 2022, 12. materials. 2020, 3, 869–876.
[7] S. N. Karadag, O. Ustun, A. Yilmaz, M. Yilmaz, Chem. Phys. 2022, 562, [48] L. Pan, S. Sun, L. Zhang, K. Jiang, H. Lin, Nanoscale. 2016, 8, 17350–
111678 17356.
[8] O. Ustun, S. N. Karadag, H. Mazlumoglu, A. Yilmaz, M. Yilmaz, Coatings [49] H. Ding, J. S. Wei, N. Zhong, Q. Y. Gao, H. M. Xiong, Langmuir. 2017, 33,
2023, 13, 456–456. 12635–12642.
[9] Y. Guo, Z. Wang, H. Shao, X. Jiang, Carbon 2013, 52, 583–589. [50] W. K. Kipnusu, C. Doñate-Buendía, M. Fernández-Alonso, J. Lancis, G.
[10] S. Chandra, V. K. Singh, P. K. Yadav, D. Bano, V. Kumar, V. K. Pandey, M. Mínguez-Vega, Part. Part. Syst. Char. 2020, 37, 2000093.
Talat, S. H. Hasan, Anal. Chim. Acta 2019, 1054, 145–156. [51] P. Shen, Y. Xia, Anal. Chem. 2014, 86, 5323–5329.
[11] H. Li, Y. Zhang, L. Wang, J. Tian, X. Sun, Chem. Commun. 2011, 47, 961– [52] H. Ding, S. Yu, J. Wei, H. Xiong, ACS Nano 2016, 10, 484–491
963. [53] N. A. Alarfaj, M. F. El-Tohamy, H. F. Oraby, Int. J. Mol. Sci. 2018, 19, 1162.
[12] T. Zhou, J. Ashley, X. Feng, Y. Sun, Talanta 2018, 190, 443–449. [54] H. Ding, J.-S. Wei, N. Zhong, Q.-Y. Gao, H.-M. Xiong, Langmuir 2017, 33,
[13] H. Liu, Q. Wang, G. Shen, C. Zhang, C. Li, W. Ji, C. Wang, D. Cui, 12635–12642.
Nanoscale Res. Lett. 2014, 9, 1–11. [55] H. Ding, J.-S. Wei, H.-M. Xiong, Nanoscale 2014, 6, 13817–13823.
[14] D. Bouzas-Ramos, J. Cigales Canga, J. C. Mayo, R. M. Sainz, J. Ruiz Enci- [56] S. Aydin, O. Ustun, A. Ghosigharehaghaji, T. Tavaci, A. Yilmaz, M. Yilmaz,
nar, J. M. Costa-Fernandez, Adv. Funct. Mater. 2019, 29, 1903884. Coatings 2022, 12, 1311–1311.
[15] A. Abbas, L. T. Mariana, A. N. Phan Carbon. 2018, 140, 77–99. [57] J. Zhou, Y. Yang, C.-y. Zhang, Chem. Commun. 2013, 49, 8605–8607.
[16] Q. Chen, L. Chen, J. Qi, Y. Tong, Y. Lv, C. Xu, J. Ni, W. Liu, Chin. Chem. [58] B. B. Chen, R. S. Li, M. L. Liu, H. Z. Zhang, C. Z. Huang, Chem. Commun.
Lett. 2019, 30, 1214–1218. 2017, 53, 4958–4961.
[17] M. Ji, Z. Zhang, J. Xia, J. Di, Y. Liu, R. Chen, S. Yin, S. Zhang, H. Li, Chin. [59] W. Wang, Y.-C. Lu, H. Huang, J.-J. Feng, J.-R. Chen, A.-J. Wang, Analyst.
Chem. Lett. 2018, 29, 805–810. 2014, 139, 1692–1696.
[18] B. Zhang, Y. Liu, M. Ren, W. Li, X. Zhang, R. Vajtai, P. M. Ajayan, J. M. [60] S. Zhu, Y. Song, X. Zhao, J. Shao, J. Zhang, B. Yang, Nano Res. 2015, 8,
Tour, L. Wang, ChemSusChem. 2019, 12, 4202–4210. 355–381.
[19] Q. Wang, H. Zheng, Y. Long, L. Zhang, M. Gao, W. Bai, Carbon. 2011, 49, [61] S. Kim, B.-K. Yoo, Y. Choi, B.-S. Kim, O.-H. Kwon, Phys. Chem. Chem. Phys.
3134–3140. 2018, 20, 11673–11681.
[20] J. Zhou, C. Booker, R. Li, X. Zhou, T.-K. Sham, X. Sun, Z. Ding, J. Amer. [62] T. Zhang, Y. Wen, Z. Pan, Y. Kuwahara, K. Mori, H. Yamashita, Y. Zhao, X.
Chem. Soc. 2007, 129, 744–745. Qian, Environ. Sci. Technol. 2022.
[21] S. Y. Lim, W. Shen, Z. Gao, Chem. Soc. Rev. 2015, 44, 362–381. [63] R. Atchudan, T. N. J. I. Edison, S. Perumal, R. Vinodh, Y. R. Lee, Mater.
[22] X. Wang, Y. Feng, P. Dong, J. Huang, Front. Chem. 2019, 7, 671. Lett. 2020, 261, 127153.
[23] Y. Wang, A. Hu, J. Mater. Chem. C. 2014, 2, 6921–6939. [64] K. Nekoueian, M. Amiri, M. Sillanpää, F. Marken, R. Boukherroub, S.
[24] R. Liu, D. Wu, S. Liu, K. Koynov, W. Knoll, Q. Li, Angew. Chem. Int. Ed. Szunerits, Chem. Soc. Rev. 2019, 48, 4281–4316.
2009, 48, 4598–4601. [65] L. Zhu, D. Shen, Q. Liu, C. Wu, S. Gu, Appl. Surf. Sci. 2021, 565, 150526.
[25] H. Li, X. He, Y. Liu, H. Huang, S. Lian, S.-T. Lee, Z. Kang, Carbon. 2011, 49,
605–609.
[26] W. Meng, X. Bai, B. Wang, Z. Liu, S. Lu, B. Yang, Energy Environ. Mater.
2019, 2, 172–192. Manuscript received: December 7, 2023

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