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A simple and green extraction of carbon dots from sugar beet molasses:
Biosensor applications

Article  in  Zuckerindustrie. Sugar industry · September 2016


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Saliha Dinç
Selcuk University


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560 Technology/Technologie

Saliha Dinç

A simple and green extraction of carbon dots

from sugar beet molasses: Biosensor applications

Einfache und umweltverträgliche Isolierung von Kohlenstoff-Nanopartikeln

aus Zuckerrübenmelasse: Anwendung in der Biosensorik

Carbon dots emerged recently as a luminescent nanopar- Kohlenstoff-Nanopartikel (C-NP) haben in ihrer Eigenschaft
ticles have received considerable attention. Carbon dots, als leuchtende Nanopartikel in letzter Zeit beträchtliche
which can be synthesized by different methods, have many Aufmerksamkeit geweckt. C-NP, die durch verschiedene Ver-
application areas such as biosensor, bioimaging etc. fahren synthetisiert werden können, finden Anwendung in
In this study, carbon dots were extracted from a sugar Bereichen wie Bioimaging, Biosensorik usw.
beet molasses without using any other synthesis methods. C-NP wurden aus Zuckerrübenmelasse ohne Verwendung
Extracted carbon dots gave strong blue fluorescence under anderer Syntheseverfahren isoliert. Die isolierten C-NP
UV light. The characterization of carbon dots was performed fluoreszierten unter UV-Licht kräftig blau. Die Charakte-
using Fourier-transform infrared spectroscopy (FTIR), scan- risierung der C-NP erfolgte unter Verwendung von Fou-
ning electron microscopy (SEM) and atomic force micros- rier-Transformations-Infrarotspektroskopie (FTIR), Raster-
copy (AFM). Optic properties were determined by UV-Vis elektronenmikroskopie (SEM) und Rasterkraftmikroskopie
and fluorescence spectroscopy. (AFM). Optische Eigenschaften wurden durch UV/Vis- und
Carbon dots were used as tool of biosensor in detection of Fluoreszenzspektroskopie bestimmt.
riboflavin and tetracycline analyses. In riboflavin detection, Die C-NP wurden für die biosensorische Bestimmung von
fluorescence resonance energy (FRET) from carbon dots Riboflavin und Tetracyclin genutzt. Bei der Riboflavin-
transferred to riboflavin and linear correlation was obtained bestimmung erfolgte ein Fluoreszenz-Resonanzenergie-
between FRET efficiency and riboflavin concentration transfer (FRET) von C-NP auf Riboflavin und es wurde eine
(R2 = 0.9904). Tetracycline decreased the fluorescence of lineare Korrelation zwischen FRET-Effizienz und Riboflavin-
carbon dots. There was a linear correlation between fluores- konzentration festgestellt (R 2 = 0,9904). Tetracyclin ver-
cence decrease and tetracycline concentration (R2 = 0.9952). ringerte die Fluoreszenz von C-NP. Es bestand eine lineare
Extracted carbon dots can successfully be used in the deter- Korrelation zwischen Fluoreszenzabnahme und Tetracyclin-
mination of riboflavin and tetracycline. konzentration (R2 = 0,9952).
Die isolierten C-NP können erfolgreich zur Bestimmung von
Riboflavin und Tetracyclin verwendet werden.

Key words: carbon dots, sugar beet molasses, fluorescence Schlagwörter: Kohlenstoff-Nanopartikel, Zuckerrübenme-
resonance energy transfer, riboflavin, tetracyline lasse, Förster-Resonanzenergietransfer, Riboflavin, Tetracyclin

1 Introduction and Baker, 2010; Wang et al., 2014). Recently, there has been
a increasing application of carbon dots in biosensor area such
Emerging as a new type of carbon nanomaterials, carbon dots as metal ions (She et al., 2016; Zhang and Chen, 2014; Sachdev
(CDs) are luminescent nanoparticles in nanoscale with dimen- and Gopinath, 2015), phytic acid (Gao et al., 2015), glucose (Shi
sion less than 10 nm. One of the most important parameters et al., 2011), bacteria, (Nandi et al., 2015) ascorbic acid (Liu et
of carbon dots is having excitation and emission characteristics al., 2016), and thiamine (Tan et al., 2015). Carbon dots can be
at different wavelengths depending on their size (Baker and produced via different routes including electrochemical syn-
Baker, 2010). In addition, carbon dots have superior features thesis, combustion, thermal, hydrothermal, acidic oxidation
such as good water solubility, biocompatibility, low toxicity and microwave. (Li et al, 2012; Himaja et al., 2014). However,
and easy synthesis compared to CdSe (cadmium selenide) and carbon dots are extracted from a variety food sources such as
CdTe (cadmium telluride) quantum dots (Yu et al., 2013; Zhou bread, caramel, Nescafe (Jiang et al., 2014; Sk et al., 2012).
et al., 2012; Zhao et al., 2015). Furthermore, carbon dots have Sugar beet molasses is a viscous, dark by product of refining
numerous applications in numerous fields such as bioimaging, at sugar beet factories containing sugars (approximately 50%),
biosensing, photoreduction of metal, photocatalysis, drug amino acids and minerals. Containing most of the nutrients
delivery and therapy (Zhao et al., 2015; Luo et al., 2013; Baker for yeast growth, it is mainly used in the production of alcohol

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Technology/Technologie 561

and yeast as well as animal feed. Its low cost and accessibility EDX, Bruker) was used for the elemental determination of car-
are another advantages of molasses for these industries. bon dots. EDX was performed using EDAX Genesis (123 ev)
With their important functions in the body, riboflavin (vita- at accelerating voltage of 20 kV and a working distance of 10
min B2) should be taken with foods. Riboflavin deficiency mm. The morphology of molasses carbon dots was analyzed by
may cause wounds in the skin, nervous system disorders, atomic force microscopy (AFM NTEGRA Solaris).
and anemia (Samur, 2008). Microbiological methods, che- Sample preparation: CDs were dispersed in water and the
miluminescence, atomic absorption spectrometry, capillary concentration of carbon dots in fluorescence cuvette was set
electrophoresis and high performance liquid chromatography to be 334 µL/mL. Riboflavin and tetracycline solutions were
(HPLC) are the conventional methods used in the detection prepared in distilled water. Firstly, carbon dot solution added
of riboflavin (Wang et al., 2015). However, these methods are to flask and then various concentrations of analytes were
time-consuming, tedious and require high cost investments. added and mixed. The concentration of analytes varied from
Therefore, the development of simple, rapid, reliable and inex- 13 µmol/L to 140 µmol/L.
pensive alternative methods is highly desirable.
Tetracycline is one of the broad spectrum antibiotic effec-
tive on most of the microorganisms including Rickettsia and 3 Results and discussion
viruses used to treat human and animal infections (Feng et 3.1 Characterization of carbon dots
al., 2015; Cinquina et al., 2003). However, monitoring of tet-
racycline residue in meat, fish, milk products and honey is an Carbon dots extracted from molasses were analyzed by UV-
important issue (Andersen et al., 2005; Schenck and Callery, Vis and fluorescence spectroscopy (Fig. 1). According to UV-
1998). Chromatographic methods, chemiluminescence, capil- Vis spectrum, carbon dots exhibit maximum absorbance at
lary electrophoresis, and dipstick colorimetric methods are 300 nm. Carbon dots showed strong blue emission under
used in tetracycline analyses. (Andersen et al., 2005; Schenck 365 nm UV lamps (Fig. 1b). Emission spectra were taken with
and Callery, 1998; Yang et al., 2014). However, since these different excitation wavelengths. The strong fluorescence emis-
methods require sophisticated instrumentation and/or com- sion band at 465 nm was recorded at excitation wavelength of
plicated sample preparation, it is necessary to develop simple, 370 nm as shown Figure 2. Excitation wavelength of 370 nm
fast, reliable and economical methods. was used during the riboflavin and tetracycline analyses.
Herein, strong luminescent carbon dots were extracted from Identification of functional groups was studied using FTIR
sugar beet molasses without using any chemical/thermal pro- spectroscopy ranged between 400–4000 cm–1 (Fig. 3). Molas-
cesses for the first time. Subsequently, extracted carbon dots ses carbon dots exhibited absorption peaks due to the presence
were applied for the fluorescence detection of riboflavin (vita- of O-H and C-H, C=C, C-C, C-O stretching’s at 3270, 1641,
min B2) and tetracycline analytes. 1496, 1453, 1364, 1312, 1253, 1192, 1149 and 1023 cm–1.
FTIR data suggested that carbon dots with above-mentioned
hydrophilic functional groups impart them water soluble char-
2 Materials and methods acteristics (Gao et al., 2015; De and Karak, 2013).
Chemical composition of carbon dots were analyses by
Materials: Sugar beet molasses was supplied from Konya SEM-EDX. The elemental composition of dots was 34.2% C,
sugar factory. Riboflavin and tetracyline are supplied from 54.8% O, 4.5% K, 1.5% Na, 2.6% Ca, 0.9% Cl, 1.0% S, 1.0% P.
Ehrenstorfer and Sigma, respectively. The EDX results revealed that carbon dots were rich in carbon
Carbon dot extraction: Approximately 5 g molasses was dis- and oxygen. AFM image of molasses carbon dot is shown
solved in 10 mL ultra-filtered water and mixed on a magnetic in Figure 4. The average particle size of carbon dots, mea-
stirrer until homogenous solution was
obtained. After 5 min centrifugation at
5000 min–1, supernatant was taken and
diluted with water in ratio of 1:25. This
solution was kept in ultrasonic bath for
15 min before use.
Characterization of carbon dots: UV-
Vis absorption spectra of carbon dots
isolated from sugar beet molasses was
performed using UV-Vis spectropho-
tometer (Biochrom Libra S22). Photo-
luminescence spectroscopy was carried
out using photoluminescence spectro-
photometer (F-7000, HITACHI, Japan).
FTIR spectra were performed using
FTIR-ATR Bruker model (Vertex70
series) at a resolution of 4 cm–1. Scan-
ning electron microscopy with Energy
Dispersive X-ray Spectroscopy (SEM- Fig. 1: UV absorbance of carbon dots (CDs). a: CDs under sunlight; b: CDs under 365 nm UV light

No. 9 (2016) Sugar Industry 141 | 560–564

562 Technology/Technologie

sured with AFM is approximately 3 nm.

According to AFM image, although the
carbon dots seems spherical, differences
in size and images were observed which
may be attributed to tendency of car-
bon dots for aggregation. Similar results
were also reported by other researchers
(Wu et al., 2013).

3.2 Riboflavin sensing

Förster resonance energ y transfer

(FRET) is the non-radiative energy trans-
fer between two luminescent molecules
Fig. 2: Emission spectra of the CDs recorded with progressively longer excitation wavelength in 10 (Clegg, 1995). In the FRET, the energy
nm increments absorbed by the donor is transferred to
the acceptor. FRET is due primarily to
a dipole-dipole interaction between the
donor and acceptor (Mitra et al., 1996).
The FRET efficiency primarily depends
on the spatial distance in nanometer
dimensions between donor and accep-
tor. Applications of FRET involve sen-
sors, molecular structure and dynamic
detections etc (Wang et al., 2015). The
FRET process, formed between ribo-
flavin and carbon dots was shown in
Figure 5. The presence of FRET process
between riboflavin and carbon dots
was also reported by other researchers
(Wang et al., 2015; Kundu et al., 2016).
In the FRET process, carbon dot acts as
the donor and riboflavin as the acceptor.
As the intensity of riboflavin increases
with the riboflavin concentration that
of carbon dots decreased (Fig. 6). This
Fig. 3: FTIR spectrum of sugar beet molasses carbon dots situation indicates the occurrence of
efficient FRET process between carbon
dot and riboflavin. The FRET distance
is provided with the hydrogen bonds between hydroxyl and
carboxyl groups (Wang et al., 2015; Kundu et al., 2016). FRET
efficiency E was calculated using following formula

E = (1 – Fda/Fd) · 100

where Fda represents the fluorescence intensity of carbon dot

in the presence of riboflavin and Fd that of carbon dot.
While the fluorescence intensity of carbon dots decreased by
8% upon addition of 13 µmol/L riboflavin, it decreased by
31% when 64 µmol/L riboflavin was added. Riboflavin can be
determined from the proportional reduction of intensity of
carbon dot upon addition of the riboflavin. Calibration curve
for riboflavin determination is given in Figure 7. Calibration
curve shows linearity in the specified concentration range
(R2 = 0.9907). In conclusion, similarly to other researches,
carbon dots isolated from sugar beet molasses can successfully
be used for the riboflavin detection (Wang et al., 2015; Kundu
Fig. 4: AFM image of molasses carbon dots et al., 2016).

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Technology/Technologie 563

ethoprim). While tetracycline addition

decreased the fluorescence intensity
of carbon dots, the above-mentioned
antibiotics did not show any response.
As a consequence, tetracycline can suc-
cessfully be detected with the molasses
carbon dots. The degree of quenching
increased by increasing concentration of
tetracycline (Fig. 8). The intensity of car-
bon dots decreased by 7% with the addi-
tion of 13 µmol/L tetracycline; while
for the 110 µmol/L, the decrement
was 62%. Tetracycline can be detected
Fig. 5: FRET process between carbon dot and riboflavin through the decreasing intensity of car-
bon dots in proportion to tetracycline
concentration. The calibration curve for
the tetracycline is presented in Figure 9.
The calibration curve showed linearity
in the range of specified concentrations
(R2 = 0.9952). The results support the
previous reports conducted with utiliza-
tion of carbon dots in tetracycline detec-
tion (Feng et al., 2015).

4 Conclusion

Strong fluorescence CDs were extracted

from sugar beet molasses without
using any tedious processes. The work
revealed the presence of CDs in sugar
beet molasses for the first time. These
Fig. 6: Fluorescence spectra of CD in the presence of different amounts of riboflavin dots had excellent fluorescence prop-
erties and good water solubility. FRET
occurred between carbon dot and ribo-
flavin facilitated the detection of ribo-
flavin as a FRET based optical sensor. Tetracycline induced
fluorescence quenching of carbon dots and this property were
used as a tool for the quantification of tetracycline. In conclu-
sion, molasses carbon dots can successfully be used in biosen-
sor or other different applications.

Fig. 7: Calibration curve for the detection of vitamin B2

3.3 Tetracycline sensing

The addition of tetracycline reduced the fluorescence intensity

of carbon dots. The selectivity of carbon dots to tetracycline
was evaluated by testing the response of other antibiotics
(penicillin G, amikacin sulfate, cefepime, cefotaxime, trim- Fig. 9: Calibration curve for the detection of tetracyline

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564 Technology/Technologie

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