Microchemical Journal 159 (2020) 105456

Contents lists available at ScienceDirect

Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

One-step synthesis of levodopa functionalized carbon quantum dots for T

selective detection of tyrosinase and inhibitor screening
Saipeng Huanga, Wenshuai Lia, Xin Zhoua, Mengyao Xiea, Qing Luoa, Huiyun Wena, Yane Luob,
Weiming Xuea,
⁎

a
School of Chemical Engineering, Northwest University, Xi’an 710069, PR China
b
College of Food Science and Engineering, Northwest University, Xi’an 710069, PR China

ARTICLE INFO ABSTRACT

Keywords: Tyrosinase (TYR), a key enzyme for melanin biosynthesis, is an important biomarker for melanoma, thus a
Tyrosinase biosensor that can specifically detect and image tyrosinase would be in urgent demand to diagnose tyrosinase
Carbon quantum dots related disease. Therefore, a novel levodopa (L-Dopa) functionalized carbon quantum dots (Dopa-CQDs) was
Tyrosinase activity detection firstly constructed and synthesized by a convenient “one-step synthesis” strategy without any modified steps,
One-step synthesis
utilizing L-Dopa and urea as precursors. The Dopa-CQDs also illustrated excellent linear relationship with a wide
Inhibitor screening
range (0–1200 U·L−1) and a low detection limit of 7.3 U·L−1. Importantly, the nanosensor exhibited high se­
lectivity for tyrosinase over other biological substances including various amino acids and intercellular pro­
teases. Moreover, the Dopa-CQDs demonstrated practicability for TYR detection in human serum samples and
inhibitor screening with satisfactory results. Particularly, the designed biosensor was successfully applied for
intracellular TYR detection with excellent biocompatibility and cellular imaging capability, which provided a
novel platform for selective detection of tyrosinase in biosystems. The novel methodology can be extended for
fabricating novel biosensor for other analytes in various field, which will illustrate great importance for both
fundamental research in biological systems and potential practical applications in clinical diagnosis.

1. Introduction for colorimetry, time-consuming synthesis and purification procedure

Melanoma, which often occurs in skin, is a kind of malignant tumor
generally originating from mutated melanocytes [1,2] with a rapidly
increased morbidity in the past decades [3–5]. As a copper-containing
monooxygenase, tyrosinase (EC 1.14.18.1, TYR) can catalyze phenolic
hydroxyl into quinones and then trigger melanin formation [6,7]. Once
malignant mutation occurs, the up-regulated level of TYR can be ob­
viously detected, indicating that TYR could be utilized as a biomarker
for melanoma diagnosis [8,9]. Owing to the biological and physiolo­
gical characteristics of tyrosinase, multiple analytical strategies have
been established to identify and quantify tyrosinase activity in the last
decade [10–14], including electrochemistry [15,16], colorimetry [17],
near-infrared probe [18,19], fluorometry [20], radiometry [21], in­
organic metal semiconductor quantum dots [22], gold/silver na­
noclusters [23] or nanoparticles [24,25]. Unfortunately, these analy­
tical methods were less than perfect and some shortcomings still
remained, such as weak selectivity for electrochemistry, less sensitivity
for near-infrared probe, high cytotoxicity for inorganic semiconductor
quantum dots as well as high cost and less stability for gold/silver na­
noclusters. Therefore, it is vital to develop a facile and accurate ana­
lytical approach for TYR activity detection with satisfied sensitivity,
selectivity and stability in biosystems.
Compared with the above analytical methods, fluorescent biosensor
is definitely a better choice for more convenient, effective and accurate
detection of TYR activity [26–34]. Carbon quantum dots (CQDs), as a
novel fluorescent biosensors and carbon nanomaterials, have attracted
much more attention recently due to their accessibility, easy prepara­
tion [35], high water solubility, good biocompatibility, outstanding
photostability, great photoluminescence [36] and other excellent op­
tical properties [37–40]. Research works related to CQDs have been
presented in a rash emerge in chemical, environmental and biological
fields [41–43], exemplified by bioimaging, drug delivery, fluorescence
sensing, analysis and so on [44–46]. However, few researches have
been focused on TYR activity detection, especially in biosystems. Due to

Corresponding author.
⁎

E-mail addresses: huangsaipeng@nwu.edu.cn (S. Huang), 1614273362@qq.com (W. Li), 601430798@qq.com (X. Zhou),
201931866@stumail.nwu.end.cn (M. Xie), 13617307198@163.com (Q. Luo), huiyunwen@nwu.edu.cn (H. Wen), luoyane@nwu.edu.cn (Y. Luo),
xue_wm@126.com (W. Xue).

https://doi.org/10.1016/j.microc.2020.105456
Received 16 June 2020; Received in revised form 21 August 2020; Accepted 23 August 2020
Available online 26 August 2020
0026-265X/ © 2020 Elsevier B.V. All rights reserved.
S. Huang, et al.
their attractive characteristics, CQDs could be strongly recommended
as a promising candidate for TYR activity detection in the biological
systems.
In this work, the innovative CQDs was firstly designed and prepared
with urea and levodopa by one-step synthesis strategy, which was much
simpler and more facile than traditional multi-step synthesis. Levodopa
is selected as the precursor due to three causes: (1) Basically, catechol is
a good carbon source and conjugate system to obtain CQDs with long-
wavelength emission and high quantum yield [47,48]; (2) Crucially,
Levodopa, as tyrosine analogue and the most commonly substrate for
TYR, can guarantee and strengthen catalytic selectivity and recognition
ability to TYR; (3) This synthesis methodology is convenient and eco­
nomical. Directly functionalized by levodopa during the particle for­
mation, the prepared Dopa-CQDs could be sensitively and selectively
catalyzed by TYR into dopaquinone derivatives, triggering photo­
induced electron transfer and subsequently fluorescence quenching.
Taking advantage of their bright green fluorescence, it would be very
helpful to effectively distinguish Dopa-CQDs from autofluorescence of
biosystem itself and enhance anti-interference ability. Based on the
excellent linear relationship and low detection limit, Dopa-CQDs illu­
strated promising application for trace TYR detection in serum samples,
real-time monitoring intracellular TYR fluctuation and TYR-inhibitor
screening. This research offered a facile synthesized procedure and
novel methodology for CQDs fabrication with high selectivity, which
illustrated potential importance for both fundamental researches in
biological system and practical applications in clinical diagnosis of
melanoma.
2. Experimental section
2.1. Chemicals and reagents.
All solvents and chemicals utilized in this work were of analytical
reagent grade and without further purification. Levodopa (C9H11NO4)
and Urea (CO(NH2)2) were purchased from Kemiou Chemical Reagent
Co., Ltd (Tianjin, China). Glucose, serine, threonine, alanine, aspartic
acid and arbutin were purchased from Aladdin Reagent Co., Ltd
(Shanghai, China). Ethanol, DMSO, Na2HPO4 12H2O, KH2PO4, NaCl
and KCl (≥99%) were all obtained from Sinopharm Chemical Reagent
Co., Ltd (Shanghai, China). Tyrosinase (TYR, 2.5 kU·mg−1), glucose
oxidase (GOx, 10 kU·mg−1), alkaline phosphatase (ALP, 3 kU·mg−1),
acetylcholinesterase (AChE, 220 U·mg−1) and lysozyme (LYS, 45
kU·mg−1) were provided by Aladdin Chemistry Co., Ltd (Shanghai,
China). Immunoglobulin G (IgG, 25 mg) and bovine serum albumin
(BSA, chromatographically pure) were purchased from Sigma-Aldrich
Trading Co., Ltd (Shanghai, China). Milli-Q-purified water was applied
for sample preparation. The human serum samples were provided by
Xi'an No. 1 Hospital (Shanxi, China). Dulbecco’s modified Eagle’s
medium (DMEM) was purchased from Sigma Aldrich Trading Co., Ltd
(Shanghai, China).
2.2. Instrumentation and Characterization.
Transmission electron microscopy (TEM) and high-resolution TEM
(HRTEM) images were obtained by Tecnai G2F20 microscope. Fourier
transform infrared spectra (FT-IR) were recorded by a Nicolet iS10 FT-
IR spectrometer. X-ray photoelectron spectroscopy (XPS) survey data
were obtained by a Thermo Scientific ESCALAB 250Xi. UV–Vis-NIR
absorption spectra were recorded on a Hitachi U-4100 spectro­
photometer. FL970 fluorescence spectrophotometer was utilized to ac­
quire excitation and emission spectra. Intracellular tyrosinase imaging
was examined by confocal fluorescence microscopy equipped with the
Olympus FV1000 with the excitation and emission wavelength at
420 nm and 530 nm, respectively. The morphology of Dopa-CQDs was
characterized by atomic force microscopy (AFM) with ScanAsyst mode,
using Bruker NanoScopeV multimode 8 at room temperature in
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Microchemical Journal 159 (2020) 105456
atmosphere, and the samples were acquired by dropping the solution
onto the substrate of mica.
2.3. Preparation and purification of Dopa-CQDs
Firstly, 0.2 g levodopa (L-Dopa, 1.01 mmol) and 0.3 g urea (5 mmol)
were dispersed in 20 mL ethanol, and then the solution was transferred
into a Teflon-lined stainless steel autoclave and heated to 160 °C for 8 h.
After the autoclave was allowed to cool to room temperature, the re­
sulting solution was centrifuged at 10000 rpm for 10 min to remove
solids. After ethanol was removed by a rotary evaporator, the acquired
product was ultrasonically dispersed in ultrapure water. Next, the ob­
tain solution was dialyzed through a dialysis membrane
(MWCO = 1000 Da) for 24 h. After filtering by microporous membrane
with pore diameter of 0.22 μm, the final Dopa-CQDs solid sample was
obtained through vacuum freeze drying.
The control sample was prepared and produced by the same ex­
periment process and procedure just without the addition of urea,
which was designed as a contrast agent to explore the contribution of
urea.
2.4. Fluorescent tyrosinase activity assay
The Dopa-CQDs was diluted with phosphate buffered saline (PBS,
10 mmol·L−1, pH 7.0) to a final concentration of 200 μg·mL−1. A series
of TYR (300 μL) ranging from 0 to 72 kU·L−1 were prepared and then
utilized to determine TYR activity, which were added into a mixture of
300 μL Dopa-CQDs solution (200 μg·mL−1) and 2.4 mL PBS. After in­
cubation for 15 min at room temperature, the fluorescence intensity
was recorded by FL970 fluorescence spectrophotometer at an excitation
wavelength of 420 nm and emission wavelength of 530 nm.
Based on the same analytical methodology and detection method,
the selectivity experiments were performed with 100 μL other inter­
fering substances instead of TYR. The fluorescence response of Dopa-
CQDs was analyzed towards different interfering anions, amino acids or
proteases. Competition and application experiments were carried out
with the subsequent addition of TYR based on selectivity experiments.
Real complex biological samples (such as human serum samples) were
utilized to evaluate the feasibility and applicability of Dopa-CQDs.
2.5. Tyrosinase inhibitor screening
Three groups of arbutin solution (300 μL, 0.2 mmol·L−1,
5 mmol·L−1, 15 mmol·L−1) were respectively added into 300 μL TYR
(72 kU·L−1) and incubated for 10 min, then 300 μL Dopa-CQDs solution
(200 μg·mL−1) and 2.1 mL PBS (pH 7.0) were added into the above
mixture, respectively. After incubation for 15 min at room temperature,
the fluorescence intensity was recorded by FL970 fluorescence spec­
trophotometer at an excitation wavelength of 420 nm and emission
wavelength of 530 nm. Each experiment was repeated three times.
2.6. TYR detection in human serum sample
Three human serum samples with different activity of TYR (90
U·L−1, 300 U·L−1 and 900 U·L−1) were directly subjected to Dopa-CQDs
analytical system, respectively. After incubation for 15 min, the fluor­
escence intensity of samples was recorded by FL970 fluorescence
spectrophotometer at an excitation wavelength of 420 nm and emission
wavelength of 530 nm. According to linear fitting equation of TYR, the
measured concentration and subsequent recovery rate of three samples
could be calculated. Each experiment was performed three times.
2.7. Cell culture and fluorescence imaging
In order to evaluate cytotoxicity of Dopa-CQDs and intracellular
TYR analysis, L929 cells were cultured in DMEM with 10% fetal bovine
S. Huang, et al.
Fig.1. Characterization of Dopa-CQDs was conducted by transmission electron microscopy, X-ray diffraction and infrared spectroscopy. (A) TEM image (B) HR-TEM
image (C) AFM images (D) The height profile of Dopa-CQDs (E) XRD and (F) FT-IR spectra of Dopa-CQDs. Panel inset on upper right of (A) is particle distribution.
serum and antibiotics (100 g·mL−1 streptomycin and 100 units/mL
penicillin) in an incubator at 37 °C with an atmosphere containing 5%
CO2 and 100% relative humidity, which were utilized to evaluate cy­
totoxicity of Dopa-CQDs and in different activity and intracellular TYR
analysis.
1.0 × 103 L929 cells grown overnight were treated with
20 μg·mL−1 Dopa-CQDs in a confocal microscope dish with a cover slip
and incubated together with at 37 °C for 4 h, subsequently removing the
medium and washing with PBS (10 mmol·L−1, pH 7.4) for twice. And
then 1.0 mL TYR (7200 U·L−1) was added into dish and incubated to­
gether with cells at 37 °C for 4 h. Washing dish with PBS (10 mmol·L−1,
pH 7.4) twice, subcellular image analysis was monitored by confocal
microscope. Dopa-CQDs Green was excited at wavelength of 420 nm
and the fluorescence was collected at 500– 550 nm.
3. Results and discussion
3.1. Characterization of Dopa-CQDs
Transmission electron microscope (TEM) were utilized to char­
acterize the morphology and microstructures of Dopa-CQDs (Fig. 1A),
which illustrated the uniform spherical nanoparticles monodispersed
well and displayed smooth surface morphology an average diameter of
4.57 nm. The particle size distribution was determined by dynamic
light scattering (DLS) and displayed normal distribution with an
average hydrodynamic size of 20.5 nm (Fig. S1), which attributed to the
excellent water absorption and solubility of Dopa-CQDs. Considerable
hydrogen bonds and aggregate generated because of surface abundant
amino and hydroxyl from Dopa-CQDs.
Meanwhile, the high-resolution TEM (HRTEM) image disclosed that
Dopa-CQDs possessed crystal structures with interlayer lattice fringes of
0.20 nm, which corresponds to the (1 0 0) plane of graphite carbon
(Fig. 1B). The ScanAsyst mode AFM image of Dopa-CQDs (Fig. 1C) il­
lustrates the presence of thin rods and spherical structures intimately
connected. The sphericity of Dopa-CQDs is intact and evenly distributed
excellent dispersion. The height profiles display that the typical topo­
graphic height difference of Dopa-CQDs approached a size range below
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Microchemical Journal 159 (2020) 105456
5 nm, as illustrated in Fig. 1D. The X-ray diffraction (XRD) of Dopa-
CQDs exhibited a strong broad diffraction peak around 2θ = 22.3°
belonging to the (0 0 2) crystal plane diffraction peak of graphite
structure [28], demonstrating Dopa-CQDs possessed a graphite-like
structure (Fig. 1E).
Compared the AFM, DLS and TEM results, nanoparticles self-ag­
gregates was speculated as the main reason for the difference. The
CQDs was easy to generate self-aggregates phenomenon when the dy­
namic light scattering (DLS) analysis of CQDs was measured in water
solution, which determined by the concentration and dispersion time of
nanoparticles [49]. Meanwhile, Dopa-CQDs was very hydrophilic and
easy to form hydrogen bonds in water because of much hydroxyl and
amino group on the surface, which promoted water absorption and
expansion, and even formed microcapsules. The cluster-cluster and
cluster-particle interactions lead to the formation of large objects with
kinetics. Hence, the centrifuged and filtered procedure also affected the
DLS result. The TEM illustrated the uniform spherical nanoparticles
monodispersed well (Fig. 1A), which presented few crystallinelike ag­
gregates phenomenon in a large number of small globular particles.
These particles seem to interact and form branched and rod structures,
disc-shaped aggregates were also observed by the AFM (Fig. 1C). The
TEM and AFM indicated that the Dopa-CQDs particles may merge
completely in a uniform self-aggregates.
The surface functional groups of Dopa-CQDs were characterized
through FTIR spectroscopy (Fig. 1F). The bands at 3250 cm−1 and the
wide peak at 3200 cm−1 represented oxygen-hydrogen (OeH)
stretching vibration and symmetric stretching vibration of primary
amino group, indicating that there were many phenolic hydroxyl and
primary amines on the surface of Dopa-CQDs. The stretching vibration
at 3100 cm−1 represented aromatic unsaturated hydrocarbon (]CeH),
while the sharp peak at 1570 cm−1 and 1520 cm−1 indicated carbon-
carbon (C]C) stretching vibration peak of the benzene ring skeleton.
The weak peak at 1650 cm−1 indicated carbon-oxygen (C]O) double
bonds stretching vibration peak, which was produced by few phenolic
hydroxide oxidation. Meanwhile, 1440 cm−1 and 1250 cm−1 re­
presented carbon-oxygen (CeO) stretching vibration peak of phenolic
hydroxyl group and carbon-hydrogen (CeH) bending vibration of the
S. Huang, et al.
Fig.2. X-ray photoelectron spectroscopy (XPS) of Dopa-CQDs (A) Full-survey, (B) C1s, (C) N 1s, and (D) O1s.
benzene ring skeleton. The FTIR spectroscopy demonstrated that the
levodopa structure was constant during the synthesis of Dopa-CQDs.
The bands at 3550 cm−1 and 1760 cm−1 represented hydroxyl group
(OeH) and carbonyl (C]O) stretching vibration peak in the carboxyl
group, respectively. Furthermore, it can be found that the FT-IR spectra
of Dopa-CQDs and its precursor levodopa were very similar and almost
completely overlapped (Fig. 1F), which confirmed that phenolic hy­
droxyl, carboxyl, primary amine and the active structure of levodopa
retained in the framework of Dopa-CQDs.
In order to further identify the functional groups and elemental
compositions of Dopa-CQDs, the as-prepared Dopa-CQDs were also
characterized by X-ray photoelectron spectroscopy (XPS). The full-
range XPS spectrum of Dopa-CQDs was shown in Fig. 2A, the three
obvious peaks located at 484 eV, 402 eV and 531 eV were attributed to
C1s, N1s and O1s, respectively. Fig. 2B displayed the high-resolution
XPS spectrum of C1s, which can be separated into three characteristic
peaks corresponding to sp2 hybridized carbon atoms in CeC/C]C
(284.4 eV), sp3 hybridized carbon atoms in CeN/CeO (285.8 eV), and
carbonyl carbons (C]O) at 289.8 eV [50], respectively. The N1s XPS
spectrum (Fig. 2C) illustrated three component peaks at 399.5 eV
(amino Ns), 400.2 eV (pyrrolic Ns), and 400.9 eV (graphite Ns) [51].
The high-resolved O1s XPS spectrum exhibited the distinctive peaks of
CeO and C]O at 531.5 and 532.6 eV, respectively (Fig. 2D) [52].
Based on the XPS analysis, it was reconfirmed that the polar functional
groups present on the surface of Dopa-CQDs were phenolic hydroxyl,
primary amine and carboxy, which was well consistent with the results
of FTIR.
3.2. Optical properties research
The optical properties of Dopa-CQDs were investigated by ultra­
violet–visible (UV–vis) and fluorescence spectroscopy. As shown in
Fig. 3A, Dopa-CQDs indicated relatively broad absorption range cen­
tered at 285 nm and 300 nm, which were attributed to the aromatic sp2
π − π* transition of the carbon–carbon (C]C) double bonds [53,54]
and n–π* transition of the carbon–oxygen (C]O) double bonds [55,56]
on the surface of Dopa-CQDs, respectively (Fig. 3A). The structural
features resulted from considerable long aromatic conjugated system
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Microchemical Journal 159 (2020) 105456
and aromatic hydrocarbons on the surface of Dopa-CQDs. Meanwhile,
the fluorescence emission spectrum exhibited a strong peak at 530 nm
with varied excitation wavelength from 390 nm to 440 nm (Fig. 3B).
Different from other carbon quantum dots, it exhibited a significant
excitation-independent behavior, which demonstrated Dopa-CQDs
possessed only one specific and dominant emission mode with uniform
emission [57,58]. Meanwhile, the control sample prepared without
urea illustrated a strong emission peak at 475 nm with a excitation
wavelength of 430 nm. The experiment result demonstrated that urea
played an important role and adulterated more nitrogen in Dopa-CQDs
preparation, which promoted and achieved strong green fluorescence
from Dopa-CQDs.
Furthermore, the fluorescence excitation spectra manifested an ex­
citation peak centered at 420 nm with the maximum emission wave­
length at 530 nm. The surface structures and functional groups were
presumably responsible for the emission at 530 nm, which was sig­
nificantly different from the spontaneous blue fluorescence of many
proteases in organisms. This demonstrated the prepared Dopa-CQDs
have potential application for tyrosinase activity analysis in biological
systems [59]. The quantum yield (QY) of Dopa-CQDs in aqueous media
was determined to be 7.8% by the comparative method of Williams
utilized quinoline sulfate as the standard [60], which demonstrated that
Dopa-CQDs exhibited bright green florescence and could potentially be
utilized as highly-sensitive nanosensors for TYR activity detection.
Photobleaching experiment was performed to examine the photo­
stability of Dopa-CQDs. As shown in Fig. S2 (Supporting Information),
the fluorescent intensity attenuated and decreased slightly after con­
tinuous and uninterrupted irradiation with a 400 W iodine–tungsten
lamp at room temperature for 60 min. Moreover, more than 95%
fluorescence intensity of Dopa-CQDs almost remained steady compared
with its original fluorescence, demonstrating that the as-fabricated
Dopa-CQDs possessed excellent photostability against photobleaching
organic probes. Besides, the photostability experiment indicated that
the fluorescence intensity of Dopa-CQDs still kept constant even after
60 days at room temperature (Fig. S3). Such outstanding photostability
attributed to the surface structure and composition of Dopa-CQDs.
Additionally, the stability of the Dopa-CQDs was also examined under
high ionic concentrations up to 1 mol·L−1 NaCl. The result illustrated
S. Huang, et al.
that the fluorescence intensity of Dopa-CQDs remained nearly constant,
which further demonstrated the high photostability and anti-interface
ability of Dopa-CQDs (Fig. S4). Therefore, the experiments implied that
most biological substances can coexist in Dopa-CQDs solution for TYR
activity detection without any interference, which indicated great po­
tential for widespread application in complex environment.
However, the fluorescence intensity fluctuated as the pH increased
from 4 to 10, possibly attributed to the deprotonation of phenolic hy­
droxyl on the surface of Dopa-CQDs. Another possible reason is the
formation of phenol negative oxygen ions with the pH increased. Under
the electrostatic repulsion, the enhancement fluorescence of Dopa-
CQDs is attributed to the enhanced monodispersity of Dopa-CQDs na­
noparticles and the p-π conjugation formed by oxygen anions and
benzene rings. The surface carboxyl and amino groups were also
speculated to be one of the reasons for fluorescence fluctuations.
Interestingly, Dopa-CQDs still possessed great fluorescence perfor­
mance in neutral and alkaline environment (Fig. S5), which demon­
strated Dopa-CQDs could be utilized in biological system and in vivo
analysis.
To determine the practicality of Dopa-CQDs as fluorescent nano­
sensors, common active substances and enzyme coexisting in biological
system were utilized to evaluate their selectivity and anti-interference
ability in biosystem, including small molecular components: glucose
(Glu), serine (Ser), threonine (Thr), alanine (Ala), aspartic acid (Asp);
macromolecular component: bovine serum Albumin (BSA), im­
munoglobulin G (IgG), glucose oxidase (GOx), alkaline phosphatase
(ALP), lysozyme (LYS), acetylcholinesterase (AChE), tyrosinase (TYR).
As is shown in Fig. 3C, the fluorescence intensity almost remains con­
stant (< 5% decrease) except for TYR (> 85% decrease), which was
attributed to the absence of specific interaction between surface func­
tional groups of Dopa-CQDs and biological substance. Meanwhile, the
high recognizability and selectivity of TYR to the surface levodopa
structure of Dopa-CQDs was identified as the most important and cri­
tical reason.
In order to evaluate the selectivity of Dopa-CQDs for TYR activity
detection, the interference factors of common inorganic ions
(100 μmol·L−1, Na+, K+, Mg2+, Ca2+, Cu2+, Al3+, CO32−, NO3−,
SO42−, and PO43−) were examined and evaluated (Fig. 3D). The ex­
periment results clearly illustrated that the fluorescence intensity
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Microchemical Journal 159 (2020) 105456
Fig.3. Spectroscopy characteristic behavior
and selectivity of Dopa-CQDs. (A) UV − vis
spectrum, (B) Fluorescent emission spectra
(530 nm) under different excitation wave­
length (from 390 nm to 440 nm); (C)
Fluorescence intensity variation of Dopa-
CQDs toward various amino acids
(100 μmol·L−1) and proteases
(5000 mg·L−1); (D) Fluorescence intensity
variation (F/F0) of Dopa-CQDs with dif­
ferent ions (100 μmol·L−1).
almost kept constant (< 4% decrease) except for TYR (> 85% de­
crease), exhibiting little specific chemical response between surface
groups of Dopa-CQDs and the common inorganic ions. The interfering
experiments indicated Dopa-CQDs possessed high selectivity toward
TYR and strong anti-interference capability to other biologically active
substance, which demonstrated a potential application for TYR detec­
tion in complex biological environment.
3.3. Tyrosinase activity detection by Dopa-CQDs
To optimize the response time for fluorescence quenching by TYR,
the time-dependent experiment of Dopa-CQDs was evaluated and re­
corded (Fig. 4A). The result indicated that fluorescence intensity of
Dopa-CQDs quenched more than 85% in 35 min and no apparent
change in emission wavelength was observed, implying the oxidation
reaction between Dopa-CQDs and tryptophan reached an equilibrium
state under a high sensitivity. Compared with those organic molecular
sensors, which achieved balance in more than 60 min, the response
time is much shorter. The reason attributed to the bisphenol structure
on the surface of Dopa-CQDs, which can be oxidized faster than
monophenol system. The quick response rate was suitable and favor­
able for real-time dynamic monitoring TYR activity, which demon­
strated Dopa-CQDs was a potentially excellent nanoprobe for TYR real-
time detection in analytical field. Similarly, the bright green cuvette can
be observed under ultraviolet lamp, while it attenuated to almost col­
orless after incubation with TYR (Fig. 4A). The obvious color change
indicates the capacity of Dopa-CQDs for sensing TYR activity through
the naked-eye.
Furthermore, the detection performance of Dopa-CQDs towards TYR
was researched and evaluated. The fluorescence of Dopa-CQDs con­
tinuously quenched with the increased concentration of TYR, and it was
almost completely quenched while TYR activity was as high as 7200
U·L−1 (Fig. 4B and Fig. S5). The result attributed to the surface bi­
sphenolic hydroxyl of Dopa-CQDs was oxidized and catalyzed into
dopaquinone derivatives by TYR (Scheme 1), which induced photo­
induced electron transfer of Dopa-CQDs and fluorescence quenching.
The detailed analyzed performance was displayed in Fig. 4C, the
fluorescence titrations with different activities of TYR (from 0 to 1200
U·L−1) exhibited a gradual quenching process. Moreover, excellent
S. Huang, et al.
linear correlation of R2 = 0.996 between the quenching efficiency (F0-
F) and activities of TYR was observed in the range of 0–1200 U·L−1
(Fig. 4C), which could be fitted into extent F0-F = 0.33 [TYR] + 5.94,
where F0 and F were the fluorescence intensity of the Dopa-CQDs at
530 nm in the absence and presence of TYR, respectively. The limit of
detection (LOD) calculated from this plot was 7.3 U·L−1 according to
the 3σ (signal-to-noise) criteria. The quantitative detection equation
revealed a wide linear range (0–1200 U·L−1) with a low detection limit
of 7.3 U·L−1, demonstrating the promising applicability of Dopa-CQDs
to sensitive and trace detection of TYR in biosystem.
TYR commonly distributed in a variety of intracellular environ­
ments and plays an important role in biological system. In this work,
L929 cell line was chosen for further cytotoxcity evaluation of Dopa-
CQDs and intracellular TYR detection. The biocompatibility over L929
cells was assessed by MTT standard assay, which indicated that L929
cells remained at a comparatively high viability (> 92%) even while
the concentration of Dopa-CQDs was up to 300 μg·mL−1. The cyto­
toxicity experiment demonstrated Dopa-CQDs possessed satisfied bio­
compatibility, which manifested exceptional potential for intracellular
TYR detection in living cell systems (Fig. 4D).
Scheme 1. The schematic illustration of possible sensing mechanism of Dopa-CQDs to TYR and inhibitors screening.
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Microchemical Journal 159 (2020) 105456
Fig.4. (A) Fluorescence response of Dopa-
CQDs in different reaction time with TYR
(7200 U·L−1) (B) Fluorescence response of
Dopa-CDs with different activity of TYR
from 0 to 7200 U·L−1; (C) The fitting curve
and regression equation between fluores­
cence intensity and TYR activity over a
linear range from 0 to 1200 U·L−1; (D)
Cytotoxicity assessment of the Dopa-CQDs
using standard MTT assay towards L929
cells.
3.4. Tyrosinase inhibitor screening
Based on the satisfied selectivity of Dopa-CQDs towards TYR, the
possibility for tyrosinase inhibitor screening was also evaluated. As
shown in Fig. 5A, arbutin was utilized as TYR inhibitor, it was experi­
mentally confirmed that the fluorescence intensity of Dopa-CQDs in­
cubated with arbutin and TYR together almost kept constant while the
control group (without arbutin) decreased more than 85%, which at­
tributed to the inhibition of arbutin over TYR and termination of oxi­
dation procedure to Dopa-CQDs. The experiments confirmed that Dopa-
CQDs have a function of selectively recognizing TYR inhibitors and can
be utilized to screen TYR inhibitors.
Fig. 5B illustrated that the fluorescence quenching of Dopa-CQDs
gradually decreased with the addition of arbutin, and seldom sig­
nificant fluorescence change was observed as the concentration of ar­
butin reached 1500 μmol·L−1, which fully demonstrated that arbutin
can effectively inhibit TYR activity and prevent the catalytic oxidation
process on the surface of Dopa-CQDs, resulting in termination of the
photoinduced electron transfer (PET) process, indicating that the pre­
pared Dopa-CQDs have good chance to be designed for TYR-inhibitors
S. Huang, et al.
Fig.5. (A) Fluorescent intensity changes of Dopa-CQDs in the presence of TYR and arbutin; (B) Fluorescent intensity changes of Dopa-CQDs in the presence of TYR
and different concentrations of arbutin.
screening by fluorescence change.
3.5. TYR detection in human serum sample
In order to evaluate the feasibility of quantitative detection under
complicated biological background, based on the satisfied detection
performance and anti-interference ability of Dopa-CQDs, a further TYR
activity detection was conducted by fresh human serum as biosystem
sample, in order to evaluate the feasibility of quantitative detection
under complicated biological background.
Taking TYR solutions with different enzyme activity levels (90
U·L−1, 300 U·L−1and 900 U·L−1) as testing samples, an analytical re­
covery test was conducted (n = 3). As reported in Table 1, the TYR
activity level determined by Dopa-CQDs was consistent with the added
sample. The analytical recovery rate was in the range of 95.5% –
104.7% with relative standard deviations (RSD) ranging from 2.5% to
3.1%, indicating the trace amount of TYR in complex biological en­
vironment can be monitored by Dopa-CQDs [61–63]. The experiment
further confirmed the reliability and applicability of Dopa-CQDs for
TYR activity monitoring in actual sample. Therefore, this proposed
fluorescent assay has potential for TYR activity detection in actual
human serum samples and provide credible evidence for clinical diag­
nosis.
3.6. Intracellular imaging of TYR
Cellular uptake ability was also investigated by L929 cells. As shown
in Fig. S7, the Dopa-CQDs initially illustrated weak fluorescence after
addition, which attributed to cellular uptake velocity limited and most
Dopa-CQDs could not be absorbed in a short time. The intracellular
green fluorescence enhanced with the incubation time extended, which
attributed to more nanoprobe were assimilated by L929 and time-de­
pendent uptake of Dopa-CQDs in cells. The fluorescence reached the
strongest and highest luminance after incubation for 4 h, which de­
monstrated the added Dopa-CQDs almost were completely uptaken by
L929 cells and the reasonable incubation time was 4 h.
The intracellular TYR activity and image were measured by con­
focal laser scanning microscopy. Firstly, the fluorescent images of L929
cells were acquired only after 4-hour incubation with 20 μg·mL−1
Dopa-CQDs. As shown in Fig. 6, the cells had complete morphology and
clear outline under bright green fluorescence, which demonstrated that
Table 1
Detection of TYR in human serum samples.
Serum Added Measured (U·L−1) Recovery (%) RSD (%)
Sample (U·L−1) n=3
1 90 94.3 ± 3.1 104.7 3.1
2 300 286.5 ± 7.1 95.5 2.7
3 900 877.5 ± 22.1 97.4 2.5
7
Microchemical Journal 159 (2020) 105456
Dopa-CQDs possessed good transmembrane permeability and accumu­
lated within cytoplasm instead of nucleus. After incubation with TYR,
the intracellular fluorescence intensity gradually decreased with the
increasing concentration of TYR from 0 U·L−1to 7200 U·L−1, which
demonstrated that the intracellular fluorescence change process was
consistent with the extracellular sample analysis (Fig. 4B). It was noted
that the green fluorescence almost disappeared after incubation with
concentration sample of 7200 U·L−1. The experiment gave a possibility
that Dopa-CQDs could be utilized for intracellular TYR activity mon­
itoring, which could be potentially utilized to provide assistance for
early diagnosis and treatment of tyrosinase-associated diseases in fu­
ture.
3.7. Possible mechanism of the optical response of Dopa-CQDs to TYR
The mechanism of expected tyrosinase-catalysed oxidation proce­
dure is illustrated in Scheme 1.
The fluorescence analysis of TYR activity performed with organic
molecules probes attracted widespread attention [64]. The catalytic
oxidation procedure [7,18] and photoinduced electron-transfer (PET)
process [10,65] were widely reported and recognized as fluorescence
response mechanisms [64,65]. Based on the reported quenching me­
chanism, we speculated that the o-diphenol hydroxyl group on the
surface of Dopa-CQDs was oxidized by TYR and effectively quenched
the fluorescence (Scheme 1). The fluorescence change was caused by
the selective catalytic oxidation reaction between surface levodopa
structure and TYR, which was estimated to generate o-diquinone pro­
duct and induce photoelectron transfer, and then resulting in fluores­
cence quenching according to the previous studies. This behavior
confirmed the feasibility of utilizing Dopa-CQDs with surface rich o-
diphenol hydroxyl group and levodopa structure as substrate for TYR
recognition and detection in high selectivity. Therefore, the preparation
of Dopa-CQDs with special carbon source was a significant factor for the
analytical performance.
Meanwhile, it has been reported that numerous fluorescent probes
and quantum dots have displayed their great inhibition effects on TYR
[66–68]. The fluorescence intensity of Dopa-CQDs (incubated with ar­
butin) almost kept constant while the control group (without arbutin)
decreased more than 85% (Fig. 5A). Based on the previous research, the
inhibitor screening mechanism was speculated that arbutin inhibited
TYR activity and terminated the catalytic oxidation process, which
stopped photoelectron transfer and the fluorescence of Dopa-CQDs al­
most kept constant. Hence, the TYR inhibitor screening mechanism
based on the high selectivity and recognized ability of Dopa-CQDs from
surface active group.
Under specific catalytic oxidation of TYR, Dopa-CQDs and the o-
diquinone product acted as an effective electron donor and acceptor,
respectively, the intraparticle photoinduced electron transfer (PET)
phenomenon inside the oxidative derivative could take place. Hence,
the fluorescence of Dopa-CQDs quenched simultaneously due to this
S. Huang, et al.
Fig. 6. Intracellular fluorescence microscopy images of L929 cells with 20 μg·mL−1 Dopa-CQDs in different concentration of TYR (1500 U·L−1, 4500 U·L−1 and 7200
U·L−1).
PET process. While an inhibitor of TYR (arbutin) is present in the
analysis system, it would inhibit TYR activity and keep Dopa-CQDs in
the reductive form, which was an electron donor rather than an elec­
tron acceptor. The PET process could be blocked and the fluorescence
was retained (Scheme 1) [69,70].
4. Conclusion
In summary, a novel fluorescent carbon quantum dots based on tyr­
osinase-catalyzed oxidation of catechol skeleton structure is firstly con­
structed and synthesized by one-step synthesized strategy for intracellular
tyrosinase detection. The prepared Dopa-CQDs are directly functionalized
by L-Dopa during nanoparticle synthesis without any extra surface
modification to introduce binding sites. Dopa-CQDs exhibited high se­
lectivity for tyrosinase over other biological substances, including various
inorganic ions, amino acid and intracellular protease. The quantitative
analysis methodology of TYR activity was established and illustrated ex­
cellent linear relationship and low detection limit. The nanosensor not
only indicated good sensitivity and selectivity toward tyrosinase but also
exhibited good performances in real human serum sample analysis and
inhibitor screening. Based on high quantum yield and photostability,
good biocompatibility, outstanding water solubility and low cytotoxicity,
our sensor also realized intracellular TYR activity real-time monitoring
and dynamic tracking. We believe that fluorescent Dopa-CQDs hold great
potential and significance for widespread application in tyrosinase-related
disease monitoring and early clinical diagnosis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ­
ence the work reported in this paper.
8
Microchemical Journal 159 (2020) 105456
Acknowledgements
This research was supported by Scientific Research Plan Projectsof
of Shaanxi Provincial Department of Education, China (16JK1770),
Shaanxi Provincial Natural Science Basic Research Project, China
(2018JM2037) and Opening Foundation of Key Laboratory of Resource
Biology and Biotechnology in Western China (Northwest University),
Ministry of Education (ZSK2018008).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.microc.2020.105456.
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