In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity

Article abstract of DOI:10.1021/acs.analchem.7b02739

As a well-known copper-containing oxidase, tyrosinase has been anticipated to serve as the biomarker of skin diseases. We describe here an exquisite label-free fluorescent and colorimetric dual-readout assay of its activity, inspired by the specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found that the tyrosinase-incubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of resorcinol and O₂, where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e., the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 min at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, cost-effective, and highly sensitive dual-readout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic research as well as in screening potential tyrosinase inhibitors.

Full text of DOI:10.1021/acs.analchem.7b02739

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Article
In Situ Fluorogenic and Chromogenic Reactions for the
Sensitive Dual-Readout Assay of Tyrosinase Activity
Jiahui Zhao, Xingfu Bao, Shuang Wang, Shasha Lu, Jian Sun, and Xiurong Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02739 • Publication Date (Web): 11 Sep 2017
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Analytical Chemistry
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In Situ Fluorogenic and Chromogenic Reactions for the Sensitive
Dual-Readout Assay of Tyrosinase Activity
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†,§
†
†,‡
†,‡
,†
,†
Jiahui Zhao , Xingfu Bao , Shuang Wang , Shasha Lu , Jian Sun* , and Xiurong Yang*
†
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun, Jilin 130022, China
University of Chinese Academy of Sciences, Beijing 100049, China
§
‡University of Science and Technology of China, Hefei, Anhui 230026, China
*Fax: +86 431 85689278. Eꢀmail: xryang@ciac.ac.cn
ABSTRACT: As a wellꢀknown copperꢀcontaining oxidase, tyrosinase has been anticipated to serve as the biomarker of skin
diseases. We describe here an exquisite labelꢀfree fluorescent and colorimetric dualꢀreadout assay of its activity, inspired by the
specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol
and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found
that the tyrosinaseꢀincubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of
resorcinol and O , where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e.,
2
the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 minutes
at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and
formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, costꢀeffective, and highly
sensitive dualꢀreadout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic researches, as
well as in screening potential tyrosinase inhibitors.
1
9,20
Tyrosinase is widely distributed in plants and animal tissues,
and plays crucial roles in many physiological processes, such
researches.
Particularly, fluorometric assays have attracted
considerable attentions due to the simple and rapid
implementation, high sensitivity and spatial resolution, as well
as realꢀtime monitoring specialties.
1
2
as plants browning reactions , melanogenesis in mammals ,
3
21ꢀ23
insect cuticle sclerotization and so on. As a copperꢀcontaining
As far as we know,
oxidase, tyrosinase possesses two binding sites for aromatic
substrates, which has activity for both monophenols and
catechols, involving two distinct reactions in melanin
synthesis. Its monophenolase activity is catalytic
hydroxylation of monophenol to oꢀdiphenol, while the
diphenolase activity is processed in the catalytic oxidation of
oꢀdiphenol to corresponding oꢀquinone in the presence of
molecular oxygen. In the clinic research, tyrosinase serves as
the biomarker of skin diseases such as melanoma cancer and
vitiligo, because of its overexpression in melanoma cancer
serval current fluorescent sensing platforms have typically
utilized the fluorescence quenching ability of oꢀquinone
moiety, the tyrosinaseꢀcatalyzed oxidation product based on
24,25
the diphenolase activity.
and catechol derivatives
In virtue of some fluorophores
(usually dopamine) as the
26,27
substrates of tyrosinase, a variety of fluorescence turnꢀoff
assays have been developed based on the electron transfer
from fluorophore to oꢀquinone and the resultant fluorescence
25,28
quenching.
In this regard, various fluorescent materials
from simple fluorescent dye or conjugated polymer to
functional nanomaterials have been prepared and employed to
4,5
6,7
cells and acting as an autoantigen in vitiligo . Thus, the
inhibitors of tyrosinase have been widely exploited as
effective constituents for skinꢀwhitening product in cosmetic
29ꢀ32
monitor the tyrosinase activity.
Such diphenolase activity
and quinoneꢀbased sensing systems have conceivable response
mechanism and certain application ability, however, the
preparation or modification processes of these fluorescent
materials are generally laborious and timeꢀconsuming.
Meanwhile, the fluorescence turnꢀoff response mechanism is
usually accompanied with the high background and relatively
low sensitivity.
8
industry. In addition, tyrosinase has implications on dopamine
9,10
neurotoxicity and neurodegeneration in Parkinson’s disease.
Therefore, the development of highly sensitive tyrosinase
activity assays and corresponding inhibitor screening is of
great value in providing effective diagnostic approaches or
therapeutic targets in biomedical research, as well as in
cosmetic industry.
On the other hand, Ma et al. has recently reported several
novel tyrosinaseꢀrecognition fluorescent small molecules by
Until now, various sensing strategies have been already
proposed to determine the tyrosinase activity, including
incorporating substrate moieties into the fluorophore
11
12ꢀ15
19,33
radiometric ,
methods
electrochemical
Among them, spectroscopic methods are
wellꢀsuited for in situ analysis and highꢀthroughput screening
,
and
spectroscopic
structures.
In presence of tyrosinase, the asꢀprepared
1
6ꢀ18
.
substrate molecules containing monophenol groups can be
catalytically oxidized to oꢀdiphenol or corresponding
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oꢀquinone, which subsequently undergo
intramolecular electron rearrangement to eliminate3 the
oꢀquinone moiety and induce a fluorescence response. By
adjusting the tyrosinaseꢀresponse moiety and fluorophore
structure, a series of nearꢀinfrared fluorescence turn on probes
based on the conversion of nonꢀfluorescent substrate
molecules to fluorescent ones have been developed and
a
spontaneous
tyrosinase activity assay in extensive bioassays and clinic
researches, as well as in screening potential tyrosinase
inhibitors.
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EXPERIMENTAL SECTION
Chemicals and Materials. Sodium carbonate, resorcinol,
tyrosine, tyramine, dopamine, levodopa, dibasic dodium
phosphate and sodium dihydrogen phosphate were purchased
from Aladdin Industrial Corporation (Shanghai, China).
Tyrosinase from mushroom (EC 1.14.18.1), EcoR Ⅰ , bovine
serum albumin (BSA), human serum albumin (HSA), alkaline
phosphatase (ALP), acetylcholinesterase (AchE), glucose
oxidase (GOX), lysozyme, and trypsin were purchased from
SigmaꢀAldrich. The ultrapure water from a Millipore system
was used in all aqueous solution. All reagents were analytical
grade and used as received without any further purification.
16
utilized into the tyrosinase detection in complex biosystems.
However, the preparation and purification processes of such
new substrates are still timeꢀconsuming and apparently require
extensive synthesis experience and formidable background of
organic chemistry. Undoubtedly, it is still highly desirable to
develop more rational, straightforward and sensitive tyrosinase
activity assays, especially taking advantage of the
commercially available common molecules with fluorescence
turnꢀon response.
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As typical catechol derivatives, catecholamines such as
dopamine and levodopa have been generally employed as the
substrate and oxidized to dopamineꢀquinone/dopaquinone in
several effective tyrosinase activity assays. However,
catecholamines should also be considered as the
tyrosinaseꢀcatalyzed oxidation product of monophenolamines
(typically tyramine or tyrosine) based on the monophenolase
activity in fact. In 1991, Prota et al. has found that the
resorcinol can be used for oxidative coupling of levodopa and
significantly inhibit the oxidative conversion of levodopa to
Apparatus and Characterization. Fluorescence excitation
and emission spectra of all samples were recorded on a Hitachi
Fꢀ4600 spectrofluorometer (Tokyo, Japan). Absorption spectra
were obtained with a CARY 500 UV−Vis−NIR Varian
spectrophotometer (CA, USA). The mass spectrum analysis was
performed on
the electrospray ionization (ESI)ꢀQꢀTOF
SYNAPT G2 High Definition Mass Spectrometer (Waters,
UK) in the negative ion mode.
Dopamine Concentration Response. In 2.0 mL
microcentrifugals, volumes of 100 ꢁL of phosphate buffer (PB,
200 mM, pH = 7.0) and 1400 ꢁL of water were injected, 200
ꢁL of dopamine aqueous solution with different dopamine
concentrations ranging from 0 to 100 ꢁM were added,
respectively. 200 ꢁL of 1.0 mM resorcinol solution and 100
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dopachrome in an aqueous solution. Acuna et al. has recently
described in detail the reactions between resorcinol and
catecholamines, and identified the rapid generation of
fluorescent azocine products at ambient temperature and
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pressure. More significantly, we have further confirmed that
similar fluorogenic reactions cannot be implemented and no
fluorescent product can be obtained if using the
monophenolamines instead of catecholamines.
ꢁ
L of 500 mM sodium carbonate solution were successively
injected into the above mixture solutions. The fluorescence
spectra and measurements were carried out after vibration at
room temperature for 5 min.
Inspired by such specific fluorogenic reactions between
resorcinol and catecholamines, we have proposed to introduce
it into the detection of tyrosinase, which is capable of
catalyzing the oxidation of monophenolamine substrates to
catecholamines. In the subsequent proof of concept of
fluorescent tyrosinase activity assay, we have first chosen
tyramine as the monophenolamine substrates of tyrosinase,
and then demonstrated that the tyrosinaseꢀincubated tyramine
solution could emit intense blue fluorescence under ultraviolet
light in the presence of resorcinol. The overall process takes
less than 100 min with exceedingly facile operation procedure
at ambient temperature and pressure conditions, and
accompanied with considerable fluorescence quantum yield of
Sensing Tyrosinase Activity. A fluorescent tyrosinase
activity assay was performed using the following procedures.
Volumes of 100 ꢁL of PB (200 mM, pH = 7.0), 200 ꢁL of 2.0
mM tyramine aqueous solution, 1250 ꢁL of water were
injected into a 2.0 mL microcentrifugal tube. Then 200 ꢁL of
tyrosinase aqueous solution with different activities ranging
ꢀ1
from 0 to 8 U mL were added into the mixtures, respectively.
After incubation at 37°C for 90 min, 200 ꢁL of 2.0 mM
resorcinol solution and 100 ꢁL of 500 mM sodium carbonate
solution were successively injected into the above mixture
solutions. The fluorescence and absorption spectra
measurements were carried out after vibration at room
temperature for 5 min. The selectivity of this sensing system
was assessed by using other control proteins/enzymes and
biological cations in the absence and presence of tyrosinase,
respectively.
3
2.5% for the product solution. At the same time, the resultant
fluorescent azocine product of the sensing system exhibits
obvious pale yellow under visible light and possesses
characteristic absorption peak centered at 420 nm. Therefore,
we have developed a fluorometric and colorimetric dual
readꢀout assay for tyrosinase activity based on the formation
of fluorophore from scratch, and the fluorescence intensities
and absorption values of the tyramineꢀresorcinol system
increased gradually with the tyrosinase concentrations. Such
innovative in situ fluorogenic reactionꢀbased sensing system
can further be extended into the inhibitor screening of
tyrosinase. Kojic acid and benzoic acid, the common inhibitors
of tyrosinase, are employed to assess the function of inhibitor
screening, and IC50 of these two inhibitors are calculated to be
approximate 155 ꢁM and 1582 ꢁM, respectively. Furthermore,
the proposed system exhibits promising prospect for
Real Sample Detection. The proposed fluorescence assay
was applied to detect the tyrosinase activity in the diluted fetal
bovine serum (1%) and human serum samples (1%),
respectively.
Tyrosinase Inhibition Assay. Kojic acid and benzoic acid,
common inhibitors of tyrosinase, were employed to assess the
function of inhibitor screening. We initially added various
concentrations of kojic acid and benzoic acid solutions into the
ꢀ
1
freshly prepared tyrosinase solution (4 U mL ), respectively.
After vibration at 37°C for 10 min, these inhibitorꢀtreated
tyrosinase solutions were injected into tyramine (200 ꢁM)
solution in PB (10 mM, pH = 7.0). After incubation at 37°C
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intensities at 460 nm enhanced with the reaction time, and the
for 90 min, the resorcinol and sodium carbonate solution were
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successively injected. The fluorescence spectra measurements
were carried out after vibration at room temperature for 5 min.
fluorescence intensities and absorption values reaches the
maximum values at 5 min and keep constant after that,
demonstrating the reactions could be drastically completed in
only 5 min. In this scheme, catecholamine oxidation, first to
semiquinone and then to the corresponding quinone, is
required for the formation of the fluorescent product, and the
nucleophilic attack of resocinol monoanion under alkaline
condition to the quinone species is the crucial initial reaction
step . Under alkaline condition, the OH group in
azamonardine became anionic, accompanied with intense
absorption band and strong fluorescence, and the reaction time
shortened considerably as well. Hence, it can be concluded
that the fluorogenic and chromogenic reaction between
dopamine and resorcinol is rapid and specific with explicit
mechanism in an alkaline aqueous solution at ambient
temperature and pressure conditions.
RESULTS AND DISCUSSION
Fluorogenic Reactions between Catecholamines and
Resorcinol. As typical catechol derivatives, catecholamines,
especially dopamine used to be employed as the substrate in
the tyrosinase activity assays, where the chemical or physical
properties between dopamine and the corresponding
enzymolysis product dopamineꢀquinone can be differentiated
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by several sensors.
However, dopamine should also be
considered as the tyrosinaseꢀcatalyzed oxidation product of
tyramine based on the monophenolase activity in fact.
Recently, it is reported that a category of biocompatible
fluorogenic
resorcinol) and 1,2ꢀdihydroxybenzene derivative (catechols or
catecholamines) would take place in the aqueous solution, to
reactions
between
1,3ꢀdihydroxybenzene
(
In contrast, we have speculated that the monophenolamines
would not be oxidated under alkaline condition by resorcinol
to yield fluorophores owing to the absence of oꢀdiphenol and
corresponding oꢀquinone moiety, which is an essential
prerequisite to the aforementioned fluorogenic reactions. In
order to verify our assumption, we have investigated the
absorption and fluorescence spectra of tyramine, dopamine,
resorcinol and the special mixture solutions, respectively
(Figure 1). After resorcinol was introduced to dopamine
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yield fluorescent oxocines or azocines. In the case of
dopamine (Scheme 1), the colorless and nonꢀemissive aqueous
solution could quickly convert into pale yellow, with bright
blueꢀemitting fluorescence owing to the generation of
azamonardine fluorophore in the presence of resorcinol under
alkaline condition at room temperature. We have further
evaluated the specificity and optimum condition of the
reaction by altering experimental parameters. It is suggested
that vigorous stirring in the open air can obviously accelerate
the fluorogenic reaction due to the ceaseless dissolution of
molecular oxygen in aqueous solution (data not shown).
solution under alkaline condition in the presence of O , a
2
typically intense absorption band centered at 420 nm appeared
(line 5), and bright blueꢀemitting fluorescence was observed
when excited at 415 nm, which is analogous to the
characteristic peak of the product of the abovementioned
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fluorogenic reactions as reported in the literature . In that
literature, the NMR and mass spectra analysis of the product
solution have further conformed the structural formula of the
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product (azamonardine) with a molecular weight of 259 Da.
Under the same reaction condition, we submitted the resultant
solution of dopamine reacting with resorcinol to ESIꢀMass
spectrum in the negative ion mode. As shown in Figure S5A,
the ion peak at m/z=258.02 (blue line) was regarded as the
quasiꢀmolecular ion peaks of azamonardine and attributed to
Scheme 1. Chemical structure of monophenolamines substrates
ꢀ
[azamonardineꢀH] , and in our opinion, the product is exactly
(tyramine, tyrosine), catecholamines (dopamine, levodopa),
azamonardine. However, if we choose tyramine instead of
dopamine, there is no typical absorption and negligible
fluorescence of the mixture solution under the similar
experimental conditions (line 4). The above distinction of
fluorescence and absorption spectra caused by tyramine and
dopamine may lay the foundation for tyrosinase assay based
on its monophenol oxidation activity.
resorcinol, and the corresponding azocine products (azamonardine,
carboxyazamonardine).
Particularly, we have carried out several experiments to
investigate the influences of pH, resorinol concentrations,
reaction time on the fluorogenic reaction between dopamine
and resorcinol. The assessment of the pH value of the reaction
system was firstly performed at different pH ranging from 5 to
12, and the results were shown in Figure S1B. It could be
indicated that the optimal pH value is 11, which happens to be
similar to the pH of Na CO solution in fact. Thus, we have
2
3
chosen to employ Na CO solution as the buffer solution in the
2
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subsequent experiments, and a concentration of 25 mM was
2
adopted after the optimization of the concentration of Na CO3
solution (Figure S1D). At a constant dopamine concentration
(
100 ꢁM), both the fluorescence intensity and absorption value
increased with resorcinol concentrations ranging from 0 to 100
M (Figure S2), implying that it is equimolar reaction between
ꢁ
dopamine and resorcinol. Furthermore, the fluorescence
spectra of the reaction solution were discontinuously measured
at room temperature to evaluate the effect of reaction time.
The results in Figure S3 displayed that the fluorescence
Figure 1. (A) Schematic representation of dopamine reacting with
resocinol in synthesis of fluorescent azamonardine. (B)
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Absorption and (C) fluorescence spectra of tyramine (1),
3 B, C) attributed to characteristic peak of azamonardine
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dopamine (2), resorcinol (3), mixture of tyramine and resorcinol
(4) and mixture of dopamine and resorcinol (5) under the same
condition, respectively. The insert photographs show the
corresponding solution under UV lamp at 365 nm.
fluorophore (blue line in Figure 3 B, C) with strong blue
fluorescence, and the ion peaks at m/z=258.02 attributed to the
quasiꢀmolecular ion peaks of azamonardine (blue line in
Figure S5C). All of above results demonstrate the
preꢀintroduction of tyrosinase essentially triggers fluorogenic
reaction through the hydroxylation of tyramine as we expected
(Figure 3A). Thus, the ignorable fluorescence and absorbance
signal in the absence of tyrosinase illustrates that tyramine
could not participate in the fluorogenic reaction, and
fluorescence “turnꢀon” assay of tyrosinase activity could be
accomplished with tyramine as the substrate specifically. Most
significantly, the distinction of fluorescence intensities and
absorbance values caused by tyrosinase demonstrates
tyrosinaseꢀdependent fluorometric and colorimetric signal,
which is expected to be exploited for tyrosinase activity assay
and its inhibitor screening.
Design and Establishment of Tyrosinase Sensing System.
Since buffer solution is usually required in clinical and
bioassay applications, we carried out the above fluorogenic
reactions in 10 mM PB to assess the influence of the buffer on
the fluorogenic reactions, and the results displayed no
significant change of the fluorescence intensity (Figure S4),
indicating the latent ability in bioanalysis and clinic diagnosis
values. On the other hand, we have investigated the effect of
dopamine concentration on the absorption and fluorescence
spectra of the resultant solution at the constant resorcinol
level. As shown in Figure 2, the absorbance value at 420 nm
and fluorescence intensity at 460 nm of the product solution
enhanced gradually with dopamine concentrations ranging
from 0 to 100 ꢁM. Besides, there was no change of the shape
of the absorbance and fluorescence spectra, reflecting the
formation of the same and unitary product with different
concentrations of dopamine. Thus, the dopamine
concentrationꢀdependent linear relationships further offer
favorable conditions for tyrosinase sensing.
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Figure 3. (A) Schematic representation of tyrosinaseꢀenabled in
situ synthesis of fluorescent azamonardine. (B) Absorption
spectra of product solution of the fluorogenic reactions using
tyramine in the absence (black line) and presence (red line) of
tyrosinase, respectively, and azamonardine (blue line),
fluorescence excitation and emission spectra of the product of
tyrosinaseꢀincubated tyramine solution in aqueous solution. The
insert photographs show the tyrosinaseꢀincubated tyramine
solution under natural light (left) and UV lamp at 365 nm (right),
respectively. (C) Fluorescence emission spectra of corresponding
product solution excited at 415 nm.
Figure 2. (A)Absorption and (C) fluorescence emission spectra at
different concentrations of dopamine. (B) Absorption values at
4
20 nm and (D) fluorescence intensities at 460 nm under different
Afterwards several experiments have been implemented to
investigate the effect of tyramine concentration, resorinol
concentration and incubation time on the fluorescence
intensity of the proposed system. The concentrations of both
tyramine and resorinol were firstly optimized to 200 ꢁM
(Figure S6). Moreover, the incubation time of tyrosinase was
evaluated with 200 ꢁM tyramine under different levels of
concentrations of dopamine
.
Inspired by the different fluorogenic reaction capacity with
resorcinol between tyramine and dopamine, we have proposed
to utilize tyramine as substrate to assay the activity of
tyrosinase. Tyramine is a common and commercial available
substrate of tyrosinase, while it has been scarcely employed in
tyrosinase activity sensing. Tyrosinase is capable of catalyzing
the hydroxylation of tyramine to dopamine based on its
ꢀ1
tyrosinase (1, 2, 4 U mL ) in 10 mM PB (pH=7), and the
results in Figure S7 illustrated that the fluorescence intensities
enhanced promptly with the incubation time in the first 90 min,
and then increased gently or keep constant between 90ꢀ150
min. In order to save time, incubation time of 90 min was
recommended in the subsequent experiment. More importantly,
resorcinol and its derivatives served as the inhibitor of
tyrosinase have been widely used to reduce the pigmentation
monophenol oxidation activity in the presence of O . After
2
resorcinol was injected into the preincubated tyramine solution
in the absence of tyrosinase, the absorption spectra displayed
simply the summation of the characteristic peak of tyramine
and resorcinol, and there was no new typical absorption peak
and negligible fluorescence (black line in Figure 3 B, C).
Besides, there was no obvious ion peak at m/z=258.02 in
ESIꢀMass spectrum of the resultant solution (Figure S5B),
which indicates azamonardine could not be generated in the
mixture solution of tyramine and resorcinol without tyrosinase
preincubation. In contrast, after resorcinol solution was added
into the tyramine solution preꢀincubated with tyrosinase, the
typical absorption band centered at 420 nm (red line in Figure
37ꢀ39
in humans , which means that the addition of resorcinol to
the tyrosinaseꢀincubated tyramine solution could terminate the
function of tyrosinase in the following fluorogenic reaction. It
has been verified by the preꢀtreatment of resorcinol with
tyrosinase, which generates negligible fluorescence under the
same condition, as shown in Figure S8.
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In a word, the proposed tyrosinaseꢀenabled fluorogenic and
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chromogenic reaction could be implemented efficiently with
200 ꢁM tyramine preꢀincubated with tyrosinase for 90 min,
followed by addition of 200 ꢁM resorcinol with reaction time
of 5 min in 25 mM Na CO solution at room temperature. The
overall process takes less than 100 minutes with easy
operation procedure. Furthermore, the fluorescence quantum
yield of the resultant mixture solution have been measured and
calculated to be as high as 32.5%, which indicates great
potential and high sensitivity in the “turnꢀon” assay of
tyrosinase and its inhibitor screening.
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Fluorescent and Colorimetric Dual-Readout Assays of
Tyrosinase Activity. Under the above optimal conditions, we
developed a fluorometric and colorimetric dual readꢀout
sensing assay for tyrosinase activity by using tyramine as the
substrate. The assay of tyrosinase activity was carried out by
using 200 ꢁM tyramine as substrate in 10 mM PB at pH 7.0
with an incubation time of 90 min, followed by adding of
resorcinol and Na CO solutions to ultimate concentrations of
2
3
2
00 ꢁM and 25 mM, respectively. The fluorescence spectra
and absorbance spectra measurements were carried out after
vibration at room temperature for 5 min. As shown in Figure
4A, the fluorescence intensity at 460 nm of the product
solution increased as tyrosinase concentrations ranging from 0
ꢀ
1
Figure 4. (A) Fluorescence emission spectra at different
tyrosinase concentrations excited at 415 nm. (B) Fluorescence
intensities at 460 nm versus different tyrosinase concentrations.
to 8 U mL . In particular, there is a linear relationship
between fluorescence intensities and tyrosinase activities over
ꢀ
1
the range from 0.005 to 3.5 U mL , where the fitted linear
equation can be described as I = − 5.06 + 365.4 C (U
(C) Absorbance spectra enhancement with different tyrosinase
tyrosinase
ꢀ
1
2
concentrations. (D) Absorbance values at 420 nm versus different
tyrosinase concentrations. (E) Corresponding photographs under
365 nm UV light (1) and natural light (2). (F) Fluorescence
responses of the proposed system against the control enzymes (2
mL ), R = 0.993, as depicted in Figure 4B. Moreover, the
absorption values at 420 nm increased as tyrosinase
concentrations ranging from 0 to 8 U mL , and there is a
linear relationship between absorption values and tyrosinase
ꢀ
1
ꢀ1
ꢀ1
U mL ) or proteins (10 ꢁg mL ) and biological cation (250 ꢁM)
ꢀ
1
activities over the range from 0.01 to 8.0 U mL , where the
2+
except for Cu (50 ꢁM) in the absence and presence of tyrosinase
2 U mL ).
fitted linear equation can be described as I = − 0.0062 + 0.107
ꢀ1
(
ꢀ1
2
Ctyrosinase (U mL ), R = 0.992, as depicted in Figure 4D. In
Similarly, the assay of tyrosinase activity can also be
realized by using tyrosine as substrate, in which tyrosine can
be transformed to levodopa by tyrosinase in the presence of O2,
addition, the fluorescent color change of the product solutions
ꢀ
1
induced by 0.1 U mL of tyrosinase activity can be detected
with nakedꢀeye readout under the UV lamp, and color change
ꢀ
1
followed
by
generating
strong
fluorescent
of the product solutions induced by 0.5 U mL of tyrosinase
activity can be detected under the natural light (Figure 4E). It
is noteworthy that fluorescent readout assay of the proposed
system displays higher sensitivity while colorimetric readout
assay exhibits wider linear range. More importantly, our
labelꢀfree fluorescent and colorimetric dualꢀreadout assay
reveals a low limit of quantitation and a wide linear range
compared to those of spectroscopic tyrosinase assay methods
in the previously reported literatures in Table S1.
36
carboxyazamonardine after the introduce of resorcinol under
similar conditions. The fluorescence intensities at 460 nm of
the product solution varied as tyrosinase concentrations
ꢀ1
ranging from 0 to 8 U mL as well, and there is a linear
relationship between fluorescence intensities and tyrosinase
ꢀ
1
activities over the range from 0.01 to 3 U mL , where the
fitted linear equation can be described as I = − 10.7+ 224.4
ꢀ
1
2
Ctyrosinase (U mL ), R = 0.98, as depicted in Figure S9. At the
same time, colorimetric readꢀout assay of tyrosinase can also
be implemented with tyrosine as substrate under the same
condition (data not shown).
To further investigate the biospecificity of the sensing
system, BSA, HSA, ALP, GOX, AchE, EcoR I, lysozyme, and
+
2+
3+
trypsin, and several kinds of biological cations (K , Zn , Fe ,
2+
2+
2+
2+
Ba , Mn , Ni , Cu ) were used as controls in the presence
and absence of tyrosinase, respectively. The results in Figure
4
F have explicitly illuminated that none of these control
proteins/enzymes/cations generates distinct fluorescence
response in the in situ fluorogenic reaction. Above all, the
proposed sensitive and selective probe for tyrosinase activity
can be fulfilled in the presence of other proteins and
biologically essential metal ions, and it exhibits great potential
in extensive applications.
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Determination of Tyrosinase in Serum Sample. In order
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to further investigate the potential bioassay applications, our
developed fluorescent tyrosinase sensing system was carried
out in the diluted fetal bovine serum (1%) and human serum
samples (1%), respectively. The fluorescence response results
in the diluted biological samples were compared with that in
the standard colorimetric assay system by using tyrosine as
substrate, in which tyrosinase causes an increase of absorbance
at A280. The comparative results in Table 1 unambiguously
illustrates our proposed labelꢀfree system possesses great
potential to monitor the tyrosinase activity in biological
samples.
0
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Table 1. Results of tyrosinase activity in the analysis of serum
samples
.
a
b
Sample
Spiked
0
Found
Found
Fetal
bovine
0.052 ± 0.003
0.143 ± 0.004
0.578 ± 0.018
1.046 ± 0.039
0.049 ± 0.002
0.146 ± 0.005
0.565 ± 0.020
1.058 ± 0.050
Figure 5. Fluorescence emission spectra at different
concentrations of benzoic acid (A) and kojic acid (C). Kinetic
plots of the fluorescence intensity of sensing system in 4 U mL
0.1
0.5
serum （
ꢀ
1
1.0
1
%）
tyrosinase against the logarithm of the kojic acid (B) and benzoic
acid (D) concentrations.
Human
serum
0
0.075 ± 0.003
0.165 ± 0.003
0.597 ± 0.024
1.12 ± 0.035
0.081 ± 0.004
0.158 ± 0.003
0.614 ± 0.033
0
.1
0.5
.0
（
1%）
CONCLUSION
1
1.06 ± 0.045
b
a
In summary, it is meticulously demonstrated that the
specific reactions between resorcinol and catecholamines can
be carried out and generates the typical fluorescent azocine
products in aqueous solution at ambient temperature and
pressure. Inspired by such fluorogenic/chromogenic reactions
and the specific oxidation ability of monophenolamine
substrates to catecholamines by tyrosinase, an exquisite
labelꢀfree fluorescent and colorimetric dualꢀreadout assay of
tyrosinase activity have been proposed and evaluated. To the
best of our knowledge, it is the first dualꢀreadout assay of
tyrosinase activity based on in situ fluorogenic and
chromogenic reactions. Under the optimum conditions, this
convenient detection system possesses the wide linear
detection range and high sensitivity that can effortlessly
obtained with proposed fluorescent readꢀout assay, obtained with
standard colorimetric assay system (U mL )
ꢀ
1
Tyrosinase Inhibition Assay. Considering the importance
of tyrosinase in melanin formation and browning process,
tyrosinase inhibitors have been widely used in skin
hyperpigmentation and food antibrowning treatment. Thus, the
development of a convenient and efficient screening method
for tyrosinase inhibitors is of great importance to both
cosmetic and food industry. Accordingly, the proposed
fluorescence assay was further extended to evaluate the
efficiency of tyrosinase inhibitor in vitro by using two
common inhibitors (kojic acid and benzoic acid) as a model.
Tyrosinase was preincubated with each inhibitor before being
submitted to the tyramine solution, and the tyrosinaseꢀenabled
fluorogenic reaction was monitored by measuring fluorescence
intensities at 460 nm. As depicted in Figure 5A, the
fluorescence intensities reduced with the kojic acid and
benzoic acid concentrations ranging from 20 ꢁM to 155 ꢁM
and from 100 ꢁM to 31.62 mM, respectively. Generally, the
concentrationꢀdependent slowing down of fluorescence
intensities of the proposed system indicates tyrosinase activity
has been efficaciously inhibited by each inhibitor. Furthermore,
representative sigmoidal curves have been obtained from the
plots of fluorescence intensities versus the logarithms of the
tyrosinase concentrations. The IC50 values (the inhibitor
concentrations where tyrosinase activity is inhibited by 50%)
of kojic acid and benzoic acid were calculated to be
approximately 155 ꢁM and 1582 ꢁM, respectively, which is of
the same order of magnitude compared with those previously
ꢀ1
evaluate 0.1 U mL of tyrosinase activity with nakedꢀeye
readout under an ultraviolet lamp. Moreover, the innovative
tyrosinaseꢀenabled fluorogenic reactionꢀbased sensing system
exhibits promising prospect for tyrosinase activity assay in
extensive bioassays and clinic researches, as well as in
screening potential tyrosinase inhibitors. We envision that the
specific and attractive reactions between resorcinol and
catecholamines could become a versatile tool for assaying
several other enzymes by rationally designing and utilizing
other novel substrate in clinical examination and diagnosis in
the near future.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
1
9,40
reported tyrosinase activity assays
.
Detailed data of parameter optimization, additional spectra,
fluorescent readꢀout assay data with tyrosine as substrate, and
data comparisons (PDF)
AUTHOR INFORMATION
Corresponding Author
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*
+
Eꢀmail: xryang@ciac.ac.cn. Phone: +86 431 85262056. Fax:
86 431 85689278
(16) Wu, X.; Li, L.; Shi, W.; Gong, Q.; Ma, H. Angew. Chem. Int.
Ed. 2016, 55, 14728ꢀ14732.
17) GarciaꢀMolina, F.; Munoz, J. L.; Varon, R.; RodriguezꢀLopez,
J. N.; GarciaꢀCanovas, F.; Tudela, J. J. Agr. Food. Chem. 2007, 55,
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* Eꢀmail: jiansun@ciac.ac.cn. Phone: +86 431 85262063. Fax:
+86 431 85689278.
9
739ꢀ9749.
18) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77,
(
Author Contributions
1
566ꢀ1571.
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
(19) Kim, T. I.; Park, J.; Park, S.; Choi, Y.; Kim, Y. Chem.
Commun. 2011, 47, 12640ꢀ12642.
20) Sun, J.; Wang, B.; Zhao, X.; Li, Z. J.; Yang, X. R. Anal.
Chem. 2016, 88, 1355ꢀ1361.
21) Li, Y.; Li, G.; Lei, K. L.; Li, M.; Liu, H. H. Chinese. J. Anal.
(
(
ACKNOWLEDGMENT
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Chem. 2016, 44, 773ꢀ778.
(22) Cui, Y. Y.; Chen, Z. P.; Yan, X. F.; Yu, R. Q. Chinese. J.
Anal. Chem. 2016, 44, 1250ꢀ1256.
We thank the financial supports by the National Key Research
and Development Program of China (2016YFA0201301), the
National Natural Science Foundation of China (No. 21435005,
(
23) Sun, J.; Hu, T.; Chen, C. X.; Zhao, D.; Yang, F.; Yang, X. R.
2
1627808, 21605139), Key Research Program of Frontier
Anal. Chem. 2016, 88, 9789ꢀ9795.
(24) Teng, Y.; Jia, X.; Li, J.; Wang, E. Anal. Chem. 2015, 87,
Sciences, CAS (QYZDYꢀSSWꢀSLH019).
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(25) Zhang, W. H.; Ma, W.; Long, Y. T. Anal. Chem. 2016, 88,
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