Detection of tyrosine and monitoring tyrosinase activity using an enzyme cascade-triggered colorimetric reaction

Article abstract of DOI:10.1039/d0ra05581f

The aromatic amino acid tyrosine is an essential precursor for the synthesis of catecholamines, including l-DOPA, tyramine, and dopamine. A number of metabolic disorders have been linked to abnormal tyrosine levels in biological fluids. In this study, we developed an enzyme cascade-triggered colorimetric reaction for the detection of tyrosine, based on the formation of yellow pigment (betalamic acid) and red fluorometric betaxanthin. Tyrosinase converts tyrosine to l-DOPA, and DOPA-dioxygenase catalyzes oxidative cleavage of l-DOPA into betalamic acid. Response is linear for tyrosine from 5 to 100 μM, and the detection limit (LOD) is 2.74 μM. The enzyme cascade reaction was applied to monitor tyrosinase activity and tyrosinase inhibition assays. Lastly, the performance of the proposed biosensor proved successful in the analysis of urine samples without the need for pre-treatment. This journal is

Full text of DOI:10.1039/d0ra05581f

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Detection of tyrosine and monitoring tyrosinase
activity using an enzyme cascade-triggered
colorimetric reaction†
Cite this: RSC Adv., 2020, 10, 29745
*
Huei-Yu Chen and Yi-Chun Yeh
The aromatic amino acid tyrosine is an essential precursor for the synthesis of catecholamines, including L-
DOPA, tyramine, and dopamine. A number of metabolic disorders have been linked to abnormal tyrosine
levels in biological fluids. In this study, we developed an enzyme cascade-triggered colorimetric reaction
for the detection of tyrosine, based on the formation of yellow pigment (betalamic acid) and red
fluorometric betaxanthin. Tyrosinase converts tyrosine to L-DOPA, and DOPA-dioxygenase catalyzes
oxidative cleavage of L-DOPA into betalamic acid. Response is linear for tyrosine from 5 to 100 mM, and
the detection limit (LOD) is 2.74 mM. The enzyme cascade reaction was applied to monitor tyrosinase
activity and tyrosinase inhibition assays. Lastly, the performance of the proposed biosensor proved
successful in the analysis of urine samples without the need for pre-treatment.
Received 26th June 2020
Accepted 30th July 2020
DOI: 10.1039/d0ra05581f
rsc.li/rsc-advances
Tyrosinase converts Tyr to L-DOPA; i.e., the substrate of
DOPA-dioxygenase (DOD). Note that DOD is an oxidase that
1. Introduction
opens the cyclic ring between carbon 4 and 5 on L-DOPA,
resulting in the synthesis of yellow betalamic acid,22–24 which is
a precursor of betalains/betaxanthins used as readouts of
optical biosensors (Scheme 1).25–27 Thus, we developed an
enzyme cascade-triggered colorimetric biosensor for Tyr
involving the synthesis of yellow betalamic acid and red betax-
anthins products. We then evaluated the proposed biosensor in
terms of Tyr detection.
Tyrosine (Tyr) is an aromatic amino acid critical to the synthesis
of compounds such as neurotransmitters and melanin.1,2
Abnormal Tyr concentrations in plasma/urine can also be used
as a biomarker in the detection of various diseases, such as
alkaptonuria, tyrosinemia, and liver disease.3–5 Therefore, an
easy quantication assay of Tyr is important and necessary.
Researchers have developed numerous methods for the
quantication of Tyr, including uorometric methods,6–10 high-
performance liquid chromatography,11 electrochemical detec-
tion,12–14 colorimetric assays,15 and chemiluminescence;16
however, implementing such methods is expensive and
complex. In this study, we sought to facilitate the quantication
of Tyr for diagnostic purposes by developing an enzyme-based
colorimetric biosensor to reduce the possible interferences
from catecholamine metabolites. The approach has proven
highly effective, particularly in real-time detection.
Tyrosinase is a copper-containing oxidase, capable of cata-
lyzing the hydroxylation of monophenol to catechol, and the
subsequent oxidation to o-quinone,17 which carries a chromo-
phoric group suitable as a biosensor readout.18 However, tyrosine
and tyrosine analogs (e.g. tyramine) are also substrates of tyros-
inase, and o-quinone is susceptible to non-enzymatic reactions
resulting in the spontaneous formation of melanin.19 We there-
fore developed a sequential cascade reaction involving multiple
enzymes to serve as a signal lter and amplier.20,21
2. Experimental methods
2.1 Chemicals
2-Aminophthalic acid (2-AIPA), epinephrine, glucose oxidase,
lysozyme, norepinephrine, para-aminobenzoic acid (PABA),
tyrosinase, and tryptophan were purchased from Sigma-Aldrich.
L-Ascorbic acid (AA), carbenicillin, iron(II) sulfate, isopropyl b-D-
1-thiogalactopyranoside (IPTG), and protease inhibitor cocktail
were purchased from Amresco. Glutamic acid (Glu), L-DOPA,
and phenylalanine (Phe) were purchased from ACROS. para-
Phenylenediamine and tyramine were purchased Alfa Aesar.
Calcium chloride, magnesium chloride hexahydrate, potassium
chloride, and tyrosine were purchased from Merck. Sodium
chloride and trypsin were purchased from VWR Life Science
and Promega, respectively. Articial urine (AU) was purchased
from GE Healthcare Life Sciences.
Department of Chemistry, National Taiwan Normal University, Taiwan. E-mail:
yichuny@ntnu.edu.tw
2.2 Expression and purication of DOPA-dioxygenase
E. coli BL21 (DE3) (Invitrogen) carrying pET21a-DOD-histag
plasmid was inoculated in LB medium containing 1 mM of
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra05581f
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Scheme 1 Enzyme cascade-triggered colorimetric reaction for Tyr analysis based on the synthesis of betalamic acid and betaxanthins.
ꢀ
carbenicillin at 37 C for 16–18 h. Then, the overnight culture tyramine, norepinephrine, epinephrine, phenethylamine,
was diluted in fresh LB (1 : 100; v%) with 1 mM of carbenicillin phenylalanine, tryptophan and ascorbic acid at 100 mM for
and incubated at 37 ꢀC until O.D.600 reached 0.4–0.6, and 1 mM interference tests respectively.
of IPTG was used to induce protein expression at 18 ꢀC for 22 h.
Next, the pellets were lysed in lysis buffer containing protease
inhibitor by sonication aer the harvest of the cells by centri-
fugation at 9000 rpm and 4 ꢀC (lysis buffer containing 25 mM of
Tris–HCl with 200 mM of NaCl). Then, the lysate was centri-
fuged at 9000 rpm and 4 ꢀC for 1 h to remove cell debris. Aer
that, HisPur™ Ni-NTA Resin Spin Columns (Thermo Scientic)
were used to purify the proteins in the supernatant, which was
ltered through a 0.22 mm syringe lter, and the puried
proteins were dialyzed against 25 mM of phosphate buffer with
100 mM of NaCl (pH 6.8) for 24 h.
2.4 Characterization and quantication of betaxanthins
The reaction solutions containing Tyr at various concentrations
were mixed with 10 mM primary amines. The uorescence
intensity of betaxanthins was detected on F-7000 uorescence
spectrophotometer (HITACHI) aer the enzymatic conversion
of Tyr to betalamic acid and further the non-enzymatic
condensation of betalamic acid with amines to synthesis
betaxanthins at room temperature for 1 h.
2.5 Preparation of articial urine samples
Articial urine was diluted 10-fold with sodium phosphate
buffer before all measurements.
2.3 Detection of tyrosine by the enzyme cascade-triggered
colorimetric reaction and interference study
This colorimetric biosensor for Tyr analysis was incubated with
various concentrations of Tyr in the reaction medium contain-
ing sodium phosphate buffer (25 mM) with NaCl (100 mM) (pH
6.8), iron(II) sulfate (0.5 mM), ascorbic acid (10 mM),27 tyrosi-
3. Results and discussion
3.1 Characterization and optimization of betalamic acid
production
nase (224 mM), and DOD (12.5 mM). The optical signal was Using absorption spectroscopy, we began by determining the
detected by measuring the absorption spectroscopy in 96-well optimal conditions for the enzyme cascade system based on the
plates in a Synergy HT microplate reader (BioTek, Winooski, VT) detection of the yellow betalamic acid. Absorption intensity at
aer the enzyme cascade-triggered reaction at room tempera- 430 nm was shown to increase with the concentration of Tyr.
ture for 0.5 h. The reaction medium without Tyr was mixed with The colorimetric sensor system was also assessed under various
Fig. 1 (A) Absorption spectra of reaction mixtures with various concentrations of Tyr (0, 5, 10, 20, 40, 80, and 100 mM). (B) The linear range for Tyr
was between 5 and 100 mM, and LOD was 2.74 mM. The corresponding photograph under the natural light in the presence of Tyr.
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Fig. 2 (A) The selectivity of the enzyme cascade-based system in the presence of other substrates at 100 mM was investigated. The intensity of
absorbance at 430 nm after 0.5 h reaction was recorded. (B) The intensities of l430 in the co-presence of Tyr at 100 mM and 1/10/50/100 mM of
tyramine.
pH values in order to determine the optimal buffer conditions completion time of proposed sensor is comparable to methods
(Fig. S1A†). Our results indicate that pH 6.8 is ideal for this which are commonly used.
system.
We then sought to optimize the concentrations of tyrosinase
3.2 Determining substrate specicity of the tyrosine sensing
system
and DOD in the proposed system. We began by varying the
concentration of tyrosinase between 0 to 224 mM in the presence
of DOD (8 mM) and Tyr (50 mM) following incubation for a period
of 0.5 h. We observed a notable increase in absorption inten-
sities at 430 nm at the highest tyrosinase concentration of 224
mM (Fig. S1B†). We then increased the concentrations of DOD
from 8 to 20 mM in the presence of tyrosinase (224 mM) and Tyr
(50 mM) (Fig. S1C†). We did not observe a notable increase in
l430 signal intensity when the DOD concentration was increased
beyond 12.5 mM. Thus, throughout the remainder of the study,
the enzyme cascade-based biosensor was implemented using
224 mM of tyrosinase and 12.5 mM of DOD.
We closely monitored the l430 signal intensity throughout
the time-course of the reaction (Fig. S1D†). Note that the initial
increase in intensity plateaued within 1 h. Thus, in all subse-
quent experiments, we recorded the absorption intensity at
430 nm aer incubation for 0.5 h in order to enable rapid
detection with reasonable incubation. The results suggest the
Under optimal experimental conditions, a dramatic increase in
the absorption intensity of betalamic acid was observed upon
the concentration of Tyr increased (Fig. 1A). The absorption
intensity of betalamic acid was shown to increase linearly with
the addition of Tyr at concentrations of 5 to 100 mM (R2 ¼ 0.99)
(Fig. 1B). According to the 3sb/slope, the detection limit (LOD)
was 2.74 mM (sb: standard deviation of background and the
slope in the calibration plot).
The selectivity of the biosensor was evaluated by mixing the
reaction medium with interfering substrates, including struc-
tural analogs (tyramine, norepinephrine, epinephrine, and
phenethylamine) and metabolic intermediates (phenylalanine,
tryptophan, and ascorbic acid) at concentrations of 100 mM,
respectively. It was found that tyramine (structurally similar to
Tyr) subtlety increased the intensity of the l430 signal (Fig. 2A).
Therefore, to test the response to interference in the co-
Fig. 3 The intensity of (A) the red fluorescence intensities of 2-AIPA–betaxanthin (F ꢁ F0) and (B) l ꢁ l0 for betalamic acid was observed as
a linear function with the concentrations of Tyr (F0 and l0 represent the background absorbance and fluorescence of blank matrices). (A) The
linear ranges for Tyr was from 2.5 to 20 mM, and LOD was 0.92 mM. The effectiveness of enzyme cascade-based biosensor in buffer and 10%
artificial urine matrix was compared (red dots). Inset: the corresponding fluorescence photograph in the presence of Tyr.
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presence of tyramine and Tyr, we compared the enhanced
intensities of l430 with that of a single substrate and a mixture of
the two. In addition, we studied the responses using various
tyramine to Tyr ratios (1, 10, 50, 100 mM) and Tyr (100 mM). As
shown in Fig. 2B, the absorbance intensity at 430 nm has not
interfered. Thus, the effect of tyramine can be considered
negligible.
3.3 Stability of the enzyme cascade-based sensor
Stability is a crucial concern in assessing any biosensor. In this
study, we compared the stability of the proposed enzyme
cascade-triggered biosensor in the detection of Tyr (based on
the formation of betalamic acid) and the tyrosinase system
(based on the formation of dopachrome). Tyrosinase could lead
to the hydroxylation of Tyr to L-DOPA and subsequently catalyze
the oxidation of L-DOPA to dopachrome leading to an increase
in absorption at 475 nm (Fig. S2A†).17,18 We therefore added Tyr
(at various concentrations) to a reaction medium containing
phosphate buffered saline (pH 7.4) and 224 mM tyrosinase. We
then observed the stability of the two systems at regular inter-
vals by measuring l430 of betalamic acid and l475 of dop-
achrome in the presence of Tyr at a concentration of 100 mM
(Fig. S2B†). The intensity of l475 (tyrosinase system biosensor)
presented a steady decrease aer 7 h incubation. By contrast,
the intensity of l430 (enzyme cascade-triggered biosensor)
remained constant for more than 24 h.
Again, the co-presence of tyramine and Tyr was examined by
monitoring intensities of l475 with that of a single substrate and
a mixture of the two using conventional tyrosinase assays. The
intensity of l475 was shown enhancement than normal in the co-
presence of tyramine at 10 mM (Fig. S2C†), indicating interfer-
ence can occur for the samples containing tyramine greater
than 10 mM.
3.4 Condensation of betalamic acid with amines
It has been shown that the color of betanin depends on the
structural motifs in the cyclo-DOPA moiety of the pigments.28,29
We therefore mixed betalamic acid with 10 mM of primary
amines phenylalanine (Phe), glutamic acid (Glu), para-amino-
benzoic acid (PABA), and 2-aminoterephthalic acid (2-AIPA),
which respectively present green (Phe-, Glu-) and red (PABA-, 2-
AIPA-) uorescence.27 We then monitored the green and red
uorescence intensity of betaxanthins at various time points
(Fig. S3A and S3B†). The green uorescence intensity plateaued
aer 21 h; however, the red uorescence intensity peaked aer
4 h. To increase detection efficiency, we selected 2-AIPA–betax-
anthin for the quantication of Tyr. The optimal concentration
for 2-AIPA was examined in Fig. S3C.† The red uorescence
intensities of 2-AIPA–betaxanthin was shown to increase line-
arly with the concentration of Tyr (from 2.5 to 20 mM) with R2 ¼
0.96 (Fig. 3A).
Fig. 4 (A) The linear relationships between the red fluorescence
intensities of 2-AIPA–betaxanthin and tyrosinase at various concen-
trations. Inset: the corresponding fluorescence photograph in the
presence of tyrosinase. (B) The selectivity of the proposed enzyme
cascade-based system in the presence of various substrates (tyrosi-
nase, trypsin, lysozyme, GOx: 6 U mLꢁ1; K+, Ca2+, Mg2+: 100 mM) was
investigated. (C) The red fluorescence intensities of 2-AIPA–betax-
anthin in the presence of Tyr (1000 mM), tyrosinase (9 U mLꢁ1), and
kojic acid at various concentrations.
3.5 The colorimetric biosensor for tyrosine detection using
articial urine matrix
biological uids of patients with tyrosine metabolism disorders.
We therefore tested the effectiveness of the proposed enzyme
cascade-based biosensor using 10% diluted articial urine
Normally, the concentration of Tyr in healthy individuals ranges
from 30 to 120 mM;30 however, it can exceed 200 mM in the
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spiked with various concentrations of Tyr. Our results revealed signals including colorimetric and uorescent method were
that the linearity, dynamic range, and sensitivity of uorescence examined. Although uorescent method of 2-AIPA–betaxanthin
and absorbance intensity in complex matrices were similar to showed a better sensitivity than colorimetric method, the
those in the buffer system (Fig. 3A and B). The calculated rela- analysis duration is relative longer than the colorimetric
tive standard deviation (RSD) and recoveries using standard method. These ndings suggest that the effectiveness and
curves were obtained (Tables S1 and S2†). The accuracy ranges simplicity of the proposed scheme make it an attractive strategy
from 88–101% and 100–120% based on absorbance and uo- for the development of biosensors for Tyr and tyrosinase
rescence assays, respectively. This is a clear demonstration that analysis.
this proposed method could be applied to the detection of Tyr
in biological specimens.
Conflicts of interest
There are no conicts to declare.
3.6 Monitoring tyrosinase activity using the enzyme cascade
reaction
Next, we employed the enzyme cascade reaction for the visual
observation of tyrosinase activity. We rst determined the
optimal concentration of Tyr for the enzyme cascade reaction.
We began by varying the concentration of Tyr between 125 to
1000 mM in the presence of DOD (12.5 mM) and tyrosinase (1.5 U
mLꢁ1) following incubation for a period of 1 h. As shown in
Fig. S4A,† the intensities of l430 were signicantly increased
when 1000 mM Tyr was introduced. Under optimal experimental
conditions, a good linear relationship was observed between the
intensities of l430 and tyrosinase concentrations (Fig. S4B†).
The red uorescence intensities of 2-AIPA–betaxanthin was
recorded as well to determine the tyrosinase activity (Fig. 4A).
The corresponding regression coefficient is 0.99, and the LOD
was 0.04 U mLꢁ1. The selectivity and activity of the proposed
sensor was investigated by using the interfering substances
including trypsin, lysozyme, glucose oxidase (GOx), and metal
ions (Fig. 4B). There is no noticeable change of red uorescence
intensity in the presence of interfering substances.
Acknowledgements
This work was funded by the Ministry of Science and Tech-
nology of Taiwan under the project number 107-2113-M-003-
013-MY3.
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