Fluorescein as a Visible-Light-Induced Oxidase Mimic for Signal-Amplified Colorimetric Assay of Carboxylesterase by an Enzymatic Cascade Reaction

Article abstract of DOI:10.1002/chem.201705980

We have found that fluorescein possesses high visible-light-induced oxidase mimetic activity and could transform colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB) without unstable and destructive H₂O₂ under visible-light illumination. Instead, fluorescein uses oxygen as a mild and green electron acceptor, and its activity can be easily controlled by the switching “on/off” of visible light. In addition, the visible-light-induced catalytic mechanism was elucidated in detail and, as the main reactive species h⁺ and O₂^.? accounted for TMB oxidation. Based on the fact that fluorescein diacetate (FDA) possessed no activity and generated active fluorescein in situ in the presence of carboxylesterase (CaE), a signal-amplified sensing platform through a cascade reaction for CaE detection was constructed. Our proposed sensing system displayed excellent analytical performance for the detection of CaE in a wide linear range from 0.040 to 20 U L^?1 with a low detection limit of 0.013 U L^?1. This work not only changes the conventional concept that fluorescein is generally considered to be photocatalytically inert, but also provides a novel sensing strategy by tailoring the enzyme mimetic activity of fluorescein derivatives with analyte.

Full text of DOI:10.1002/chem.201705980

A Journal of
Accepted Article
Title: Fluorescein as a visible-light-induced oxidase mimic for signal-
amplified colorimetric assay of carboxylesterase by enzymatic
cascade reaction
Authors: Li Liu, Chaoqun Sun, Juan Yang, Ying Shi, Yijuan Long, and
Huzhi Zheng
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201705980
Link to VoR: http://dx.doi.org/10.1002/chem.201705980
Supported by

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Fluorescein as a visible-light-induced oxidase mimic for signal-
amplified colorimetric assay of carboxylesterase by enzymatic
cascade reaction
Li Liu,[a,b]Chaoqun Sun,[a] Juan Yang,[a] Ying Shi[a], Yijuan Long[a], and Huzhi Zheng*[a]
Abstract:We found that fluorescein possessed high visible-light- mimics that their activities can be easily modulated without the
induced oxidase mimetic activity and could transform colorless
3,3′,5,5′-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB)
aid of H2O2.
In response to demand, Wang et al.[5-9] have exploited several
without unstable and destructive H2O2 under visible light illumination. nanomaterials to mimick oxidase that make use of visible light
Instead, fluorescein uses oxygen as a mild and green electron as a simple and rapid mean to tune the oxidase-mimicking
acceptor, and its activity can be easily controlled by the switching activity, which ignites the exploration of photocatalytic enzyme
“on/off” of visible light. In addition, the visible-light-induced catalytic mimics. Unfortunately, most previous pioneering works focus on
mechanism was elucidated in detail and, as the main reactive nanozymes whose preparation involve organic solvents or
species h+ and O2•- accounted for TMB oxidation. Based on the fact surfactants as toxic sources, rigorous conditions (e.g. a high
that fluorescein diacetate (FDA) possessed no activity and
reaction temperature, high pressure) or complex synthesis
procedures, which may hinder in the large-scale production and
generated active fluorescein in situ in the presence of
carboxylesterase (CaE), a signal-amplified sensing platform through widespread practical applications. Thus, it is imperative to
cascade reaction for CaE detection was constructed. Our proposed develop new types of photoinduced oxidase mimics possessing
sensing system displayed excellent analytical performance for the new properties and potentials.
detection of CaE in a wide linear range from 0.040 to 20 U/L with a
Fluorescein has been exploited in various areas for instance
low detection limit of 0.013 U/L. This work not only changes the ophthalmology, bioimaging and biosensing for their versatile
conventional concept that fluorescein is generally considered to be fluorescent properties.[10] Nevertheless, the applications of
photocatalytic inert, but also provides a novel sensing strategy by fluorescein are mainly concentrated on their fluorescent
tailoring the enzyme mimetic activity of fluorescein derivatives with properties[11] and enhancer effect on the chemiluminescence[12]
.
analyte.
To the best of our knowledge, there has been not any study
devoted to the photocatalytic property as mimetic oxidase so far.
In this paper, excitingly, the novel visible-light-induced
oxidase-mimicking activity of fluorescein was unprecedentedly
found. Upon visible light irradiation, fluorescein can catalyze the
oxidation of typical enzyme catalytic chromogenic substrates
Introduction
Recently, considering the intrinsic drawbacks of natural
enzymes (e.g. horseradish peroxidase,HRP), such as high cost
in large scale production, poor stability of catalytic activities
against denaturation, digestion or the variations of
environmental conditions,[1] researchers have therefore
endeavoured to develop artificial enzymes such as nanozymes[2]
such
as
3,3’,5,5’-tetramethylbenzidine
(TMB),
o-
phenylenediamine
(OPD) and 2,2’-azino-bis
(3-
ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS)
leading to the typical color changes, and its function was the
same as natural oxidase. What is more, fluorescein exhibits a
higher affinity to TMB in comparison with HRP.
,
metal-organic frameworks[3] and small molecule-based mimic
peroxidises[4], as viable alternatives. Although these artificial
peroxidases exhibit some advantages over HRP, for example,
low in cost, easy to prepare, high stability and so on,[1-4] most of
them need destructive and unstable H2O2 as an oxidant to
obtain high catalytic activity, which may become obstacle to
further applications. More significantly, owing to lack of simple,
green and cost-effective means to modulate the catalytic activity
of nanozymes, it is highly desired to exploit new class of enzyme
On the basis of fluorescein diacetate (FDA) without oxidase-
like activity and fluorescein, in situ generated by
carboxylesterase (CaE) catalyzing FDA hydrolysis, possessing
excellent photocatalytic capability to transform colorless TMB
into blue oxidized TMB (oxTMB), a signal amplification sensing
strategy through cascade reaction for CaE detection was
elaborated (illustrated in Scheme 1). The limit of detection
reached to 0.013 U/L and the practicability of the sensing
platform was also validated in biological samples. In the view of
low cost, commercial availability FDA and robust, excellent
photocatalytic fluorescein, our proposed protocol is simple,
cheap, and easy to perform. To the best of our knowledge, for
the first time we discovered that organic small molecules
possess intrinsic visible-light-induced oxidase-mimicking activity
and this is the first instance of colorimetric strategy for CaE
detection. Meanwhile, this work also offers a new insight into the
application of fluorescein derivatives to develop colorimetric
biosensors based on the analyte tuned visible-light-induced
oxidase-mimicking activity.
[a] Dr. L. Liu, Dr. Y. Shi, M. Li, Y. Pang, C. Sun, Prof. Y. Long, Prof. H.
Zheng
The Key Laboratory on Luminescent and Real-Time Analytical
Chemistry, Ministry of Education, College of Chemistry
andChemical Engineering, Southwest University,
Chongqing 400715 (P. R. China)
E-mail: zhenghz@swu.edu.cn
[b]Dr. L. Liu
Department College of Chemistry and Environmental Science,
Qujing Normal University, Qujing, 655011 (P. R. China)
Supporting information for this article is given via a link at the end of
the document.
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further validating its oxidase-mimicking property. Different from
the characteristic that peroxidase always requires the help of
[13]
high concentration H2O2 to achieve high catalytic activity,
fluorescein employs greener dioxygen as oxidant to replace
destructive H2O2 and the photocatalytic activity can be easily
tuned by the on/off state of the light.
To further verify the visible light induced oxidase-mimicking
activity of fluorescein, we carried out the experiments using
other enzymatic substrates, i.e., OPD and ABTS to replace TMB,
producing the typical yellow or green color with characteristic
absorption peak at 448nm or 417 nm (Figure S1), respectively,
which indicated that the oxidase-mimicking activity of fluorescein
was not only restricted to TMB.
Influence factors on the visible-light-induced oxidase-
mimicking activity of fluorescein
The relative catalytic activity of fluorescein was, like natural
enzymes, dependent on pH and substrate concentration. The
optimal pH and the substrate (TMB) concentration were 4.0 and
0.50 mM, respectively (Figure S2A and B). As shown in Figure
S3C, the absorbance increased depending on the added
amount of fluorescein, suggesting that the catalysis was
catalyst-concentration-dependent. Figure S2D showed a good
linear relationship between absorbance and fluorescein
concentration over the range from 1.0 nM to 1.0 μM.
Scheme
reaction.
1 The proposed principle for CaE detection based on cascade
Results and Discussion
Visible-light-induced
fluorescein
oxidase-mimicking
activity
of
Catalytic mechanism of fluorescein as artificial oxidase
In order to elucidate the reactive species generated in the
catalytic reaction induced by visible light, different quenchers
that can scavenge the relevant reactive species including
Firstly, the visible-light-induced oxidase-mimicking activity of
fluorescein was evaluated by the catalysis of a typical oxidase
chromogenic substrate TMB. In the presence of visible light
irradiation, fluorescein could catalyze the oxidation of TMB and
produce a deep blue color, with maximum absorbance at 370nm
and 652 nm (Figure 1A, red line). In contrast, neither the mixture
of fluorescein and TMB without visible light irradiation nor TMB
alone irradiated by visible light without fluorescein exhibited any
color variation (Figure 1A, black line and blue line). Apparently,
these results above confirmed that fluorescein possessed
visible-light-induced oxidase-mimicking activity.
hydroxyl radicals (•OH), singlet oxygen (1O2), superoxide anions
•−
(O2
)
and photo-generated holes (h+) were added to the
catalytic system. As illustrated in Figure S3, almost no change in
the absorbance of the oxTMB can be observed after adding •OH
scavenger isopropanol (IPA) or tert-butanol (TBA)[8]. It indicated
that •OH could not be generated in the catalytic reaction.
Catalase, an efficient enzyme for decomposing of H2O2 to H2O
and O2,[8] could not affect the photocatalytic activity, indicating
that H2O2 was not produced by illuminated fluorescein. On the
contrary, the catalytic activity of fluorescein is apparently
suppressed in the presence of h+ scavenger like
ethylenediaminetetraacetic acid (EDTA)[8],ammonium oxalate
(AO)[14] or KI[8], indicating that h+ is one of the main oxidative
species. N2 was purged in the reaction system to perform TMB
oxidation under an oxygen free atmosphere. The catalytic
activity of fluorescein decreased obviously under the oxygen
free condition, indicating dissolved oxygen in solution plays an
important role in the photostimulated catalytic oxidation of TMB
by fluorescein (Figure 1B and S3A). As illustrated in Figure S3A,
the absorbance didn’t decrease obviously in the presence of
NaN3, which was utilized to scavenge 1O2 [15]. It indicated that
1O2 was not mainly reactive oxygen species (ROS) in the
catalytic reaction. In addition, the absorbance dramatically
Figure 1. (A) UV-vis absorbance spectra of different reaction systems: (a)
0.50 mM TMB (black line) under visible light irradiation, (b) 1.0 μM fluorescein
+ 0.50 mM TMB without visible light irradiation (blue line) and (c) 1.0 μM
fluorescein + 0.50 mM TMB under visible light irradiation (red line) in HAc-
NaAc buffer (0.10 M, pH 4.0). The insert shows the corresponding optical
photographs. (B) The absorbance spectra of TMB oxidation catalyzed by
illuminated fluorescein conducted in air saturated solution (black line) and
nitrogen saturated solution (red line).
decreased after adding superoxide dismutase (SOD), which was
•- [16]
utilized to effectively scavenge O2
,
showing that O2•- radicals
may be also main reactive specie during the catalytic process.
Because TMB, OPD or ABTS reacts with p-benzoquinone (BQ),
Oxidase can utilize O2 as oxidant to catalyze an oxidation-
reduction reaction. As showed in Figure 1B, the catalytic activity
of fluorescein was inhibited in the N2-saturated reaction system,
•−
a typical O2 scavenger,[17,18] methyl orange (MO) in place of
these substrates is usually used to evaluate the catalytic
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performance of photocatalysts[19]. The visible-light-induced MO
oxidative degradation catalyzed by fluorescein was considerably
suppressed after the introduction of BQ (Figure S3B),
response to on/off cycles (Figure S4) is indicative of the effective
charge generation, separation, and transfer process. On the
basis of the above results, the schematic illustration of catalytic
oxidation of TMB by photoactivated fluorescein is shown in
Scheme 2.
•−
suggesting that O2 plays a key role in this catalytic system. As
•−
the above results demonstrated that h+ and O2 as dominant
reactive species could be responsible for the TMB oxidation
catalyzed by fluorescein under visible light illumination.
Electron spin resonance (ESR) spectroscopy accepted as
powerful and direct detection method of ROS was used to
further confirm the ROS involved in the oxidation, unequivocally
•−
with DMPO as spin traps for O2 [20]. ESR signal cannot be
observed from the reaction between fluorescein and DMPO
without visible light irradiation (Figure 2). Due to concomitant
•−
exposure of DMPO and fluorescein to visible light for 5 min, O2
was generated, as verified by a strongly characteristic peak of
•−
the typical DMPO-O2
adduct with an intensity ratio of
1:1:1:1[20](Figure 2), demonstrating that fluorescein could
catalyze O2 to generate O2 radicals, similar to the case of
•−
oxidase[21]. The intensity of these ESR signals enhanced when
visible light irradiation time increased to 10 min (Figure 2). All
experimental results mentioned above provide a direct evidence
•−
to support that fluorescein catalytically generates O2 under
•−
Scheme 2 Proposed mechanism for visible light stimulated oxidase-mimicking
activity of fluorescein
visible light illumination and the quantity of O2 generated
depends on the irradiation time.
The oxidation and reduction potential of fluorescein is 0.87
and -1.18 V vs. saturated Ag/AgCl, respectively,[23] thus HOMO
and LUMO level was calculated to be 1.07 and -0.98 V versus
standard hydrogen electrode (NHE). Because the HOMO level
of fluorescein was more positive than that of TMB (rang from
0.22 to 0.70 V vs. NHE), [24,25] the photogenerated h+ can oxidize
TMB directly. In addition, the LUMO level of fluorescein was
more negative than that of oxygen (-0.046 V vs. NHE).[26]
Therefore, dissolved oxygen can accept the excited electrons of
fluorescein to generate O2•−. All these results provided direct
evidence supporting that fluorescein acted as a functional mimic
of oxidase.
Steady-state kinetic analysis
Next, the visible-light-stimulated oxidase-mimicking activity of
fluorescein was investigated according to steady-state kinetic
analysis. As shown in Figure 3, typical Michaelis-Menten curves
Figure 2. Time-dependent changes in the ESR spectra of 10 μM fluorescein
with 50 mM spin trap DMPO after being photoirradiated with visible light for 0,
5 or 10 min.
were observed and
a series of the steady-state kinetic
parameters (Km: Michaelis-Menten constant; Vmax: maximum
initial velocity; Kcat: catalytic constant, where [E] is fluorescein
concentration; and Kcat/Km: catalytic efficiency) were calculated
using the Lineweaver-Burk equation. The Km is an indicator of
the binding affinity between enzymes and substrates, and a high
Km value represents a weak affinity whereas a low value
suggests a high affinity.[27] As summarized in Table 1, the Km
value of fluorescein as peroxidase mimic was 8.3 fold larger
than that of it as oxidase mimic, indicating that fluorescein as
visible light induced artificial oxidase has a higher affinity to TMB
than that of it as artificial peroxidase. Quite impressively, the Km
value of fluorescein as mimic oxidase, was much lower than that
of HRP (Km = 0.434 mM−1)[13], indicating that it had a higher
affinity to TMB than that of HRP. Additional, the maximum initial
velocity (16 fold), catalytic constant (1.6×103 fold) and catalytic
Fluorescein has high molar extinction coefficient (ε490 = 8.8 × 104
cm-1 M-1) and absorbs light in the visible regime.[22] When
fluorescein is irradiated by visible light, it absorbs photons and
results in the corresponding generation of electrons in the lowest
unoccupiedmolecular orbital (LUMO) and leaving h+ in the
highest occupied molecular orbital (HOMO) to react with TMB
directly. On the other hand, the photogenerated electrons on
LUMO of fluorescein might be trapped by dissolved oxygen (as
•−
an electron acceptor), forming O2 radicals. To validate the
photoinduced charge transfer of fluorescein, the experiment of
photoelectrochemistry was conducted. The rapid rise of the
photocurrent of fluorescein upon illumination with a reproducible
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efficiency (1.3×104 fold) of the visible-light-induced oxidase
mimetic fluorescein was higher than that of peroxidase mimetic
fluorescein, demonstrating that fluorescein has high catalytic
activity under visible light irradiation.
Figure 3. Steady-state kinetic assay (A) and double reciprocal plots (B) of
fluorescein as oxidase mimic under visible light illumination using TMB as the
substrate. The velocity (v) of the reaction was measured using 1.0 μM
fluorescein and TMB concentration was varied in HAc-NaAc buffer (0.10 M) at
pH 4.0. Error bars represent the standard deviation of three repeated
measurements.
Table 1.Comparison of the Km, Vmax, Kcat and Kcat/Km of fluorescein as oxidase mimic, fluorescein as peroxidase mimic.
Catalyst
Concentration[μM]
Km[mM]
V
max[10-8 M·s-1]
K
cat[10-2 s-1]
Kcat/Km[M-1·s-1]
Reference
1.0
0.158±0.009
1.31±0.129
6.717±0.117
0.418±0.011
6.717
0.425
This work
[4]
Fluorescein as oxidasemimic
100
4.18×10-3
3.19×10-5
Fluorescein as peroxidase mimic
The working principle of CaE sensing
resulting absorbance changes at 652 nm relates directly to CaE
concentration (Figure 5). The above-mentioned results verified
the feasibility of the sensing strategy. Notably, both visible light
irradiation and in situ produced fluorescein through enzymatic
cascade reaction are very vital to endow fluorescein with
enhanced catalytic efficiency, thus our proposed sensing
platform can exhibit powerful signal amplification performance.
We found the functional group changes of fluorescein may
significantly impact on its catalytic performance. After
introducing carboxylic ester groups into xanthene ring of
fluorescein skeleton, the visible light-induced catalytic activity is
lost (Figure S5A) due to the lack of light absorption in the visible
region (Figure S5B). In virtue of these attractive features, it is
easy and flexible to take advantages of functional groups-
mediated photocatalytic activity to rationally design some facile,
label-free colorimetric platforms. Based on this scenario, CaE,
an enzyme specific for cleavage of the carboxylic ester bond,
and its substrate FDA were employed as proof-of-concept to in
situ regulate the catalytic capability of fluorescein derivatives.
Typical routes to detect CaE have been established by virtue of
fluorescein-release to induce the recovery of fluorescence
signal.[28-34] In our work, we proposed an enzymatic cascade
reaction colorimetric strategy for CaE detection using FDA as an
analyte-active probe, which could initiate the fluorescein-
catalyzed chromogenic reaction of TMB to act as an amplifier.
Thus, a high sensitivity towards CaE could be anticipated.
As illustrated in Scheme 1, CaE catalytically hydrolyzes
inactive FDA to yield active fluorescein and then the in situ
Figure 4. Absorption spectra of TMB-FDA systems in the absence (a) and
presence (b) of 10 U/L CaE after 10 min visiblelight irradiation. Reaction
condition: 0.50 mM TMB, 5.0 μM FDA, 0.10 MHAc-NaAc buffer (pH 4.0). Inset
showed the color change of the corresponding samples.
produced
fluorescein
under
visible
light
irradiation
catalyzescolorless TMB oxidation to form blue oxTMB, which
allows visual or spectroscopic detection. In order to examine the
feasibility of the designed enzymatic cascade reaction for CaE
sensing, the absorption spectra of the mixture of TMB and FDA
in the absence and presence of CaE under visible light
irradiation were investigated. Just as expected, the solutions
without CaE show almost colorless solution and no obvious
absorption (inset and curve “a”, Figure 4). Upon addition of CaE,
the color of TMB changes from colorless to blue, which is
reflected by absorption spectra (inset and curve “b”, Figure 4).
On account of the fact that the linear relationship between
absorbance and fluorescein concentration (Figure S2D) and the
content of fluorescein controlled by CaE concentration, the
Optimization of the sensing conditions.
In order to obtain the optimal performance of the CaE sensing
system, the effect of pH and FDA concentration was
investigated. As depicted in Figure S6A, absorbance first
enhanced accompanied by the increase of pH from 1.0 to 4.0,
and got a maximum at pH 4.0, then decreased with further
increasing pH value. So, pH 4.0 was selected as the optimal pH
value for subsequent experiments. As displayed in Figure S6B,
absorbance showed a rising tendency and achieved a plateau
once the FDA concentration was higher than 5.0 μM. Therefore,
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the optimal concentration of FDA was 5.0 μM for CaE detection. Conclusions
While for convenience, the subsequent experiments were
conducted at room temperature (25 °C).
Analytical performance of CaE detection
In summary, fluorescein possesses intrinsic visible-light-
included oxidase-mimicking activity and exhibits higher affinity to
TMB than HRP. In addition, we elucidated the photocatalytic
As can be seen from Figure 5, along withthe augment of CaE
concentration the absorbance increases. As expected, the blue
color deepened as the CaE concentration increased, which
could be observed directly by the naked eye (inset in Figure
S5B,). Figure S7 depicted the corresponding calibration plot and
indicated that absorbance is linear with the logarithm of CaE
concentration. The linear equation is A = 0.7801 lg[CaE] +
1.4595 (R2 = 0.9971) in the range from 0.040 to 20 U/L with a
low detection limit (LOD) of 0.013 U/L (3σ, n=11). The relative
standard deviation (RSD) was 3.0% for detecting 10 U/L CaE (n
=11), implying the good reproducibility of the assay. As shown in
Table S1, the proposed method exhibits wider linear range and
lower LOD than that of the existing optical methods.[28-35] The
high sensitivity can be attributed to the enzymatic cascade
reaction through in situ generation fluorescein as signal amplifier.
•-
mechanism in detail and found h+ and O2 were the main
reactive species account for TMB oxidation. Fluorescein uses
oxygen as a mild and green electron acceptor (not relies on
destructive H2O2), and the high catalytic activity can be attain
fast and simply only through 10 min visible light irradiation.
Significantly, its activity is easily switched on by visible light and
tuned by introduction of groups. As found, induction of carboxylic
ester groups caused the loss of activity. On basis of this novel
property of fluorescein, we have successfully constructed a
facile, economical, fast and green colorimetric sensing strategy
through enzymatic cascade reaction as a signal amplification
technique for highly sensitive and selective CaE detection. With
the benefit of the versatility in varieties and functionalities of
fluorescein derivatives, as well as the schemes of functional
groups-mediated photocatalytic activity, such integration will be
promising in future sensor developments and the application of
fluorescein as enzyme mimics. We also develop a simple
strategy for biosensing by tailoring the enzymcatic activity with
analyte, which might guide the design of other artificial enzymes
for different biological applications.
Experimental Section
Figure 5. (A) Absorption spectra of the sensing system in the presence of CaE
with different concentrations. (B) CaE concentration-dependent absorbance
response, the insert shows the corresponding optical photographs. a→k: 0,
0.04, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 U/L. Error bars represent the standard
deviation of three repeated measurements.
Reagents and chemicals
Fluorescein (F, Reagent Ph. Eur. grade), 3,3′,5,5′-tetramethylbenzidine
(TMB, 99%), methyl orange (MO, ACS reagent, 85 %), sodium azide
(NaN3,99.5%), carboxylesterase from porcine liver (CaE, EC 3.1.1.1, 18
units/mg), bovine serum albumin (BSA,98%),human serum albumin
(HSA,96%),cholesterol oxidase from cellulomonas species (ChOx, EC
1.1.3.6, 31 units/mg), L-glutamic acid (Glu, TLC grade, 99%),lysozyme
from chicken egg white (E.3.2.1.17, 63628 units/mg) were purchased
from Sigma-Aldrich. Fluorescein diacetate (FDA, HPLC grade, 98.0%)
were purchased from TCI Chemical Industry (Tokyo, Japan). Trypsin (EC
3.4.21.4, 213 units/mg) was purchased from Worthington Biochemical
Corporation (Lakewood, USA). Sodium acetate anhydrous (99.99%
metals basis), 2-methoxyethanol (99.7%, GC), L-tryptophan (Try, 99%),
L-glutamine (Gln, 99%), L-serine (Ser, 99%), catalase (CAT, 35000
units/mg ), p-benzoquinone (BQ, 99%), potassium iodide (99%),
Next, the selectivity of the method was evaluated and
presented in Figure S8. The results from interference assays
demonstrated that various endogenous molecules in serum
including other enzymes (trypsin, lysozyme and ChOx), serum
albumin (BSA and HSA), glucose, common amino acids (Glu,
Arg, Tyr, Lys, Gly, Ser, Try, Gln, Cys), GSH and metal ions (K+,
Zn2+, Mg2+, Ca2+, Co2+ and Al3+) showed negligible effect on the
CaE sensing system, indicative of satisfactory selectivity to CaE
(Figure S8A,). The results presented in Figure S8B indicated
that the method could provide good ability to resist interference
from common metal ions and biological species. The
aforementioned findings clearly indicated that our newly
constructed approach is reliable, sensitive and suitable for CaE
determination in complex real samples.
To validate the suitability and reliability of the sensing platform
in biological samples, CaE activity in FBS was measured using
standard addition method. As shown in Table S2, the recovery
for all the samples ranged from 95.4-108.2%, and RSD ranged
from 2.6-3.8%, which revealed the practicability and feasibility of
the method in complex biological samples.
ammonium
oxalate
monohydrate
(AO,
99.8%),
ethylenediaminetetraacetic acid (EDTA, 99.5%), tert-butyl alcohol (TBA,
99.5%) and ispropyl alcohol (IPA, 99.5%) were obtained from Aladdin
Reagent Co., Ltd. (Shanghai, China). L-glycine (99%) was from Alfa
Aesar. Tris (99.9%), reduced glutathione (GSH, 99%) L-cysteine (Cys,
99%), L-lysine (Lys, 99%), L-tyrosine (Tyr, 99%) and L-arginine (Arg,
99%) were from Genview (USA). Superoxide dismutase from bovine
erythrocytes (SOD, 2500 uints/mg), 2,2’-azino-bis (3-ethylbenzthiazoline-
6-sulfonic acid) diammonium salt (ABTS, reagent grade) and o-
phenylenediamine (OPD, reagent grade) were purchased from Sangon
Co., Ltd. (Shanghai, China). Acetate (HPLC grade, ≥99.9%), hydrochloric
acid (HCl, Guaranteed reagent) and H2O2 (30%) was obtained from
Macklin biochemical Co. Ltd. (Shanghai, China). Fetal bovine serum
(FBS) was purchased from Gibco (Australia). All other reagents were of
analytical
grade
and
were
used
directly
without
further
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10.1002/chem.201705980
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purification.Ultrapure water (18.2 M) was prepared with a Milli-Q system
(Millipore, USA) and used throughout the experiments.
for 10 min under visible-light (λ ≥ 400 nm). Finally, the absorbance of the
oxTMB at 652 nm was measured by a UV-Vis spectrophotometer.
Visible-light-induced oxidase-mimicking activity of fluorescein
Determination of CaE in FBS
The visible-light-induced oxidase-mimicking activity of fluorescein was
monitored by the catalytic oxidation of TMB, ABTS and OPD. In a typical
experiment, 200 μL of fluorescein (10.0 μM) and 200 μL of chromogenic
substrate (5.0 mM TMB, 5.0 mM OPD or 50 mM ABTS) were added into
1.0 mL acetate buffer solution (0.20 M, pH 4.0) and the mixture was
diluted with ultrapure water to a volume of 2.0 mL. The reaction solution
was irradiated for 10 min with a 150 W halogen tungsten lamp (CTTH-
150W, Crown Tech. Inc, USA) equipped with a long-pass cutoff filter (λ ≥
400nm) to provide visible light at room temperature and then the
Firstly, 20 μL FBS (2%) was spiked with different concentration of CaE
solution and diluted to 500 μL using Tris-HCl buffer (pH 8.0, 1 mM). Next,
200 μL of 50 µM FDA solution was added into as-prepared samples of
FBS containing various concentration of CaE. After incubation at 37 °C
for 30 min, 1.0 mL NaAc buffer (0.2mM, pH 4.0) was added and the
above mixture diluted to 1.8 mL using ultrapure water. Finally, 200 μL of
TMB stock solution (5.0 mM) were added. The absorbance at 652 nm
was recorded after visible light illuminating for 10 min.
absorbance at 652 nm was recorded using
a UV-2450 UV-Vis
spectrophotometer (Shimadzu, Japan). The light intensity of the light
source was fixed at 213.4 mW/cm2 and measured using a radiant power
meter (91150V, Newport Corporation, Stratford, CT USA).
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No.21405124, No.21175110). We thank
Prof. Yuan Ruo and Dr. Zheng Yingning (Southwest University)
for their kind assistance with the photocurrent measurements.
Kinetic assay
The steady kinetic assays were performed by monitoring the absorbance
change at 652 nm with a 1 min interval. 5.0 mL of 0.10 M acetate buffer
(pH 4.0) containing 1.0 μM fluorescein and varied TMB concentrations
was illuminated by visible light for 1 min, and then the absorbance was
recorded. The Lineweaver-Burk plot: 1/v = Km/ (Vmax[S]) +1/Vmax, was
used to calculate kinetic parameters, where v is the initial velocity, Vmax is
the maximal reaction velocity, Km is the Michaelis constant and [S]
corresponds to TMB concentration.
Keywords:colorimetric method• visible-light-induced oxidase
mimic• fluorescein• carboxylesterase• cascade reaction
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Fluorescein can catalyze TMB
oxidation under visible light irradiation
to produce colored reaction and be
applied to carboxylesterase
detectionby enzymatic cascade
reaction
Li Liu,[a,b]Chaoqun Sun,[a] Juan Yang, [a]
Ying Shi[a], Yijuan Long[a], Huzhi
Zheng*[a]
Page No. – Page No.
Fluorescein as a visible-light-induced
oxidase mimic for signal-amplified
colorimetric assay of
carboxylesterase by enzymatic
cascade reaction
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Author(s), Corresponding Author(s)*
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