A two fluorescent signal indicator-based ratio fluorometric alkaline phosphatase assay based on one signal precursor

Article abstract of DOI:10.1039/d1cc00244a

Two fluorescent signal indicators were simply converted from one organic precursor system by using the superior oxidation capability of manganese dioxide (MnO₂) nanosheets for the first time, finally resulting in the successful fabrication of a ratio fluorometric bioassay of alkaline phosphatase (ALP).

Full text of DOI:10.1039/d1cc00244a

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A two fluorescent signal indicator-based ratio
fluorometric alkaline phosphatase assay based
on one signal precursor†
Cite this: Chem. Commun., 2021,
7, 4444
5
Received 14th January 2021,
Accepted 26th March 2021
a
a
b
a
a
Rongji Wang, Zhihua Wang,* Honghong Rao,
Xin Xue, Mingyue Luo,
a
a
Zhonghua Xue * and Xiaoquan Lu
DOI: 10.1039/d1cc00244a
rsc.li/chemcomm
Two fluorescent signal indicators were simply converted from one fluorescence strategy (ratio-fluorescent sensor) armed with two
organic precursor system by using the superior oxidation capability fluorescent signal indicators was proposed in the past decade
of manganese dioxide (MnO
2
) nanosheets for the first time, finally and has been widely reported in great successes to obtain more
1
7–19
resulting in the successful fabrication of a ratio fluorometric bioassay accurate results in various sensing applications.
Typically, in
of alkaline phosphatase (ALP).
a ratio fluorometric sensor, target analytes can be designed to
sensitively and selectively induce the signal change of two
Alkaline phosphatase (ALP), one of the most important phos- fluorescent signal indicators by presenting two independent
phate hydrolases in the human body, plays an important role in fluorescence emission peaks. More significantly, this analyte-
1
many physiological reaction processes. The concentration of dependent asynchronous change of two fluorescent signal indi-
À1
À1
20
ALP in normal adults ranges from 40 U L to 150 U L , and its cators is in close correlation to the target content. To this end,
abnormal level is closely related to many diseases such as the significant signal amplification for analyte analysis can be
2
3
hepatobiliary diseases, skeletal system diseases, and tumors.
well demonstrated by integrating the specific two fluorescent
Therefore, the development of a highly sensitive, selective, and signals of ‘‘turn-on’’ and ‘‘turn-off’’ mode. For ALP determina-
reliable method to monitor ALP activity is of great significance tion, Xian et al. developed a ratiometric fluorescent bioassay by
4
21
for biological systems.
2
using Ag S and calcein as the two fluorescent signal indicators.
To date, many efforts have been made towards the quantita- However, this sensing strategy requires the necessary synthesis
5
,6
7,8
tive analysis of ALP, such as colorimetry, electrochemistry,
of complex fluorescent probes and the sensing principle is
9
,10
11,12
fluorometric
and so on.
Among these strategies, fluoro- mainly due to the incubation interaction between the added
metric strategies have been widely used due to their extremely fluorescent probes and the target, which limits its further
high sensitivity and excellent real-time detection performance. application. Therefore, it is still a challenge to develop a simpler
Though fluorometric sensors have demonstrated more advan- ratio-fluorescence method for detecting ALP.
tages in various sensing events, most of them usually operate
2
More recently, manganese dioxide (MnO ) nanosheets as
based on the single output readout of fluorescent indicators, two-dimensional inorganic nanomaterials have exhibited more
1
3–16
such as ‘‘turn-off’’ or ‘‘turn-on’’ mode.
It, therefore, often potential in MnO -based sensing applications, mainly due to
2
results in the limitation of poor spatial resolution and uncom- their superior light absorption capability, large specific surface
2
2
prehensive information for accurate analysis, because of the area, and easy to form hybrid-based nanoprobes. Unfortu-
influences of some unavoidable factors such as the background nately, most previously developed MnO -based fluorescence
2
signal, fluorescent substance concentration, surrounding methods for ALP detection are almost used for single-mode
environment and so on. To solve these drawbacks, a ratiometric detection, and therefore usually result in poor spatial
2
3
resolution. Only very few methods related to ratiometric
fluorescent sensing performance toward ALP have been
explored. So in this study, we attempt to develop a ratio
fluorometric ALP biosensor based on a simple system with
a
Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu
Province, College of Chemistry & Chemical Engineering, Northwest Normal
University, Lanzhou, 730070, China. E-mail: wangzh@nwnu.edu.cn,
xzh@nwnu.edu.cn
2
4
MnO nanosheets and o-phenylenediamine (OPD). As illustrated
b
2
School of Chemistry & Chemical Engineering, Lanzhou City University, Lanzhou,
in Scheme 1, the colorless OPD substrate could easily be
7
30070, China
2
catalyzed by MnO nanosheets to form a yellow oxidized product
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cc00244a
of 2,3-diaminophenazine (oxOPD), a typical fluorescent indicator
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the typical folds, which is extremely consistent with the pre-
2
3,25
vious reports.
Whereas the confirmation of Mn and O
elements in the MnO nanosheets was achieved by using the
2
energy-dispersive X-ray spectroscopy (EDX) spectrum (Fig. 1B),
and the FT-IR results (Fig. S1A, ESI†) also confirm the success-
ful formation of the Mn–O bond. Furthermore, the UV-vis
absorption spectrum (Fig. 1C) displays a peak at 380 nm of
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6
MnO
2
nanosheets. Furthermore, XPS analyses were per-
2
7
formed to verify the preparation of MnO
2
nanosheets. A wide
Scheme 1 Schematic illustration of the signal generation process of the
scan XPS spectrum shown in Fig. S1B (ESI†) clearly indicates
ratio fluorometric bioassay for ALP detection.
the binding energy peaks of Mn 2p, O 1s, N 1s, and C 1s at
6
41.6, 528.6, 401.6, and 284.6 eV, respectively. Two character-
with a strong yellow fluorescence at 563 nm. This indicator
exhibits a sensitive response toward ALP content in the proposed
system. With the introduction of ALP, L-ascorbic acid 2-
phosphate trisodium salt (AAP) substrate could be hydrolyzed
to produce ascorbic acid (AA), and the product AA could be
further oxidized by MnO nanosheets to produce dehydroascor-
2
bic acid (DHAA). Subsequently, the generated DHAA could
rapidly react with OPD to form 3-(1,2-dihydroxy ethyl) furo[3,
istic peaks located at 641.2 and 652.95 eV could be attributed to
Mn 2p3/2 and Mn 2p1/2 (Fig. 1D). All of the above observations
indicate the successful preparation of MnO nanosheets.
2
To further evaluate the feasibility of this ratio fluorometric
bioassay for ALP detection, we investigated the fluorescence
characteristics of the proposed two fluorescent signal
indicator-based system. As shown in Fig. 2A, in the presence
of MnO nanosheets, OPD can easily be oxidized to produce
oxOPD with a strong yellow fluorescence emission peak at
2
4-b]quinoxalin-1(3H)-on (DFQ), a sensitive fluorescent probe with
a strong blue fluorescence at 430 nm. As a result, a ratiometric
fluorescence sensing platform comprising dual-emission peaks
of oxOPD at 563 nm (yellow fluorescence) and DFQ at 430 nm
5
63 nm. On the other hand, it is well known that AA has two
2
8,29
isomers: enol-form and keto-form.
that AA could be oxidized to DHAA in the presence of the
appropriate oxidant of MnO nanosheets, and with the
We found in this system
(blue fluorescence) was successfully developed for visual identifi-
2
cation of ALP. A small variation in the ratio of the two different
fluorescence intensities can lead to an obvious change in the
fluorometer color, which can be easily observed with the naked
eye and accurately measured with a fluorometer. More impor-
presence of OPD, the product DHAA could further react with
OPD through a condensation process to form an N-heterocyclic
compound with the large conjugated p-bond system, named
DFQ, which can emit a strong blue fluorescence at 430 nm
3
0
2
tantly, because of the high catalytic activity of MnO nanosheets,
(
Fig. 2B). However, as displayed in Fig. S2 (ESI†), no fluorescent
substance was observed for the reaction system of AA and OPD
in the absence of MnO nanosheets. The UV-vis spectrum shown
the generation of both fluorescent signals in this work is more
direct and easy by only using an OPD chromogenic substrate, a
special readout generation mode that converts from one signal
precursor to two fluorescent signal indicators.
2
The obtained MnO
TEM, FT-IR, XPS, and UV-vis, respectively. As shown in Fig. 1A,
MnO samples are two-dimensional nanosheet structures with
2
nanosheets were confirmed by using
2
Fig. 2 Schematic illustration of the fluorescent signal generation. (A) oxOPD
from the fluorogenic reaction between OPD and MnO nanosheets, (B) DFQ
from the fluorogenic reaction of AA + MnO + OPD systems. (C) Schematic
diagram of ALP induced two fluorescent signal indicator. (D) Fluorescence
emission spectra of (a) AAP+ ALP + MnO + OPD; (b) AAP + MnO + ALP;
(c) AAP + MnO ; (d) ALP + MnO + OPD; (e) AAP + OPD; (f) ALP + OPD in the
Tris-HCl buffer solution (20 mM pH = 7.4).
2
2
Fig. 1 Characterization of the as-synthesized MnO
2
nanosheet samples.
2
2
(
A) TEM image, (B) EDS spectrum, (C) UV-vis absorbance spectra and the
2
2
photo of the solution (inset), and (D) XPS spectra of Mn 2p3/2 and Mn 2p1/2
.
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in Fig. S3 (ESI†) also supports the above speculative process. also optimized, respectively. As shown in Fig. S10 (ESI†), at a
Additionally, we further verified the above reaction features by reaction time of 50 min, the optimal contents of 0.8 mM OPD,
À1
using a mass spectrometric approach. As displayed in Fig. S4 5 mg mL MnO nanosheets, and 0.5 mM AAP were chosen for
2
(ESI†), it is clear that the reaction system of AA and OPD only ALP sensing. To ensure the applicability of the proposed system
demonstrated a characteristic mass spectrum peak at 109.07 of in the practical bioanalysis, a temperature of 37 1C and the Tris-
OPD and 175.02 of AA, whereas no corresponding mass spectral HCl buffer of pH 7.4 were used. The fluorescent photos and the
response of oxOPD, DHAA and DFQ was observed. However, fluorescence spectra of the proposed ratiometric sensing plat-
these reaction products and intermediate substances were form with varied ALP contents are obtained. As shown in
significantly demonstrated in the presence of MnO
as shown in Fig. S5 and S6 (ESI†). This may be attributed to the 200 mU mL , the color of the system photographed under
2
nanosheets Fig. 3A, with the increase of the ALP concentrations from 0 to
À1
unique feature of this proposed reaction system, in which AA 365 nm UV irradiation was gradually changed from yellow to
may be oxidized by MnO nanosheets more easily than OPD as yellowish and then to white and blue, indicating a sensitive
2
supported by some validation experimental results based on zeta colorimetric response of this fluorescent system toward ALP con-
potential and electrochemical measurements shown in Fig. S7 tent. It is clear that the fluorescence intensity of DFQ at 430 nm
(
ESI†). Inspired by the previous reports that ALP could exclu- gradually increased but that of oxOPD at 563 nm correspondingly
3
1,32
À1
sively produce AA by hydrolyzing its substrate AAP,
we decreased with increasing ALP from 0 to 200 mU mL (Fig. 3B).
believe that ALP could also indirectly trigger the construction It was found that the F563/F430 was proportional to the concen-
À1
of the two fluorescent signal indicator-based fluorescent sys- tration of ALP within a range of 0.25–10 mU mL with the linear
tems, in which oxOPD and DFQ provide the sensitive fluorescent regression equation of F = 21.25–2.092 Â C
and the square of
ALP
response. As illustrated in Fig. 2C, firstly, ALP can hydrolyze its the correlation coefficient of 0.9983 (Fig. 3C). The calculated
À1
substrate AAP to produce AA, and then the redox reaction LOD was 0.06 mU mL based on 3s/k (k of 2.092 and s of 0.0418
2
between AA and MnO nanosheets will happen and produce are the slope of the obtained regression equation and the
DHAA. Subsequently, the generated DHAA will react with OPD standard deviation of blank signals of 20 measurements, respec-
through a specific condensation process to produce DFQ along tively). To our knowledge, this ratio fluorometric bioassay has a
with a strong emission peak at 430 nm. Meanwhile, the satisfactory LOD compared to many reported methods (Table S1,
2
reduction of both MnO nanosheets and OPD leads to a decrease ESI†), suggesting a good sensing performance of our developed
in the intensity of the emission peak of oxOPD at 563 nm. Thus, method toward ALP detection. In addition, it is worth noting
a ratio fluorescent platform could be fabricated and used for ALP that this proposed ratio sensing platform has high selectivity
detection based on the readout change of the fluorescence toward ALP detection. As shown in Fig. 4, in the presence of
intensity ratio (F563/F430). To verify the above mechanism, the typical potential substances such as HRP, Trypsin, AChE, Lyso-
fluorescent properties of the mixed solvent system containing zyme, Cellulase, Gox, BSA, and Protease with a higher content
À1
À1
2
MnO nanosheets, OPD and AAP were evaluated. As shown in (80 mU mL ), only target ALP (8 mU mL ) caused a significant
Fig. 2D, with the addition of ALP, both the fluorescence emission decrease in the fluorescence intensity ratio (F563/F430) and no
peaks corresponding to the oxOPD at 563 nm and the DFQ at obvious readout change for the system without ALP addition.
4
3
30 nm were clearly observed at the excitation wavelength of More significantly, we subsequently found that some reducing
75 nm (Fig. 2D, a), indicating that ALP can successfully trigger substances like cysteine and glutathione did not interfere with the
the formation of the two fluorescent signal indicator based
fluorescent system. Because for the reaction system without
ALP introduction, only one fluorescence emission peak corres-
ponding to the oxOPD at 563 nm (Fig. 2D, d) was observed, the
fluorescence intensity of oxOPD was significantly higher than
that of the ALP added reaction system. In particular, for the
reaction system of AAP, ALP and OPD without MnO nanosheets,
2
no obvious fluorescence response could be observed (Fig. S8,
ESI†). All these observations suggest a successful construction of
this proposed fluorometric system with an ALP-dependent two
fluorescent signal response, indicating a potential application of
this platform for sensitively sensing ALP.
The fluorometric performance of these developed two fluor-
escent signal indicators was also estimated at varied excitation
light sources (355, 365, 375, 385 nm). As shown in Fig. S9 (ESI†),
the wavelength of 375 nm was chosen as the optimal excitation
light source to investigate the ALP sensing performance in the
subsequent experiments. Other conditions that influenced the
sensing performance such as the enzymatic incubation time,
Fig. 3 (A) Photos of the corresponding sensing system under 365 nm
ultraviolet light upon different ALP concentrations. (B) Fluorescence
emission spectra of the sensing system in the presence of different
concentrations of ALP. (C) Linear relationship between F563/F430 and ALP
the concentration of OPD, MnO
2
nanosheets, and AAP were concentration.
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Conflicts of interest
There are no conflicts to declare.
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