In situ formation of fluorescent silicon-containing polymer dots for alkaline phosphatase activity detection and immunoassay

Article abstract of DOI:10.1007/s11426-019-9690-7

The discovery and application of analyte-triggered fluorophore generation or fluorogenic reaction are significant and beneficial to the development of novel fluorescence (FL) analysis method. In this study, for the first time, we have reported a fluorogenic reaction to prepare fluorescent silicon-containing polymer dots (Si-PDs) by simply mixing N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) and hydroquinone (HQ) in aqueous solution at ambient temperature. Inspired by the alkaline phosphatase (ALP)-catalyzed hydrolysis of the substrate sodium 4-hydroxyphenyl phosphate (4-HPP) into HQ and the resultant HQ-controlled intense green Si-PDs generation, we have established a straightforward ALP activity assay by innovatively employing commercially available 4-HPP as the substrate. More significantly, the specific preparation method, clear formation mechanism and excellent performance enable the Si-PDs as well as its generation process to develop facile and attractive FL immunoassay. With the help of the universal ALP-based enzyme-linked immunosorbent assay (ELISA) platform and corresponding antibody, a convenient and conceptual ALP-based fluorescent ELISA has been constructed and applied in sensing cardiac troponin I (cTnI), a well-known biomarker of acute myocardial infarction. Our research via in situ formation of fluorescent nanomaterials has great potential application in ALP activity assay, inhibitor screening, and disease diagnosis.

Full text of DOI:10.1007/s11426-019-9690-7

SCIENCE CHINA
Chemistry
•ARTICLES•
https://doi.org/10.1007/s11426-019-9690-7
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In situ formation of fluorescent silicon-containing polymer dots for
alkaline phosphatase activity detection and immunoassay
Guoyong Liu1,2, Jiahui Zhao1,3, Mengxia Yan1,2, Shuyun Zhu1,4, Wenchao Dou5,
Jian Sun1* & Xiurong Yang1,2*
1State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, China;
2University of Science and Technology of China, Hefei 230026, China;
3University of Chinese Academy of Sciences, Beijing 100049, China;
4College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China;
5Food Safety Key Laboratory of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University,
Hangzhou 310018, China
Received November 27, 2019; accepted January 19, 2020; published online March 12, 2020
The discovery and application of analyte-triggered fluorophore generation or fluorogenic reaction are significant and beneficial
to the development of novel fluorescence (FL) analysis method. In this study, for the first time, we have reported a fluorogenic
reaction to prepare fluorescent silicon-containing polymer dots (Si-PDs) by simply mixing N-[3-(trimethoxysilyl)propyl]ethy-
lenediamine (DAMO) and hydroquinone (HQ) in aqueous solution at ambient temperature. Inspired by the alkaline phosphatase
(ALP)-catalyzed hydrolysis of the substrate sodium 4-hydroxyphenyl phosphate (4-HPP) into HQ and the resultant HQ-
controlled intense green Si-PDs generation, we have established a straightforward ALP activity assay by innovatively employing
commercially available 4-HPP as the substrate. More significantly, the specific preparation method, clear formation mechanism
and excellent performance enable the Si-PDs as well as its generation process to develop facile and attractive FL immunoassay.
With the help of the universal ALP-based enzyme-linked immunosorbent assay (ELISA) platform and corresponding antibody, a
convenient and conceptual ALP-based fluorescent ELISA has been constructed and applied in sensing cardiac troponin I (cTnI),
a well-known biomarker of acute myocardial infarction. Our research via in situ formation of fluorescent nanomaterials has great
potential application in ALP activity assay, inhibitor screening, and disease diagnosis.
fluorescent, polymer dots, ELISA, alkaline phosphatase, cardiac troponin I
Citation:
Liu G, Zhao J, Yan M, Zhu S, Dou W, Sun J, Yang X. In situ formation of fluorescent silicon-containing polymer dots for alkaline phosphatase activity
detection and immunoassay. Sci China Chem, 2020, 63, https://doi.org/10.1007/s11426-019-9690-7
1 Introduction
an important immunoassay format, ELISA mainly depends
on the efficient catalytic amplification of enzyme and the
specific recognition of antigen-antibody [2]. In typical
ELISA, alkaline phosphatase (ALP) acts as one of the most
commonly utilized labeling enzymes because of its easy
conjugation to antibody, good stability, and high catalytic
activity [3–5]. In addition, ALP can hydrolyze numerous
phosphorylated substrates, especially phosphate-containing
metabolites in an alkaline environment [6]. Abnormal ex-
Enzyme-linked immunosorbent assay (ELISA) is ex-
tensively employed in laboratory research, food safety con-
trol, environmental analysis, clinical diagnosis, and
biomedical application [1], owing to its quantification ac-
curacy, easy operation, low cost, and convenient readout. As
*Corresponding authors (email: jiansun@ciac.ac.cn; xryang@ciac.ac.cn)
© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
chem.scichina.com link.springer.com

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Liu et al. Sci China Chem
pression of ALP is linked with many diseases, such as he-
patobiliary disease and bone disease [7]. Thus, accurate de-
termination of ALP activity is not only beneficial for clinical
diagnosis, but also conducive to the development of novel
ALP-based ELISA platform.
[25]. Here, we try to provide a facile method for preparing
fluorescent CDs at room temperature without any external
energy supply. It is demonstrated that the fluorescent silicon-
containing polymer dots (Si-PDs) can be synthesized in
80 min by simply mixing the solutions of hydroquinone
(HQ) and N-[3-(trimethoxysilyl)propyl]ethylenediamine
(DAMO). The Si-PDs display monodisperse dots with in-
tense green FL, as evidenced by further characterizations.
Compared to previous reports, our approach is simple and
green, especially can be rapidly completed in aqueous so-
lution at ambient conditions. Such cheap, mild and con-
venient reaction inspires us to develop an in situ approach for
biological sample detection.
Herein, we have constructed a simple and convenient
fluorescent ELISA based on in situ generation of green-
emission Si-PDs. The commercially available sodium 4-
hydroxyphenyl phosphate (4-HPP) is selected as the ALP
substrate. Most significantly, HQ, the enzymatic hydrolysate
of 4-HPP, can react with DAMO to form green-emission
Si-PDs in situ, while 4-HPP hardly reacts with DAMO. The
FL intensity directly associates with ALP activity. With the
help of the universal ALP-based ELISA platform and cor-
responding antibody, we construct a novel ALP-based
fluorescent ELISA. In an ELISA platform, the concentration
of the antigen is related to the ALP-labeled secondary anti-
body. Thus, the FL intensity can indirectly reflect the con-
centration of the target antigen. Our approach can be used not
only to measure biomarkers with the help of the corre-
sponding antibodies, but also to broaden the potential ap-
plication of FL nanoparticles.
There have been many analytical approaches for ALP
activity detection, such as colorimetry, fluorometry, elec-
trochemistry, chemiluminescence, and surface enhancement
Raman scattering [8]. Correspondingly, a number of im-
munoassays have been established on the basis of these
analytical approaches [9]. Among these immunoassays,
colorimetry and fluorometry are more popular methods due
to their low cost, convenience, simple operation, and easy
readout. Nevertheless, conventional colorimetric ELISA is
suffering from a high detection limit. The introduction of
plasmonic ELISA, a new-type colorimetric immunoassay
based on noble metal nanoparticles aggregation, etching, and
deposition increases the sensitivity of detection [10,11].
However, noble metal nanoparticles are unstable and are
easily interfered by the external environment. In addition, the
synthesis or surface modification of nano-sized noble metal
particles takes time and effort. As a substitute or supplement
for colorimetric immunoassay, fluorescence (FL) im-
munoassay draws people’s attention [12]. In this regard,
ALP-based FL immunoassays are mostly established based
on the substrate ascorbic acid 2-phosphate (AA2P) [13–17],
and p-nitrophenyl phosphate (pNPP) [18–20], by means of
the high reaction activity of corresponding enzymatic
hydrolysates (ascorbic acid and orthophosphate, respec-
tively). And then, exploring new ALP substrates and novel
ALP-enabled fluorogenic reaction will undoubtedly enrich
and promote the development of FL immunoassays. Re-
cently, our group [21] has synthesized a new ALP substrate,
called m-hydroxyphenyl phosphate sodium salt (m-HPP), but
the synthesis process is ineluctably time-consuming and la-
borious. Therefore, it is necessary and important to construct
an efficient, simple, and convenient fluorescent ELISA
platform by employing a new commercially available sub-
strate.
Nowadays, fluorescent carbon dots (CDs), mainly in-
cluding graphene quantum dots (GQDs), carbon nanodots
(CNDs), and polymer dots (PDs), have attracted widespread
attention due to their superior optical properties, stability,
and biocompatibility [22]. A number of approaches for
preparing CDs have been developed, such as chemical oxi-
dation, electrochemical etching, hydrothermal or solvother-
mal synthesis, microwave-assisted synthesis, and so on [23].
At the same time, a variety of heteroatoms (nitrogen,
phosphorus, sulfur, boron, silicon, etc.) have been success-
fully doped into the CDs to modulate their fluorescent
properties [24]. However, these synthetic or doping ap-
proaches may have to involve hazardous solvents, high
temperature and high pressure, and microwave radiation
2 Experimental
2.1 Chemicals
Sodium 4-hydroxyphenyl phosphate (4-HPP) was acquired
from Bidepharm Corporation (China). Alkaline phosphatase
(ALP), sodium orthovanadate (Na3VO4), human serum al-
bumin (HSA), EcoR I, trypsin, lysozyme (LZM), glucose
oxidase (GOx), and bovine serum albumin (BSA) were
supplied by Sigma-Aldrich. Hydroquinone (HQ), Tris-Buf-
fered Saline Tween-20 (TBST), and human immunoglobulin
G (IgG) were purchased from Sangon Biotechnology Cor-
poration (China). DAMO was purchased from Aladdin In-
dustrial Corporation (China). Human serum samples were
obtained from the Second Hospital of Jilin University
(China).
Cardiac troponin T (cTnT), cardiac troponin I (cTnI),
mouse anti-cTnI antibody (capture antibody), rabbit anti-
cTnI antibody (Ab1), ALP-labeled goat anti-rabbit second-
ary antibody (ALP-Ab2), carcino-embryonic antigen (CEA),
and human alpha-fetoprotein (AFP) were purchased from
Abcam Corporation (UK).

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Liu et al. Sci China Chem
2.2 Apparatus
lowed by washing again. Afterward, a mixed solution of
45 μL of Tris-HCl buffer solution (pH 8.5, 50 mM) con-
taining 50 μM MgCl2, 56.3 μL of 4-HPP (2.0 mM), and
123.7 μL of water was injected into each well and main-
tained at 37 °C for 1 h. Subsequently, 25 μL of DAMO
(50%, v/v) was introduced into the wells. After incubation for
80 min at room temperature, FL spectra measurements were
performed.
Fluorescence and absorption spectra were carried out on
Hitachi F-4600 spectrofluorometer (Japan) and CARY 500
UV-Vis-NIR Varian spectrophotometer (USA), respectively.
Transmission electron microscopy (TEM) image was ob-
tained on a Tecnai G2 F20 instrument (FEI, USA). Fourier
transform infrared spectroscopy (FTIR) spectra were con-
ducted on a Bruker Optics VERTEX 70 spectrometer (Ger-
many). X-ray diffraction (XRD) pattern and X-ray
photoelectron spectroscopy (XPS) measurements were ac-
quired by Bruker D8 ADVANCE and Thermo ESCALAB
VG Scientific 250 (UK), respectively.
3 Results and discussion
3.1 Characterization of the fluorescent Si-PDs
A previous article had reported a similar reaction that HQ
and DAMO could act as precursors to prepare fluorescent
nanoparticles. However, the synthesis of fluorescent nano-
particles was carried out at 200 °C for 4 h [26]. To our sur-
prise, we found the fluorescent Si-PDs could be easily
synthesized at room temperature in 80 min by simply mixing
the aqueous solutions of HQ and DAMO. The optical
properties of the as-prepared Si-PDs were studied by ab-
sorption and FL spectra. As seen from Figure 1(A), com-
pared with the absorption spectra of DAMO and HQ, the Si-
PDs show two new absorption peaks at 370 nm (n-π* tran-
sitions of C=O/C=N) [27] and 500 nm (surface state transi-
tions) [28], respectively. The maximum excitation and
emission wavelength of Si-PDs are located at 370 nm and
around 520 nm (Figure S1, Supporting Information online),
respectively. The Si-PDs solutions display light orange in
daylight and strong green FL under the 365 nm UV lamp
(inset of Figure 1(A)). The morphology of the Si-PDs was
investigated by TEM. The Si-PDs exhibit a spherical struc-
ture with a particle size of 20–200 nm (Figure 1(B) and its
insert). The XRD pattern shows a broad distinct peak at 20.3°
(Figure S2), illustrating that Si-PDs are amorphous nature
[29,30]. The TEM image and XRD pattern indicate that Si-
PDs are polymer dots (PDs). The surface functional groups
of the Si-PDs were studied by FTIR. As shown in Figure 1
(C), compared with the FTIR spectra of DAMO and HQ, the
Si-PDs appear a new absorption peak at 1655 cm−1, re-
presenting stretching vibration of C=O/C=N [31]. The broad
absorption peak around 3400 cm−1 is attributed to the
stretching vibration of O–H and N–H [32]. The absorption
peaks at 2938, 1471, and 1571 cm−1 belong to C–H stretch-
ing vibration [33], C–H bending vibration [34], and N–H
bending vibration [35], respectively. The stretching vibration
of C=C, C–N, and C–O can be observed at 1603, 1350, and
1195 cm−1, respectively [36]. The absorption peaks at 1123
and 1034 cm−1 can represent stretching vibration of Si–O
[37], indicating that silicon has been successfully doped into
the polymer dots. To confirm the elemental composition and
chemical bonding of the Si-PDs, XPS measurements were
carried out. As seen from Figure 1(D), the Si-PDs contain
2.3 Synthesis of fluorescent Si-PDs
100 mg HQ and 5 mL of DAMO were dispersed in 95 mL of
deionized water. The resultant solution was allowed to stand
at room temperature for 80 min and then lyophilized. The
lyophilized crude product was washed thrice with ethanol.
The precipitate was collected and dried at room temperature
for the further characterization.
2.4 ALP activity measurement
ALP activity was detected as follows. First of all, 180 μL of
50 mM Tris-HCl buffer solution (pH 8.5) containing 50 μM
MgCl2, 225 μL of 4-HPP (2.0 mM), 405 μL of water, and
90.0 μL of ALP with different concentrations were mixed
and incubated for 60 min at 37 °C. Subsequently, 100 μL of
DAMO (50%, v/v) was added to the mixed solution. Then,
the resultant solution was allowed to stand at room tem-
perature for 80 min. Finally, the FL spectrum was recorded
with an excitation wavelength of 370 nm. To demonstrate the
specificity of our assay, interfering substances (including
non-specific enzymes and proteins) were assayed in the
presence and absence of ALP.
2.5 FL immunoassay for cTnI
The cTnI detection was carried out by a sandwich format
immunoassay procedure. First of all, 100 μL 5 μg/mL of
mouse monoclonal antibody in carbonate buffer (100 mM,
pH 9.6) was introduced into each well and maintained at 4 °C
for 12 h. After discarding the solution, the wells were rinsed
three times with TBST, and then blocked with 200 μL 3%
BSA for 1 h at 37 °C. Then, the wells were washed thrice
with TBST again. Subsequently, 100 μL of cTnI with various
concentrations was injected into the wells and maintained for
1 h at 37 °C. The washing steps were repeated. Next, 100 μL
2 μg/mL of Ab1 was added into each well and incubated for
1 h at 37 °C. After washing, 100 μL of ALP-Ab2 (1:5000)
was injected into the wells and kept at 37 °C for 1 h, fol-

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Liu et al. Sci China Chem
Figure 1 (A) Absorption spectra of DAMO (a), HQ (b), and Si-PDs (c), and the maximum excitation (d) and emission (e) spectra of Si-PDs. Insets are
optical photographs of Si-PDs solutions in daylight (left) and 365 nm UV light (right). (B) TEM image of Si-PDs. Inset is the distribution histogram of
particle size. (C) FTIR spectra of HQ (black), DAMO (red), and Si-PDs (blue). (D) The full survey of the XPS spectrum of Si-PDs (color online).
four non-hydrogen elements of C (284 eV), N (398 eV), O
(531 eV), and Si (102 and 152 eV) [38]. The spectra of C 1s,
N 1s, O 1s, and Si 2p are shown in Figure S3. The C 1s
spectrum is divided into four parts at 284.0, 284.6, 285.7, and
288.5 eV, which can be associated with C–Si, C–C/C=C, C–
O/C–N, and C=O/C=N bonds, respectively [39]. The N 1s
spectrum displays three peaks located at 398.3, 399.0, and
400.1 eV, corresponding to C=N/N–Si, C–N, and N–H
bonds, respectively [40,41]. O–Si (531.0 eV), C–O
(531.9 eV), and C=O (533.4 eV) bonds can be seen from O
1s spectrum [42]. The Si 2p spectrum implies the existence
of Si–C (101.0 eV), Si–N (101.6 eV), and Si–O (102.2 eV)
bonds, respectively [43]. The results of XPS characterization
are consistent with those of FTIR characterization. The
comprehensive characterization results indicate that we have
successfully synthesized fluorescent Si-PDs with abundant
functional groups on the surface.
Figure 2(a, b), the FL intensity at 520 nm exhibits con-
centration-dependent increase with increasing HQ con-
centration. A good linearity (R2=0.992) between the
concentration of HQ (1 to 80 μM) and FL intensity can be
obtained, indicating that we may monitor HQ-related targets.
In this regard, HQ could be considered as the depho-
sphorylated enzymatic product of 4-HPP through ALP-cat-
alyzed cleavage of the phosphoester bond in 4-HPP. To
verify the feasibility of 4-HPP as a substrate for ALP, we
performed absorption and FL spectra measurements. As
depicted in Figure 3(B, C), 4-HPP solution (line a), the
mixed solution of 4-HPP and DAMO (line b), and the mixed
solution of ALP and DAMO (line c) display no absorption
peak at 370 nm and FL signal at 520 nm. The mixed solution
of 4-HPP and ALP (line d) has an absorption peak at 350 nm,
but no FL signal at 520 nm. The mixed solution of 4-HPP
and ALP after adding DAMO (line e) shows two char-
acteristic absorption peaks at 370 and 500 nm. Meanwhile, a
FL signal at 520 nm can be observed in the FL spectra. The
absorption and FL spectra of in situ formation of products
catalyzed by ALP coincide with those of as-prepared Si-PDs
(line f). Thus, 4-HPP can be used as the substrate for ALP
activity sensing based on the ALP-enabled produce of FL
signal (Figure 3(A)).
3.2 Establishment and optimization of ALP sensing
system
To obtain favorable sensing performance, the reaction
parameters of HQ and DAMO were optimized. As illustrated
in Figure S4, as the DAMO concentration and reaction time
increase, the FL intensities at 520 nm of the Si-PDs also
increase. To save reagents and time, the DAMO concentra-
tion and reaction time were chosen to be 5% and 80 min,
respectively. When the concentration of DAMO and reaction
time were fixed, we studied the relationship between HQ
concentration and FL intensity in detail. As presented in
To acquire optimal sensing performance, we investigated
the experimental parameters in the presence and absence of
ALP. As shown in Figure S5, in the absence of ALP, the
background signals (red bar) are negligible. Meanwhile, in
the presence of ALP, the favorable conditions of the pH, the
4-HPP concentration, the incubation time of ALP, and

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Liu et al. Sci China Chem
Figure 2 FL emission spectra (a) and corresponding FL intensities (b) of HQ with various concentrations in the presence of 5% (v/v) DAMO (color online).
Figure 3 (A) Principle of ALP-catalyzed in situ generation of Si-PDs. (B) Absorption and (C) FL emission spectra of 4-HPP (a), 4-HPP+DAMO (b), ALP
+DAMO (c), 4-HPP+ALP (d), 4-HPP+ALP+DAMO (e), as-prepared Si-PDs (f). The concentrations of 4-HPP, DAMO, and ALP are 0.5 mM, 5%, and
20 mU/mL, respectively. The Si-PDs are prepared by reaction of HQ (50 μM) and DAMO (5%) at room temperature for 80 min (color online).
the fluorogenic reaction time of ALP-incubated 4-HPP
with DAMO are 8.5, 0.5 mM, 60 min, and 80 min, respec-
tively.
the effects of interfering substances can be neglected. In the
absence of ALP, the interfering substances cannot produce
FL signals. These results demonstrate the good selectivity of
the assay for ALP detection.
Inhibitor studies of enzymes have an important impact on
drug design/discovery. By using a common ALP inhibitor
(Na3VO4), the proposed assay was utilized to assess the ef-
ficiency of enzyme inhibitor.
3.3 FL assay for ALP activity and its inhibitor
Under optimal experimental conditions, the relationship
between ALP concentration and FL intensity was in-
vestigated in detail. As presented in Figure 4(a), as the ALP
concentration increases, the FL intensity of the sensing
system increases gradually. ALP concentrations between 0.7
and 50 mU/mL have a good linear relationship (R2=0.996)
with FL intensities at 520 nm (F1), as depicted in Figure 4(b).
As the concentrations of Na3VO4 increase from 1 to
1,500 μM, the ALP activity (20 mU/mL) is gradually sup-
pressed, resulting in a decrease in the FL intensity of the test
solution (Figure S7(a)). There is a sigmoidal curve re-
lationship between the FL intensities and the logarithm of
Na3VO4 concentrations (Figure S7(b)). The IC50 value (the
concentration of an inhibitor that is required for 50% in-
hibition of enzyme activity) is approximately 25.9 μM. This
value is consistent with previously reported value [44], in-
dicating that our assay could be utilized for the screening of
ALP inhibitor.
The linear relationship can be express as F1=19.12CALP
–
4.25. The limit of detection is 0.1 mU/mL, which is lower
than the previously reported assays for ALP (Table S1,
Supporting Information online). Using non-specific proteins
and enzymes as controls, we investigated the selectivity of
the assay. As indicated in Figure S6, in the presence of ALP,

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Liu et al. Sci China Chem
3.4 ALP-based fluorescent ELISA for cTnI detection
sandwiched immunocomplex. ALP connected to the Ab2
catalyzes the hydrolysis of 4-HPP to form HQ, which could
react with DAMO to produce intense green-emission Si-PDs
in situ. Therefore, the FL intensity establishes an indirect
relationship with the cTnI concentration. As shown in Figure
5(b, c), the FL intensities exhibit a concentration-dependent
increase with the increase of cTnI concentration. This re-
lationship between FL intensities (F2) and cTnI concentra-
Encouraged by the good results of ALP detection and the
widespread use of ELISA, we attempted to develop a novel
ALP-based fluorescent ELISA for the determination of an-
tigens. cTnI, a biomarker of acute myocardial infarction
(AMI), is chosen as a model. When AMI occurs, the cTnI
concentration increases, even reaching to 550 ng/mL or
above. Thus, accurate determination of cTnI concentration is
of great significance in the early diagnosis of AMI [45]. With
the help of commercial antibodies to cTnI, we investigated
the ability of our proposed fluorescent ELISA to detect cTnI.
Figure 5(a) schematically depicts the fluorescent ELISA for
cTnI detection via ALP-catalyzed in situ generation of Si-
PDs. In this strategy, the capture antibody is first immunized
to the well. The cTnI is then captured by the capture anti-
body. Subsequently, the Ab1 recognizes the cTnI and the
Ab2 recognizes the Ab1, resulting in the formation of a
tions (1–300 ng/mL) can be expressed as F2=9.06+0.77CcTnI
,
R2=0.997. Compared with previously reported im-
munoassays for cTnI (Table S2), a comparable detection
limit of 0.1 ng/mL is obtained. To verify the selectivity of the
immunoassay for cTnI, some non-specific proteins, includ-
ing cTnT, trypsin, IgG, CEA, LZM, and AFP, were chosen as
controls. The effects of various interfering substances on the
sensing system are negligible, as depicted in Figure 5(d). The
above results demonstrate that our fluorescent ELISA ex-
hibits excellent sensitivity and specificity for cTnI. Fur-
Figure 4 (a) FL emission spectra and (b) the corresponding FL intensities of Si-PDs formed by in situ catalysis of ALP with different concentrations (color
online).
Figure 5 (a) Schematic illustration of the fluorescent ELISA. (b) The FL emission spectra and (c) the corresponding FL intensities of the fluorescent ELISA
in the presence of various concentrations of cTnI (inset: the linear relationship from 1 to 30 ng/mL). (d) Selectivity investigation of the fluorescent ELISA for
cTnI. The cTnI and other proteins/enzymes are 100 ng/mL (color online).

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In summary, we provide a facile method for preparing strong
green-emission Si-PDs at room temperature by simply
mixing the solutions of HQ and DAMO. Owing to the ALP-
catalyzed specific conversion of 4-HPP into HQ and the
difference in the reaction with DAMO between HQ and
4-HPP, we develop a simple strategy to detect ALP activity
and its inhibitor based on in situ formation of green-emission
Si-PDs. Inspired by the widespread use of ELISA and ALP-
catalyzed in situ generation of green-emission Si-PDs, we
have constructed a novel fluorescent ELISA for cTnI de-
tection. The results of the proposed fluorescent ELISA for
cTnI detection in diluted human serum coincide with those of
standard colorimetric ELISA. Under the unambiguously re-
cognition mechanism and easy signal generation approach,
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assay, inhibitor screening, and disease diagnosis.
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Acknowledgements This work was supported by the National Key Re-
search and Development Program of China (2016YFA0201301), the Na-
tional Natural Science Foundation of China (21435005, 21627808,
21974132), the Youth Innovation Promotion Association, CAS (2018258)
and Open Project of State Key Laboratory of Supramolecular Structure and
Materials (sklssm2019023).
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Conflict of interest The authors declare that they have no conflict of
interest.
Supporting information The supporting information is available online at
http://chem.scichina.com and http://link.springer.com/journal/11426. The
supporting materials are published as submitted, without typesetting or
editing. The responsibility for scientific accuracy and content remains en-
tirely with the authors.
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