In Situ Fluorogenic Reaction Generated via Ascorbic Acid for the Construction of Universal Sensing Platform

Construction of Universal Sensing Platform

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In Situ Fluorogenic Reaction Generated via Ascorbic Acid for the

Yongchao Fan, Mengmeng Lv, Yuan Xue, Jing Li,* and Erkang Wang*

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sı Supporting Information

ABSTRACT: A highly ﬂuorescent emission reaction between

terephthalic acid (PTA) and ascorbic acid (AA) via simple control

of the reaction temperature was ﬁrst revealed with the detailed

formation mechanism and various characterizations including

electron paramagnetic resonance and mass spectrometry. Based

on the AA-responsive emission, the alkaline phosphatase (ALP)

triggered the transformation of L-ascorbic acid 2-phosphate

trisodium salt to AA was integrated with the present system for

developing a sensitive, selective, and universal platform. The

monitoring of the activity of ALP and the fabrication of ALP-based

enzyme-linked immunoassay (ELISA) with carcinoembryonic

antigen (CEA) as the model target was performed. The

ﬂuorescence intensity correlated well to the CEA concentration

in the ranges of 0.25−30 ng/mL, with a detection limit of 0.08 ng/mL. Such a facile protocol based on the ﬂuorescent reaction

between PTA and AA without the assistance of catalysis of nanomaterials avoided the laborious synthesis procedure and provided a

direct strategy for the early clinical diagnosis coupled with ALP-related catalysis.

INTRODUCTION

developed a ﬂuorescent and colorimetric dual-readout mode

for the ALP activity assay based on the chromogenic and/or

ﬂuorescent reactions between N-[3-(trimethoxysilyl)propyl]

ethylenediamine and AP in situ generated from the ALP-

■

Alkaline phosphatase (ALP or AKP) is a biological enzyme

closely related to human health, which is widely distributed in

human liver, bone, intestine, kidney, and other tissues and

1,2

catalyzed hydrolysis of APP.

To further make the

organs. Clinically, abnormal levels of ALP in serum are often

determination of ALP more accurate, functional nanomaterials

were often employed the catalyze the substrate-related

reactions. However, the synthesis or surface modiﬁcation of

used in the diﬀerential diagnosis of bone and hepatobiliary

diseases, especially the diﬀerential diagnosis of jaundice.

Considering the importance of ALP detection in disease

diagnosis, many analytical methods such as colorimetry,

nanomaterials is inevitably complicated, laborious, and time-

19−24

ﬂuorescence, chemiluminescence, surface-enhanced Raman

consuming.

Meanwhile, the introduction of these nano-

10,11

spectroscopy (SERS), electrochemical methods,

and

materials will also increase the cost of detection, the

complexity of the system and reduce the reproducibility of

the experiment. Therefore, it is very urgent to explore a simple,

fast, and low-cost ﬂuorescence method to accurately detect

ALP.

In this work, an interesting ﬂuorescence reaction between

AA and terephthalic acid (PTA) with a high ﬂuorescent

emission was ﬁrst disclosed by simple control of the reaction

temperature in the presence of dissolved oxygen. The strong

blue ﬂuorescence centered at 430 nm was due to the formation

chromatography have been developed for monitoring of

the ALP activity. Among these methods, the ﬂuorescence

method has the merits of high reliability, high sensitivity,

convenient operation, fast response speed, and convenient

instrument requirements, and it is widely used for the analysis

and detection of the samples.

In most case, the determination of the ALP activity is

achieved indirectly by monitoring the ﬂuorescence response of

hydrolysates such as L-ascorbic acid (AA), p-aminophenol

(

AP), phosphate (Pi), and 4-nitrophenol (NP), which were

produced by ALP hydrolyzing the substrate such as L-ascorbic

acid 2-phosphate trisodium salt (AA2P), 4-aminophenol

Received: March 4, 2021

Accepted: April 15, 2021

Published: April 26, 2021

−

phosphate (APP), pyrophosphate (P O₇, PPi), and 4-

13−17

nitrophenyl phosphate (NPP) in the presence of ALP.

Therefore, developing or exploring the hydrolysate-related

ﬂuorescent reaction with a facile procedure is of vital

importance. For example, Sun and Lu et al. envisioned and

2021 American Chemical Society

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Anal. Chem. 2021, 93, 6873−6880

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Article

Scheme 1. Schematic Diagram of the Fluorescence Detection of Carcinoembryonic Antigen (CEA) by Coupling AA2P/PTA-

Participated Fluorescence Turn-on ALP Activity and the Conventional ELISA Platforms

of 2-hydroxyterephthalic acid (PTA-OH), as conﬁrmed by

electron paramagnetic resonance (EPR) and mass spectrom-

etry (MS). The ﬂuorescence intensity (F₄₃₀_n_m) is directly

related to the concentration of AA. On the basis of AA-

dependent FL response and low background from the

substrate of AA2P, the proposed system was utilized for the

activity monitoring of ALP, inhibitor screening, and ALP-

related enzyme-linked immunoassay (ELISA) via the ALP-

triggered transformation of AA2P (Scheme 1). The practical

application of the present ﬂuorescence ELISA was demon-

strated using serum samples with satisfactory recovery. This

convenient and speciﬁc reaction between PTA and AA enabled

the present platform with good selectivity and high sensitivity

for ALP-related assays.

Detection of ALP Activity. Fifty microliters of AA2P (25

mM), 50 μL of ALP with various activities, and 400 μL of

Tris−HCl (25 mM, pH 9.8) buﬀer solution were incubated at

37 °C for 60 min. Subsequently, 100 μL of the above solution

and 20 μL of PTA (10 mM) were added to 500 μL of

phosphate-buﬀered saline (PBS) (0.2 M, pH 6.0) and

incubated at 80 °C for 70 min. At last, the ﬂuorescence

intensities of mixed solutions were recorded directly.

Fluorescence Immunoassay for CEA. First, 50 μL of

mouse monoclonal anti-CEA antibody (1:200) in a coating

solution was added into a 96-well polystyrene plate and

incubated at 4 °C overnight. After discarding the solutions, the

plate was washed three times with 250 μL of washing buﬀer

(PBST) and blocked with 1% BSA at 37 °C for 1 h to block

nonspeciﬁc binding sites. After washing, 50 μL of CEA

standard solutions with diﬀerent concentrations (0, 0.1, 0.2,

EXPERIMENTAL SECTION

■

0.5, 1.0, 2.0, 5.0, 10, 15, 20, 25, 30, 40, 50, and 80 ng/mL)

Chemicals and Reagents. All of the chemicals are used as

received without any further puriﬁcation. Phthalic acid, m-

phthalic acid, PTA, trimesic acid, AA, AA2P, tris-(hydrox-

ymethyl) aminomethane (Tris), sodium vanadate (Na VO ),

were added into each well to incubate at 37 °C for 1 h,

followed by washing steps and the addition of Ab1 (1:500, 50

μL). After incubation at 37 °C for 1 h, the wells were washed

three times and 50 μL of Ab2 (1:3000) was added and

subsequently incubated at 37 °C for 1 h. After washing, we

performed the enzymatic reaction as follows: 100 μL of 2.5

mM of AA2P (dissolve in Tris−HCl 25 mM, pH 9.8) was

added into each well and then incubated at 37 °C for 1 h.

Afterward, the above reaction solution (50 μL) was mixed with

thiourea, and isopropanol were obtained from Aladdin

Industrial Corporation (Shanghai, China). Alkaline phospha-

tase from bovine intestinal mucosa (ALP) was purchased from

Sigma-Aldrich. Mouse monoclonal anti-CEA antibody, carci-

noembryonic antigen (CEA), rabbit polyclonal anti-CEA

antibody (Ab1), and ALP-conjugated goat antirabbit secon-

dary antibody (Ab2) were purchased from Beijing Biosynthesis

Biotechnology Company. Bovine serum albumin (BSA),

glucose oxidase (GOx), trypsin, lysozyme, acetylcholinesterase

20 μL of PTA (5 mM) and added into 550 μL of PBS (0.2 M,

pH 6.0) buﬀer solution at 80 °C for 70 min. The ﬂuorescence

intensities of the resultant solutions were directly recorded

ultimately.

(

AChE), horseradish peroxidase (HRP), and exonuclease were

purchased from Sigma-Aldrich. NaCl, KH PO , and K HPO ·

RESULTS AND DISCUSSION

H O were bought from Beijing Chemical Reagent Company

■

(

Beijing, China). The deionized water (18.2 MΩ/cm) puriﬁed

Fluorescence Reaction between PTA and AA. In the

present work, the sensing mechanism was based on the ALP-

triggered formation of AA; therefore, exploring the AA-

dependent FL response was vital. PTA was ﬁrst introduced

and incubated with AA and the FL spectra were collected. To

our surprise, the mixture of PTA and AA triggered the

generation of a strong emission centered at about 430 nm

when excited at 315 nm and the strong blue ﬂuorescence was

by a Milli-Q system (Millipore, Bedford, MA) was used to

prepare all of the solutions.

Apparatus. The ﬂuorescence spectra were collected on a

Fluoromax-4 spectroﬂuorometer (Horiba Jobin Yvon Inc.,

France) with excitation and emission slit widths of 10 and 10

nm, respectively. UV−vis spectra are recorded using a Cary

00 scan UV−vis spectrometer (Varian).

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Anal. Chem. 2021, 93, 6873−6880

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Figure 1. (A) UV−vis absorption peak, ﬂuorescence excitation, and emission spectra of PTA/AA. Insets are the photographs of PTA/AA in visible

light (a) and under 302 nm UV light (b). (B) UV−vis absorption peak of PTA, AA, and PTA/AA. (C) Fluorescence emission spectra of phthalic

acid/AA, m-phthalic acid/AA, PTA/AA, and trimesic acid/AA. (D) Fluorescence emission spectra of the individual PTA (a), AA (b), PTA/ALP

(

c), PTA/AA2P (d), PTA/AA2P/ALP (e), and PTA/AA (f). Also, the inset is the picture of the corresponding sample (a−f) under 302 nm UV

light.

•

observed under a 302 nm UV light lamp in Figure 1A. UV−vis

spectra were employed to monitor the reaction between AA

and PTA. Apparent adsorption at 312 nm occurred, which

diﬀers from the individual AA (262 nm) or PTA (240 nm),

indicating the formation of products. Meanwhile, the UV−vis

absorption peaks of both PTA and AA decreased, indicating

that both were partially consumed as reactants (Figure 1B).

Interestingly, under the same reaction conditions, this kind of

ﬂuorescent reaction can be extended to the derivatives of PTA

OH and exhibit the typical 1:2:2:1 signal consistent with an

OH spin adduct of DMPO. In addition, the FL responses

after the addition of thiourea and isopropanol (scavenger of

•

OH) were also recorded (Figure 2B). During the reaction

process, the addition of thiourea and isopropanol led to an

obvious decrease in the FL signal, while control tests with

individual thiourea or isopropanol could not induce the

generation of the ﬂuorescent product. All of the above results

disclosed that the formed OH in the presence of AA and

dissolved O under high temperature was captured by PTA

•

(phthalic acid, m-phthalic acid, trimesic acid) (Figure 1C). The

strongest emission was induced by the reaction between PTA

and AA and the maximum emission wavelength blue shifted.

We deduced that the diﬀerent FL responses originated from

the diﬀerent molecular structures. Therefore, the PTA/AA

system was chosen for the investigation of the detailed

formation mechanism. According to the previous litera-

and the blue-ﬂuorescent product of PTA-OH was obtained. To

verify the role of dissolved O in this system, diﬀerent gases

(

such as N , air, and O ) were introduced into the system in

2 2

advance for 30 min, and the ﬂuorescence signal obtained under

the high-temperature reaction showed a signiﬁcant change.

The ﬂuorescence emission with purged N was obviously

5,26

ture,

PTA was often employed to capture the hydroxyl

reduced while that with O was greatly enhanced compared to

•

radicals ( OH) to produce the ﬂorescent PTA-OH for the

indirect monitoring of the amount of the OH or the synthesis

that in the air atmosphere (Figure S1 ). Therefore, we can infer

•

that dissolved O in the solution itself is involved in the

•

of the ﬂorescent MOF. For example, PTA as the bridging

ﬂuorescence reaction and converted to OH by AA under

high-temperature conditions. Subsequently, the resulting OH

•

ligands of MIL-53(Fe) has been applied for the synthesis of

•

MIL-53(Fe) with FL emission due to the generation of OH in

is captured by PTA to form PTA-OH. The MS of the products

with the PTA/AA reaction system had the strongest peak at

m/z = 181.04, conﬁrming the formation of PTA-OH (Figure

2C). Based on the above discussion, the reaction mechanism is

given in Figure 2D. For the ALP-based sensing, AA2P was

often adopted as the common substrate of ALP to generate

AA. Therefore, the blank signal from the AA2P and ALP

should be distinguished. As depicted in Figure 1D, replacing

AA with individual AA2P and individual ALP did not induce

the presence of H O . Therefore, we inferred that the

formation of the ﬂuorescent product was due to the generation

•

of OH induced by AA in the presence of O . Then, the

•

production of OH was ﬁrst characterized by the EPR spin-

trapping techniques (Figure 2A) via the introduction of 5,5-

dimethyl-1pyrroline-N-oxide (DMPO) as radical scavengers to

•

elucidate the formation of OH in the process. Typical EPR

spectra were obtained due to the reaction between DMPO and

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Anal. Chem. 2021, 93, 6873−6880

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•

Figure 2. (A) EPR spectrum of OH in the reaction system (PTA/AA at 80 °C). (B) Fluorescence emission spectra of PTA/AA, PTA/AA/

isopropanol, PTA/AA/thiourea, PTA/isopropanol, and PTA/thiourea (at 80 °C). (C) MS spectra (acquired in the negative ion mode) of PTA/

AA after PTA and AA reﬂect 70 min at 80 °C. (D) Schematic illustration of the ﬂuorescence reaction between PTA and AA.

any ﬂuorescence emission, indicating that the diﬀerent results

between AA2P and AA conﬁrmed the pivotal role of hydroxyl

from enol, which provided the necessary prerequisite for the

determination of ALP-based targets via the enzyme-catalyzed

dephosphorylation of AA2P for the generation of AA.

(Figure 3A,B). Then, the catalytic behavior due to the

introduction of Fe or Fe was investigated. The FL emission

enhanced (ca. 3−4-fold) greatly with the introduction of Fe

or Fe under 37 °C with an incubation time of 30 min to

achieve the original emission intensity obtained at 80 °C for 20

min. To exclude the inﬂuence of Fe or Fe on the emission

of generated PTA-OH, the kinetic ﬂuorescence curves at 430

Optimization of Experimental Conditions. Scheme 1

illustrates the enzyme-triggered assays for monitoring the ALP

activity based on the generation of AA, and the feasibility is

discussed in Figure 1D. Only in the co-presence of ALP and

AA2P, the intense blue ﬂuorescence was induced (inset in

Figure 1D). To obtain optimal performance, the reaction pH,

reaction time, temperature, and concentration of AA2P were

studied in detail (Figure S2). The reaction pH had an

important inﬂuence on the blue ﬂuorescence response ranging

from 4 to 10. Considering the best reaction medium and the

alkaline environment for the transformation of AA2P to AA,

the optimal pH for the reaction between PTA and AA was

determined to be 6.0. Since the detection range and sensitivity

of the sensing system are directly related to the substrate of

AA2P, we evaluated the eﬀect of the amount of AA2P, and the

optimized concentration of AA2P was 2.5 mM. During the

reaction process, it was found that the reaction temperature

and time played an important role in the generation of PTA-

OH. With the increase of the temperature and time, the FL

response increased obviously, and the best FL emission was

observed at 80 °C for 70 min. According to the available

nm were recorded by introducing Fe or Fe into the reaction

mixture of PTA and AA at 37 °C for 30 min (Figure 3C).

Negligible FL change was observed, further indicating that Fe

or Fe had no eﬀect on the emission on the generated PTA-

OH but promoted the formation of OH induced by AA and

•

produced more PTA-OH. Notably, the metal-ion-induced

catalytic reaction was dependent on the reaction temperature.

When the reaction temperature was shifted from 37 to 80 °C,

the catalytic activity decreased (Figure 3D). This may be due

to the fact that high temperature is more favorable to produce

•

OH induced by AA compared to metal ions. Therefore,

improving the temperature or introducing Fe or Fe can be

used for the sensitive assays. In the present work, to simplify

the determination process, control of the temperature was

adopted for the determination of ALP-related targets.

Under optimal experimental conditions, the ﬂuorescence

response of ALP with various activities from 0 to 100 mU/mL

was studied and is depicted in Figure 4A. As shown in Figure

4B, with an increase in the generation of AA, the ﬂuorescence

emission spectrum centered at 430 nm increases linearly, and

the ﬂuorescence intensity correlates well with the ALP

activities in the range of 0.05−20 mU/mL (F = 13.81 +

•

literature, hemin can promote the generation of OH due to

the presence of ferriporphyrin in its structure. Therefore, we

expected that the proposed reaction can be catalyzed by

introducing Fe or Fe , which enabled the reaction to occur

at a suitable temperature with a shortened incubation time

19.25 [ALP] (mU/mL), R = 0.998). The limit of detection

(LOD) was calculated to be 0.016 mU/mL based on 3σ/S,

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Anal. Chem. 2021, 93, 6873−6880

Article

Figure 3. (A) and (B) Catalytic eﬀect of diﬀerent metal ions on the PTA/AA system (from left to right: blank, Na , Mg , Zn , Co , Ni , Ag ,

Mn , Fe , Fe ) at 37 °C, 30 min (metal ions were 0.1 mM). (C) Kinetic ﬂuorescence (430 nm) curve of responses to metal ions on the PTA/AA

system (blank, Fe , Fe ) at 37 °C. (D) Catalytic eﬀect of metal ions (blank, Fe , Fe ) on the PTA/AA system at 20, 37, and 80 °C.

which was comparable or superior to those of other existing

methods (Table S1). According to the previous reports, the

range of ALP in the serum of normal adults is generally 40−

values for ﬂuorometric detections was estimated to be 125.8

μM, which are in good accordance with the reported values in

33,34

other works.

These results indicate that our method has

90 mU/mL, so the ﬂuorescence detection method

great application prospects for screening ALP inhibitors.

ALP-Triggered ELISA Strategy for CEA Detection.

ELISA as a powerful detection technology that combines

antigen−antibody-speciﬁc recognition capabilities and enzyme

high-eﬃciency biocatalytic capabilities has been widely used in

drug development, screening, disease diagnosis, food testing,

constructed is suﬃciently sensitive for the actual detection of

ALP activity in biological samples. To prove the speciﬁcity of

the proposed ﬂuorescence method for the determination of

ALP (20 mU/mL), we compared it with several common

proteases, including BSA, GOx, trypsin, lysozyme, AChE,

HRP, and exonuclease (10 μg/mL). The results showed that

several other proteases could not cause signiﬁcant changes in

the ﬂuorescence intensity of the sensing system (Figure 4D).

Therefore, the ﬂuorescence detection of the ALP method has

high speciﬁcity, which is beneﬁcial to the determination of

ALP in biological samples.

35−38

and other ﬁelds.

Among them, ALP is often used as a

marker for ELISA because of its high catalytic activity, high

stability, and easy coupling with antibodies. To further increase

the practicability of the present ﬂuorescence system,

combining with the enzyme-linked immunoassay, CEA, a

tumor marker widely used in the diagnosis of the gastro-

Inhibitor Screening for ALP. Due to the important eﬀect

of the ALP activity on human physiological activities, the study

of inhibitors of this enzyme is of great signiﬁcance in drug

intestinal tract, breast cancer, and lung cancer was analyzed.

The concentration of CEA in normal human serum is 2.5−5

ng/mL. When this concentration is surpassed, the possibility of

1,32

design.

Here, we took a well-known ALP inhibitor Na VO

human disease is greatly increased. However, the existing

as an example to further verify the proposed ﬂuorescence

detection method to evaluate the inhibitory eﬃciency of the

enzyme. As shown in Figure 5A, as the concentration of

Na VO increases from 0 to 5000 μM with ﬁxed ALP (20 mU/

methods for detecting CEA have many shortcomings such as

complicated detection conditions, expensive detection equip-

41−43

ment, and introduction of nanomaterials.

Thus, a cost-

eﬀective, sensitive, and rapid detection of CEA concentration

in the blood is of great signiﬁcance to human health. At

present, immunoassay has become the most widely used

method for detecting CEA and other cancer markers. Among

them, ELISA has attracted wide attention due to the signal

ampliﬁcation eﬀect of enzyme catalysis. In this experiment, we

designed a ﬂuorescent sandwich enzyme-linked immunosorb-

ent assay to achieve CEA detection. As shown in Scheme 1, the

mL), the ﬂuorescence intensity of the system displays a

concentration-dependent decrease, indicating that Na VO has

a signiﬁcant inhibitory eﬀect on the ALP activity. When

plotting the logarithmic relationship between the ﬂuorescence

intensity and Na VO concentration, a typical sigmoidal curve

was obtained (Figure 5B). The IC₅₀(the inhibitor concen-

tration required for 50% inhibition of the enzyme activity)

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Anal. Chem. 2021, 93, 6873−6880

under 302 nm UV light. (D) Anti-interference ability of the sensing platform in the absence and presence of ALP 20 mU/mL.

Article

Figure 4. (A) Fluorescence responses of the sensing system upon addition of diﬀerent ALP activities. (B) Fluorescence intensities at 430 nm vs

various low ALP activities. Inset: ﬂuorescence intensities vs the concentrations of ALP from 0 to 100 mU/mL. (C) The corresponding photographs

Figure 5. (A) Fluorescence emission spectra with various concentrations of Na VO -treated 20 mU/mL ALP. (B) The corresponding ﬂuorescence

intensity vs the logarithm of the Na VO concentration.

mouse anti-CEA monoclonal antibody, rabbit anti-CEA, and

goat antirabbit IgG labeled with ALP were employed as the

capture antibody, primary antibody, and secondary antibody,

respectively. We ﬁxed the antigens and antibodies used in 96-

well polystyrene plates in turns, added AA2P to generate AA,

and then reacted with PTA at 80 °C to record the ﬂuorescence

signal. As shown in Figure 6A, as the concentration of CEA

increases, the ALP captured on the 96-well plate increased. As

a result, as more AA was produced, the stronger the

ﬂuorescence readout signal became. Figure 6B shows a

quasi-linear relationship between the ﬂuorescence intensity

and CEA concentrations from 0.25 to 30 ng/mL, and then the

ﬁtted linear data could be expressed as F = 72.6 + 4.95 C_C_E_A

(ng/mL), R = 0.996. The LOD as low as 0.08 ng/mL CEA

was obtained, which was comparable to those of the previous

reports for CEA detection (Table S2). To demonstrate the

practical application of this ALP-triggered ELISA in actual

blood samples, we diluted the normal human blood sample by

20 times and adopted the standard addition method for the

CEA blood sample detection. As revealed in Table S3, the

experimental results obtained are consistent with the added

CEA concentration, which proved that our analytical methods

have high accuracy and excellent performance in actual sample

analysis.

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Anhui 230026, P. R. China; orcid.org/0000-0001-9843-

Article

Figure 6. (A) Fluorescence responses of the sensing system upon addition of diﬀerent concentrations of CEA. (B) Fluorescence intensities at 430

nm vs diﬀerent CEA concentrations. (C) Linear relationship of F₄₃₀_n_mwith diﬀerent concentrations of CEA from 0 to 30 ng/mL. (D) Anti-

interference ability of the sensing platform for CEA.

CONCLUSIONS

AUTHOR INFORMATION

■

We disclosed an interesting ﬂuorescence reaction between

PTA and AA to form PTA-OH in the absence of enzymes for

the ﬁrst time with the detailed and clear formation mechanism.

Based on the distinguished response between AA2P and AA,

ALP-triggered transformation was integrated to develop a

simple, universal platform for the assays of ALP activity, ALP

inhibitor, and immunoassay of CEA. Beneﬁting from the

excellent sensing performance for ALP with a LOD of 0.016

mU/mL, the ﬂuorescence immunoassay achieved a detection

limit of as low as 0.08 ng/mL CEA. The proposed ﬂuorescence

sensing platform combined with the metal-ion-speciﬁc catalytic

behavior oﬀered an eﬀective strategy for obtaining high

sensitivity for ﬂuorescence immunoassay in the future.

Corresponding Authors

Jing Li − State Key Laboratory of Electroanalytical Chemistry,

Changchun Institute of Applied Chemistry, Chinese Academy

of Sciences, Changchun, Jilin 130022, P. R. China; University

of Science and Technology of China, Hefei, Anhui 230026, P.

R. China; orcid.org/0000-0002-9828-7569 ;

Phone: +86-431-85262003; Email: lijingce@ciac.ac.cn

Erkang Wang − State Key Laboratory of Electroanalytical

Chemistry, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun, Jilin 130022, P. R.

China; University of Science and Technology of China, Hefei,

1834 ; Email: ekwang@ciac.ac.cn

Authors

Yongchao Fan − State Key Laboratory of Electroanalytical

Chemistry, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun, Jilin 130022, P. R.

China; University of Science and Technology of China, Hefei,

Anhui 230026, P. R. China

ASSOCIATED CONTENT

■

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.analchem.1c00967.

Mengmeng Lv − State Key Laboratory of Electroanalytical

Chemistry, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun, Jilin 130022, P. R.

China

Yuan Xue − State Key Laboratory of Electroanalytical

Chemistry, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun, Jilin 130022, P. R.

China; University of Science and Technology of China, Hefei,

Anhui 230026, P. R. China

The eﬀects of diﬀerent dissolved gases such as N , air,

and O₂on the ﬂuorescence system (Figure S1),

optimized experimental conditions of pH, concentration

of AA2P, temperature, and time (Figure S2), compare

our work with other reported methods for the detection

of ALP and CEA (Tables S1 and S2), and analytical

results for CEA in the human serum samples (Table S3)

(PDF )

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Anal. Chem. 2021, 93, 6873−6880

Analytical Chemistry

Complete contact information is available at:

Article

https://pubs.acs.org/10.1021/acs.analchem.1c00967

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Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

This work was supported by the MOST, China

2020YFB2009004 and 2019YFA0709202) and the Youth

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