Alkaline Phosphatase Assay Based on the Chromogenic Interaction of Diethanolamine with 4-Aminophenol

Article abstract of DOI:10.1021/acs.analchem.8b01371

Diethanolamine (DEA) has been extensively utilized as an alkaline buffer in current assays of alkaline phosphatase (ALP) activity in the past decades. While playing the role of a buffer, the chemical reactivity of DEA has been widely ignored in such assays. Herein, we report an interesting chromogenic interaction between DEA and 4-aminophenol (AP) in the presence of H₂O for the first time, which inspires us to develop a novel DEA-participated ALP activity assay by using 4-aminophenyl phosphate (APP) as a substrate. This APP/DEA-based colorimetric approach has been proved to be comparable and even superior to the conventional p-nitrophenyl phosphate (pNPP)-based one, especially in the low ALP activity region, due to its higher sensitivity. The clear response mechanism and excellent sensing performance ensure that it can be further applied to determining ALP activity in real biological samples, screening potential ALP inhibitors in vitro, establishing ALP-enabled ELISA, and even fluorophore-assisted fluorescent ALP activity assay. It is demonstrated that this strategy not only possesses a good feasibility, but also exhibits a promising outlook for a series of ALP-related and -extended detections.

Full text of DOI:10.1021/acs.analchem.8b01371

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Alkaline Phosphatase Assay Based on the Chromogenic
Interaction of Diethanolamine with 4-Aminophenol
Jian Sun, Jiahui Zhao, Xingfu Bao, Qifeng Wang, and Xiurong Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01371 • Publication Date (Web): 23 Apr 2018
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Analytical Chemistry
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Alkaline Phosphatase Assay Based on the Chromogenic Interaction
of Diethanolamine with 4-Aminophenol
Jian Sun†, Jiahui Zhao†,§, Xingfu Bao†, Qifeng Wang*,‡, and Xiurong Yang*,†
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†State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciꢀ
ences, Changchun, Jilin 130022, China.
‡ International Joint Research Laboratory of NanoꢀMicro Architecture Chemistry (NMAC), Department of Organic Chemisꢀ
try, College of Chemistry, Jilin University, Changchun, Jilin 130012, China.
§University of Chinese Academy of Sciences, Beijing 100049, China.
*Fax: +86 431 85689278. Eꢀmail: xryang@ciac.ac.cn, wangqifeng@jlu.edu.cn.
ABSTRACT: Diethanolamine (DEA) has been extensively utilized as an alkaline buffer in current assays of alkaline phosphatase
(ALP) activity in the past decades. While playing the role of a buffer, DEA has been widely ignored about its chemical reactivity in
such assays. Herein, we report an interesting chromogenic interaction between DEA and 4ꢀaminophenol (AP) in the presence of
H2O for the first time, which inspires us to develop a novel DEAꢀparticipated ALP activity assay by using 4ꢀaminophenyl phosꢀ
phate (APP) as a substrate. This APP/DEAꢀbased colorimetric approach has been proved to be comparable and even superior to the
conventional pꢀnitrophenyl phosphate (pNPP)ꢀbased one, especially in the low ALP activity region due to its higher sensitivity. The
clear response mechanism and excellent sensing performance ensure that it can be further applied in determining ALP activity in
real biological samples, screening potential ALP inhibitors in vitro, establishing ALPꢀenabled ELISA and even fluorophoreꢀassisted
fluorescent ALP activity assay. It is demonstrated that this strategy not only possesses a good feasibility but also exhibits a promisꢀ
ing outlook for a series of ALPꢀrelated and ꢀextended detections.
The ubiquitous alkaline phosphatase (ALP, EC 3.1.3.1) is a
homodimeric digestive enzyme that is involved in phosphate
metabolism and widely distributed in various biological organꢀ
isms from bacteria to mammalian.1ꢀ3 ALP exhibits an optimal
enzyme reaction rate in an alkaline environment and can speꢀ
cifically catalyze the hydrolysis of phosphate ester groups.
Owing to its widespread prevalence, the level of serum ALP
serves as a significant indicator of certain diseases, such as
rickets, osteomalacia, hepatitis, periodontal diseases and even
prostatic cancer et al.4,5 Moreover, ALP is frequently emꢀ
ployed as a signal enzyme in the enzymeꢀlinked immunoꢀ
sorbent assay (ELISA), dairy industry and so on.6ꢀ8 Therefore,
developing a remarkable and convenient assay for ALP activiꢀ
ty is necessary and important to the related disease diagnosis,
drug screening and other extended applications.9ꢀ11
phate and several other phosphate functionalized molecules
have been already developed and used as fluorogenic subꢀ
strates.19 With rapid development of plasmonic and fluorescent
nanomaterials, a series of booming colorimetric and fluorimetꢀ
ric ALP assays have been also established based on the differꢀ
ential influence of certain substrates and corresponding prodꢀ
ucts on the property of such nanomaterials.9,10,14
Generally, higher enzyme reaction rate of ALP can be obꢀ
served in alkaline solution (typical pH = 9.8), therefore, a
common buffer reagent, diethanolamine (DEA), is employed
as the main component in most current assays of ALP activiꢀ
ty.20,21 In fact, DEA is prone to provide a proper buffer pH for
ALP detection while it does not influence the enzymatic reacꢀ
tion and signal generation owing to the favorable chemical
stability and biocompatibility.22 Thus, it is relatively scarce to
pay attention to the DEA molecule and there is no report conꢀ
cerning the chemical reactivity of DEA except for the pHꢀ
buffering effect in ALP assay to the best of our knowledge.
It is particularly noteworthy that ALP has broad substrate
specificity and ability to dephosphorylate or transphosphoryꢀ
late a large number of phosphated molecules.12 On the basis of
the clearly catalytic principle and enough desirable substrates,
various analytical methods including electroanalytical, fluoꢀ
rometric and colorimetric assays have been developed for deꢀ
termining ALP activity.13ꢀ15 In this regard, the phenyl phosꢀ
phate compounds have been used in the colorimetric determiꢀ
nation of ALP activity for more than seventy decades.16,17 Esꢀ
pecially, pꢀnitrophenyl phosphate (pNPP) is one of the most
understood and commercially available chromogenic subꢀ
strates in the conventional colorimetric assay for ALP activity,
in which pNPP can be converted into a colored product, pꢀ
nitrophenol (NP).16,18 Moreover, 4ꢀmethylumbelliferyl phosꢀ
As a typical and desired substrate, 4ꢀaminophenyl phosphate
(APP) can be hydrolyzed and transformed into 4ꢀaminophenol
(AP) in the presence of ALP.23,24 In comparison with APP, the
enzymatic product AP exhibits better electrochemical activity
and can be oxidized more easily.25 Therefore, both APPꢀbased
electrochemical ALP activity assay and resultant ALPꢀenabled
electrochemical immunoassay have been proposed using redox
cycling by a reducing agent.26ꢀ28 Furthermore, Lin et al. have
developed an ALPꢀtriggered colorimetric ELISA based on the
fact that ALP could catalyze the hydrolysis of APP into AP,
which could reduce silver ions to generate silver shell onto the
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Analytical Chemistry
Page 2 of 9
gold nanobipyramids (Au NBPs) and thus resulted in a signifiꢀ
aqueous solutions. The absorption spectrum measurements
were carried out after incubation at 37 °C for 60 min.
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cant impact on the localized surface plasmon resonance propꢀ
erties of Au NBPs.29 On the other hand, however, it is very
limited in the direct colorimetric assays for ALP by using APP
as the substrate, due to the indistinguishable differences in the
colors and absorption spectra between the colorless APP and
AP aqueous solutions, unlike traditional pNPP/NP subꢀ
strate/product pair.20
APP/DEA-Based Assay for ALP Activity. The ALP activꢀ
ity assay was performed with the following procedures. Volꢀ
umes of the 400 ꢂL of APP (2 mM), 400 ꢂL of DEA (400 mM,
pH 9.8), 80 ꢂL of MgCl2 (1 mM) and 320 ꢂL of water were
injected into a 2.0 mL microcentrifugal tube. Next, the 400 ꢂL
of freshly prepared ALP standard aqueous solutions with difꢀ
ferent activities ranging from 0 to 200 mU/mL were added
into the solutions, respectively. The above mixture solutions
were incubated at 37 °C for 60 min before the absorption specꢀ
trum measurements. The APPꢀbased and the control pNPPꢀ
based assays for ALP activity in diluted fetal bovine serum
sample (FBS, 2%) were carried out under the same conditions.
For the first time, however, we discovered a very interesting
phenomenon that the absorption spectrum of AP in DEA buffꢀ
er solution had a new absorption peak centered at about 365
nm after incubation, which was not obtained if using APP
instead of AP. We then tried to explain this phenomenon by
demonstrating the mechanism and behavior of the chromogenꢀ
ic interaction between DEA and AP. Inspired by such disꢀ
criminability and difference between APP and AP, we report
here an original colorimetric assay for ALP activity by emꢀ
ploying APP as the substrate, in which DEA is not only a pHꢀ
buffering agent, but also a reactant in the aforementioned
chromogenic interaction. More significantly, such facile and
exclusive interaction between DEA and AP demonstrated high
sensitivity, good selectivity, and universal applicability in
ALP assay. According to the clear response mechanism and
excellent sensing performance, such APP/DEAꢀbased ALP
assay is extended into determining ALP activity in real biologꢀ
ical samples, screening potential ALP inhibitors and establishꢀ
ing ALPꢀenabled ELISA. In addition, an original fluorescent
ALP assay is also rationally developed through the combinaꢀ
tion of the proper fluorophore (silver/gold bimetallic nanoclusꢀ
ters capped with 11ꢀMUA, AgAuNCs@MUA as an example).
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ALP Inhibition Assay. Volumes of the 400 ꢂL of APP (2
mM), 400 ꢂL of DEA (400 mM, pH 9.8), 80 ꢂL of MgCl2 (1
mM), and 80 ꢂL of freshly prepared ALP standards (100
mU/mL) and 240 ꢂL of water were injected into a 2.0 mL
microcentrifugal tube. Then the 400 ꢂL of Na3VO4 or
Na2MoO4 with various concentrations were added into the
solutions, respectively. The above mixture solutions were inꢀ
cubated at 37 °C for 60 min before the absorption spectrum
measurements.
APP-Based ELISA for Human AFP. First, volumes of the
100 ꢂL of AFP standards (0 to 100 ng/mL) were added into
the microplates coated with antiꢀhuman AFP and the microꢀ
plates were incubated overnight at 4°C. After incubation, the
removal of AFP solutions and washing processes were executꢀ
ed sequentially. Next, 100 ꢂL of 500ꢀfold diluted AFP detecꢀ
tion antibody was added, followed by incubation at room temꢀ
perature for 60 min with gentle shaking. After removal and
washing again, 100 ꢂL of 3000ꢀfold diluted ALPꢀconjugated
secondary antibody was added, followed by incubation for 60
min with gentle shaking. After removal and washing processes,
the 62.5 ꢂL of APP (2 mM), 62.5 ꢂL of DEA (400 mM, pH
9.8), 12.5 ꢂL of MgCl2 (1 mM) and 112.5 ꢂL of water were
added. The absorption spectrum measurements were carried
out after gentle shaking at 37 °C for 60 min.
EXPERIMENTAL SECTION
Reagents and Apparatus. Diethanolamine (DEA), 4ꢀ
aminophenol (AP), 4ꢀaminophenyl phosphate monosodium
salt hydrate (APP), 4ꢀnitrophenyl phosphate disodium salt
hexahydrate (pNPP), silver nitrate (AgNO3), alkaline phosphaꢀ
tase (EC 3.1.3.1) from bovine intestinal mucosa, 11ꢀ
mercaptoundecanoic acid (MUA), hydrogen tetrachloroaurate
trihydrate (HAuCl4·3H2O) were purchased from Sigmaꢀ
Aldrich (St. Louis, MO). Magnesium chloride (MgCl2), sodiꢀ
um molybdate dehydrate (Na2MoO4), trisodium tetraoxovanaꢀ
date dodecahydrate (Na3VO4) and sodium hydroxide (NaOH)
were purchased from Aladdin Industrial Corporation (Shangꢀ
hai, China). Fetal bovine serum was purchased from Sanꢀ
gonBiotech Co. Ltd. (Shanghai, China). The microplates coatꢀ
ed with antiꢀhuman AFP, lyophilized AFP protein standard,
AFP detection antibody, sample diluent buffer and wash buffꢀ
er were purchased from RayBiotech (Norcross, GA). ALPꢀ
conjugated secondary antibody was purchased from Abcam
(Cambridge, MA). Ultrapure water (18.2 Mꢁ cm) was used in
all aqueous solution.
AgAuNCs@MUA-Assisted Fluorescent Assay for ALP
Activity. The silver/gold bimetallic nanoclusters capped with
11ꢀMUA (AgAuNCs@MUA) were synthesized and purified
according to a modified strategy reported previously.30,31 In
brief, MUA (13.1 mg) and NaOH (6.0 mg) were dissolved in
water (8.5 mL), followed by addition of the solution of AgNO3
(0.5 mL, 10 mM) and HAuCl4 (1.0 mL, 10 mM) at room temꢀ
perature. The thorough mixture was left to stand for 5 h at
room temperature, and then was dialyzed with an 8−14 kDa
cutoff dialysis bag, against ultrapure water for more than 24 h
with four changes.
The fluorescent ALP activity assay was performed using the
following procedures. Volumes of the 200 ꢂL of APP (2 mM),
200 ꢂL of DEA (400 mM, pH 9.8), 40 ꢂL of MgCl2 (1 mM)
and 160 ꢂL of water were injected into a 2.0 mL microcentrifꢀ
ugal tube. Next, the 200 ꢂL of freshly prepared ALP standard
aqueous solutions with different activities ranging from 0 to
80 mU/mL were added into the above solutions, respectively.
After gentle shaking at 37 °C for 60 min, 800 ꢂL of 100ꢀfold
diluted AgAuNCs@MUA was added, followed by the fluoresꢀ
cence spectrum measurements.
Absorption and fluorescence spectra were recorded on
CARY 500 UV−VisꢀNIR Varian spectrophotometer (CA) and
Hitachi Fꢀ4600 spectrofluorometer (Tokyo, Japan), respectiveꢀ
ly, by using a 1 cm path length quartz cell at room temperature.
Transmission electron microscopy (TEM) images were acꢀ
quired by a FEI Tecnai G2 F20 SꢀTWIN (OR).
The Interaction Between DEA and AP. The 400 mM
DEA stock solution were prepared by dissolving DEA into the
ultrapure water and adjusting pH values to 9.8. The interaction
between DEA and AP was performed by adding different
amounts of DEA stock solution into freshly prepared AP
RESULTS AND DISCUSSION
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Analytical Chemistry
The Chromogenic Interaction Between DEA and AP. As
First, we investigated the concentration effect of AP to the
resultant mixture. As shown in Figure 2A and 2B, by adding
different concentrations of AP into the aqueous solution of
constant DEA (100 mM, pH 9.8), the absorption spectra of the
mixture brought a continuous enhancement and the absorbꢀ
ance values at 365 nm presented a quasiꢀlinear relationship as
AP concentrations ranging from 0 to 200 ꢂM. Second, the
absorption spectra in Figure 2C demonstrated that pH values
of the solution could influence the AP/DEA interaction and
the optimum pH value was around 10 according to the maxiꢀ
mum absorbance. This strong alkaline pH value was similar to
that of DEA buffer, which was commonly used in the ALP
activity assay. We next evaluated the concentration effect of
DEA to the resultant mixture. By adding different concentraꢀ
tions of DEA (pH 9.8) into the aqueous solution of constant
AP (100 ꢂM), the corresponding absorption spectra in Figure
2D had a quick to slow absorption enhancement under inꢀ
creased concentrations (typically > 100 mM). Incubation time
and temperature could also affect the interaction between AP
and DEA. As presented in Figure 2E and 2F, the timeꢀ
dependent absorption spectra of the mixture solutions and
plots of absorbance values at 365 nm as a function of time
showed that the AP/DEA interaction accelerated over the iniꢀ
tial stage and then reached a plateau after 60 min. In addition,
higher incubation temperature would be only beneficial to the
rapid completion of the reaction under current conditions (data
not shown).
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a typical phosphated molecule, APP is frequently employed as
the substrate in ALP activity assays, in which the oxidationꢀ
reduction properties between APP and the corresponding enꢀ
zymolysis product AP can be differentiated by electrochemical
measurements.25ꢀ27 Besides the better electrochemical activity,
AP has no obviously differences in absorption or reactivity
compared to APP in previous reports. On the other hand, owꢀ
ing to its buffer capacity in alkaline environment, the colorless
DEA solution used to serve as a pH mediator in ALP activity
assay, as well as in ALPꢀenabled ELISA.20 In fact, the aqueous
solution of sole AP or DEA kept colorless under the incubaꢀ
tion at 37 °C for 60 min, where the representative absorption
spectrum of AP exhibited an absorption peak around 305 nm
and that of DEA lacked a characteristic peak (Figure 1). Howꢀ
ever, we were surprised that the mixture solution of AP and
DEA displayed an apparent absorption peak centered at about
365 nm. Such an interesting phenomenon as well as the new
characteristic peak has not been reported in the previous reꢀ
search as far as we know. Therefore, we further evaluated this
unusual interaction between AP and DEA.
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Insight into the Interaction of DEA with AP in H2O. The
absorption peak at 365 nm was proposed to be related to the
reaction between DEA and AP. From the viewpoint of synꢀ
thetic organic chemistry, the synthesis and isolation of the
corresponding product is impossible under our current condiꢀ
tions because the stoichiometry of DEA to AP is at least 200
equiv. excess and both of the substrates have to be under low
concentrations for a reasonable rate of transformation. Neverꢀ
theless, we performed the reaction of DEA with AP under
Figure 1. Absorption spectra of DEA (black), AP (red) and DEA
+ AP (blue) aqueous solution after incubation at 37 °C for 60 min.
Figure 2. (A) Absorption spectra and (B) absorbance values at 365 nm of the solutions by adding different concentrations of AP (0−200
ꢂM) into DEA (100 mM, pH 9.8) after incubation at 37 °C for 60 min. (C) Absorption spectra of the solutions by adding AP (100 ꢂM) into
DEA (100 mM) at different pH values from 7 to 12 after incubation at 37 °C for 60 min. (D) Absorption spectra of the solutions by adding
AP (100 ꢂM) into different concentrations of DEA (0−500 mM, pH 9.8) after incubation at 37 °C for 60 min. (E) The timeꢀdependent abꢀ
sorption spectra of the mixture solutions of AP (100 ꢂM) and DEA (100 mM, pH 9.8) at 37 °C. (F) Absorbance values at 365 nm of the
solutions by adding different concentrations of AP (0−500 ꢂM) into DEA (100 mM, pH 9.8) as a function of incubation time.
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Analytical Chemistry
literature reported condition32 and obtained a thermodynamiꢀ
Page 4 of 9
In order to achieve the optimal performance of our proposed
assay, several experimental conditions were investigated and
optimized. Obviously, DEA was not only a buffer component
but also served as a reactant in the chromogenic reaction. Thus
the adopted concentration of DEA here was 100 mM, which
was prone to provide a proper buffer pH and enough to react
with the enzymatic product AP according the investigation on
the AP/DEA interaction. It is suggested that magnesium ions
(Mg2+) act an important role in accelerating the enzymatic
reaction of phosphatase.33 Our results in Figure S1 showed
that higher concentrations of Mg2+ could induce a more marꢀ
velous enhancement of the absorption at 365 nm. However,
the absorbance values had little change when adding Mg2+
concentration more than 50 ꢂM, and such concentration was
chosen in the following assay experiments. In addition, the
concentrations of APP also influenced the absorption spectrum
in the assay process. As shown in Figure S2, under constant
DEA and Mg2+ ions concentrations, the absorption values at
365 nm exhibited a gradual enhancement with an increased
concentration of APP. Comprehensive considering the signal
accuracy and reagents consumption, we selected APP (500 ꢂM)
in 100 mM DEA buffer (pH 9.8) containing Mg2+ (50 ꢂM) as
the standard sensing solution to determine the activity of ALP.
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cally stable product, 1ꢀ(4ꢀhydroxyphenyl)piperazine (HPPA)
(Figure 3A). HPPA was then characterized under a series of
solution conditions. As shown in Figure 3B, to avoid the influꢀ
ence from DEA and AP, HPPA was characterized immediateꢀ
ly after dissolved in pure H2O and ethyl acetate, respectively.
Interestingly, no absorption peak at 365 nm appeared under
both conditions (line a and c in Figure 3B). In sharp contrast,
after incubation for 60 min, the corresponding absorption apꢀ
peared in aqueous solution (line b) while be absent in ethyl
acetate solution (data is very similar to line c and not shown),
indicating the absorption peak at 365 nm is originated from the
specific interaction between HPPA and H2O. By extracting the
resultant mixture of HPPA and H2O with ethyl acetate, we
found the absorption peak at 365 nm was maintained. It should
be noted that the interaction between HPPA and H2O is quite
incomplete, with most of HPPA remained. Although the low
conversion rate of HPPA in H2O led to a difficulty in isolation
and full characterization of the resultant, the high UVꢀ
absorbanceꢀsensitivity and detectability of the resultant made
it an observed advantage for potential ALP activity assay.
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APP/DEA-Based Colorimetric Assay for ALP Activity.
Under similar conditions, no chromogenic product with an
absorption peak at 365 nm could be obtained when we used
APP to incubate with DEA (Figure 4A). These results further
demonstrated the high chemical selectivity of the interaction
between AP and DEA. On the other hand, it is not surprising
that the incubation of sole ALP in DEA aqueous solution
could not induce any featured absorption. As expected, the
resultant mixture exhibited an apparent absorption peak cenꢀ
tered at 365 nm by simultaneously adding APP and ALP into
DEA aqueous solution followed by incubating at 37 °C for 60
min. Inspired by such a phenomenon and the catalytic capabilꢀ
ity of ALP in the hydrolysis of APP to AP, we proposed an
APP/DEAꢀbased colorimetric assay for ALP activity by using
APP as the substrate and the AP/DEA interaction as a signal
generation unit. More significantly, the most commonly used
buffer in the enzymatic reaction by ALP is just alkaline DEA
buffer (pH = 9.8), which is also very beneficial to the subseꢀ
quent chromogenic interaction between AP and DEA. Thereꢀ
fore, both the enzymatic and chromogenic processes of our
ALP assay can be performed in one pot at the same time.
In the presence of serial diluted ALP standards, the aforeꢀ
mentioned sensing solutions were incubated at 37 °C for 60
min and the absorption spectra recorded in Figure 4B showed
that the absorbance at 365 nm of the mixture solution had a
continuous enhancement with increasing ALP activities (0 –
50 mU/mL). In particular, a quasiꢀlinear relation between abꢀ
sorbance values and ALP activities were presented between
0.01 to 10 mU/mL and the fitted data could be expressed as
A365 = 0.0033 + 0.144 CALP (mU/mL), R2= 0.995 (Figure 4C
and 4D). Moreover, the inset graph of Figure 4C showed that
0.01 mU/mL of ALP could clearly result in an obvious inꢀ
crease in absorbance values and be easily identified by the
absorption spectrophotometer.
Figure 4. (A) Absorption spectra of the solutions by adding APP
(black), ALP (red), APP + ALP (blue) and AP (green) into DEA
after incubation at 37 °C for 60 min. (B) Absorption spectra, (C)
absorbance values at 365 nm and (D) expanded linear response
region of the sensing system towards ALP standards with various
activities (0−50 mU/mL).
Figure 3. (A) Chemical structures and proposed reaction of AP,
DEA and HPPA. (B) Absorption spectra of HPPA in aqueous
solution (a), in aqueous solution after incubation for 60 min (b), in
ethyl acetate solution after incubation for 60 min (c) and the ethyl
acetate (EA) extraction from b (d), respectively.
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Figure 5. (A) Schematic representation of the APPꢀbased and pNPPꢀbased assays for ALP activity. Absorption spectra of the APPꢀbased
(B) and pNPPꢀbased (D) sensing systems towards ALP standards with 2% fetal bovine serum. (C) Plots of absorbance values at 365 nm for
APPꢀbased assay (black) and at 405 nm for pNPPꢀbased assay (red) as a function of ALP activities (0−50 mU/mL). Inset: expanded region.
Figure 6. (A) Schematic representation of the immunoassay based on the APPꢀbased ALP assay and conventional ELISA platform. (B)
Absorption spectra and (C) absorbance values at 365 nm of the ELISA system toward AFP standards (0.01 – 100 ng/mL).
As described in Figure 5A, the conventional colorimetric asꢀ
say for ALP activity usually employs a commercially available
compound pNPP as the substrate and pNPP can be converted
into a colored product NP in DEA buffer. Alternatively, our
proposed ALP assay needs to add commercially available APP
into DEA buffer. To evaluate the feasibility and potential bioꢀ
application of our developed assay, we carried out the
APP/DEAꢀbased and conventional pNPPꢀbased sensing sysꢀ
tems, respectively, in determination of ALP activity in 2%
fetal bovine serum (Figure 5). Absorption spectra reveal that
both sensing systems are promising in real biological sample
analysis based on the high specificity in terms of the phenolic
phosphates. However, our developed APP/DEAꢀbased assay is
superior in the sensitivity to pNPPꢀbased one, especially in low
ALP activity region, due to its higher sensitivity in absorption
with increasing ALP activities (Figure 5C). On the other hand,
the different detection window (absorbance values at 365 nm
for APP/DEAꢀbased assay and at 405 nm for pNPPꢀbased one)
might make the APP/DEAꢀbased assay as a substitute or supꢀ
plement for the conventional pNPPꢀbased one.
applying the assay for inhibitor screening and ALPꢀenabled
immunoassay. First, considering the integral role in metaboꢀ
lism, it is necessary to develop a facile inhibitor screening
method for ALP, where molybdate (MoO42–) and orthovanaꢀ
date (VO43–) are commonly used as the model inhibitors in
vitro.34ꢀ36 By premixing increasing concentrations of MoO42ꢀ or
3ꢀ
VO4 with constant activity of ALP (5 mU/mL), as shown in
Figure S3 and S4, the enzymatic activity were efficaciously
inhibited and the absorption peak centered at 365 nm exhibited
a gradual reduction. Plotting the absorbance values at 365 nm
2–
3–
versus the logarithm of the MoO4 or VO4 concentration, a
typical sigmoidal profile were obtained and the IC50 values (50%
maximal inhibitory concentration) were calculated to be apꢀ
2–
proximate 1.56 mM for MoO4 and 63.3 ꢂM for VO43–. Both
of the values were in the same order of magnitude as those
previously reported by other assays for ALP activity.34,35
Next, being one of most frequently used enzymes in ELISA,
ALP activity assay has usually been extended into the ALPꢀ
enabled immunoassay.6 As descripted in Figure 6A, we used
AFP as a model antigen target, combining with commercially
available antibodies and antibodyꢀlabeled ALP to demonstrate
the analytical behavior of ALPꢀlabeled ELISA for a proofꢀofꢀ
concept.37 Under the controls, the addition of individual antiꢀ
Additional Application of the ALP Activity Assay. To
further confirm the availability of our developed APP/DEAꢀ
based ALP activity assay, we demonstrated the possibility of
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human AFP (capture Ab), AFP standard and detection antiꢀ
Page 6 of 9
including APP, DEA and AgAuNCs@MUA clearly exhibited
a characteristic emission of the AgAuNCs. With the activities
of incoming ALP standards increasing from 0 to 20 mU/mL,
the fluorescence emission intensity decreased gradually.
Moreover, a quasiꢀlinear relationship between the logarithm of
the intensity values at 630 nm and ALP activities could be
obtained from 0 to 5 mU/mL, in which the fitted data could be
expressed as Log (I630) = 3.091 – 0.370 CALP (mU/mL), R2=
0.991. Such fluorescent sensing performance were similar to
that in the colorimetric response, undoubtedly suggesting
fluorophoreꢀassisted fluorescent assay possessed considerable
operability and making the detection results more convincing.
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body (ab1), respectively, had no influence on absorption specꢀ
trum of the aforementioned ALP sensing solution (data not
shown). Following the routine immobilization of the antigens
and antibodies on a 96ꢀwell plate, our proposed sensing soluꢀ
tion was introduced and the absorption spectra were recorded.
The results in Figure 6 demonstrated that the absorption specꢀ
trum and absorbance values at 365 nm of the reaction solutions
exhibited a gradual enhancement with the AFP concentrations
increasing from 0 to 100 ng/mL. In particular, a quasiꢀlinear
relationship between double logarithmic absorbance values
and AFP concentration could be obtained from 0.1 to 20
ng/mL and the fitted data could be expressed as Log (A365) = –
0.867 + 0.370 Log (CALP) (ng/mL), R2= 0.994. In such sensiꢀ
tive APP/DEAꢀbased ELISA, 0.1 ng/mL of AFP could be easiꢀ
ly identified by the absorption spectrophotometer and this valꢀ
ue was lower than those in previously reported ELISA.22,38
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CONCLUSION
In summary, an interesting chromogenic interaction between
DEA and AP has been discovered and studied in detail. The
absorption spectrum of the AP and DEA in the presence of
H2O has a new absorption peak centered at about 365 nm,
which is not presented if using APP instead of AP. Inspired by
the ALPꢀtriggered hydrolysis of APP into AP, a wellꢀdefined
colorimetric assay for ALP activity has been rationally develꢀ
oped based on such discriminability between APP and AP. For
the first time, DEA behaves as not only a buffer component
but also a reactant in a chromogenic reaction for assaying ALP.
By means of the clear response mechanism and excellent sensꢀ
ing performance, our developed DEAꢀparticipated ALP activiꢀ
ty assay is successfully applied in measuring the real biologiꢀ
cal samples, screening potential inhibitor, constructing the
ALPꢀenabled immunoassay and even fluorophoreꢀassisted
fluorescent assay. Thus, this promising assay will not only be
useful in direct detection of ALP activity but also in other
ALPꢀrelated detection and sensing systems. More significantly,
the APP/DEAꢀbased assay is superior to pNPPꢀbased one in
terms of the sensitivity. On the other hand, the different detecꢀ
tion window might make such assay as a substitute or suppleꢀ
ment for the conventional pNPPꢀbased one.
Figure 7. (A) Fluorescence spectra of the AgAuNCs@MUAꢀAPP
sensing system towards ALP standards with various activities
(0−20 mU/mL). (B) Plots of the corresponding logarithm of fluoꢀ
rescence intensities at 630 nm as a function of ALP activities.
AgAuNCs@MUA-Assisted Fluorescent Assay for ALP
Activity. Besides colorimetric method, the fluorescent assays
have several advantages and are also well exploited due to
their intrinsic high sensitivity and fast response time. Inspired
by the fluorescence quenching phenomenon of some fluoroꢀ
phores with certain chromogenic reagents, a series of coloriꢀ
metric responses has been further extended into fluorescent
assays by means of proper fluorescent materials.39ꢀ41 In fact,
adding another signal sensing mode is conducive to providing
more accurate and convincing detection results. On the basis of
the unique optical properties and large stokes shift,42,43 we
introduced a type of promising fluorescent noble metal
nanoclusters (e.g. silver/gold bimetallic nanoclusters capped
with 11ꢀMUA, AgAuNCs@MUA) into APP/DEAꢀbased ALP
activity assay.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Effect of Mg2+ and APP concentrations on the colorimetric
ALP assay, detailed absorption spectra of the assay for inꢀ
2–
hibitor screening (MoO4 and VO43–), typical TEM image
and
fluorescence
excitation/emission
spectra
of
AgAuNCs@MUA and the effect of AP in DEA solution on
the fluorescence of AgAuNCs@MUA (PDF)
As shown in Figure S5, the typical TEM image and fluoresꢀ
cence excitation/emission spectra confirmed the generation of
AgAuNCs@MUA. More significantly, AgAuNCs@MUA
exhibited an intense excitation band with a broad range around
350 nm, which was partially overlapped with the absorption
spectrum of AP/DEA mixture. By adding APꢀDEA mixture
solution into AgAuNCs@MUA solution, the fluorescence
emission peak around 630 nm of AgAuNCs@MUA was reꢀ
mained, while the fluorescence intensity exhibited a gradual
decrease owing to the quenching effect of the resultant HPPA
derivative (Figure S6). In addition, the presence of individual
ALP, APP, AP and even strong alkaline DEA solution did not
affect the fluorescence of AgAuNCs@MUA (data not shown).
Therefore, it encouraged us to develop a fluorescent ALP acꢀ
tivity assay by means of ALPꢀenabled generation of chromoꢀ
phore and its quenching effect on the fluorescence of
AgAuNCs@MUA (Figure 7). The fluorescent sensing solution
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: xryang@ciac.ac.cn; Fax: +86 431 85689278
*Eꢀmail: wangqifeng@jlu.edu.cn
Author Contributions
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Financial supports by the National Natural Science Foundation of
China (Grant Nos. 21605139, 21435005, 21627808), the National
Key Research and Development Program of China
(2016YFA0201301) and Key Research Program of Frontier Sciꢀ
ences, CAS (QYZDYꢀSSWꢀSLH019).
6
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(23) Tang, H. T.; Lunte, C. E.; Halsall, H. B.; Heineman, W. R. pꢀ
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