Fluorescence Immunoassay Based on the Phosphate-Triggered Fluorescence Turn-on Detection of Alkaline Phosphatase

Article abstract of DOI:10.1021/acs.analchem.7b05325

A simple and cost-effective fluorescence immunoassay for the sensitive quantitation of disease biomarker α-fetoprotein (AFP) has been developed based on the phosphate-triggered fluorescence turn-on detection of alkaline phosphatase (ALP), with the reversi

Full text of DOI:10.1021/acs.analchem.7b05325

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Article
A Fluorescence Immunoassay Based on the Phosphate-Triggered
Fluorescence Turn-on Detection of Alkaline Phosphatase
Chuanxia Chen, Jiahui Zhao, Yizhong Lu, Jian Sun, and Xiurong Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05325 • Publication Date (Web): 02 Feb 2018
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Analytical Chemistry
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A Fluorescence Immunoassay Based on the Phosphate-Triggered
Fluorescence Turn-on Detection of Alkaline Phosphatase
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†,‡
‡,§
,†
,‡
‡
Chuanxia Chen, Jiahui Zhao, Yizhong Lu,* Jian Sun,* and Xiurong Yang
†
School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, China
‡
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciꢀ
ences, Changchun, Jilin 130022, China
§
University of Chinese Academy of Sciences, Beijing 100049, China
*Fax: +86 431 85689278. Eꢀmail: mse_luyz@ujn.edu.cn, jiansun@ciac.ac.cn
ABSTRACT: A simple and costꢀeffective fluorescence immunoassay for the sensitive quantitation of disease biomarker αꢀ
fetoprotein (AFP) has been developed based on the phosphateꢀtriggered fluorescence turnꢀon detection of alkaline phosphatase
3+
(
ALP), with the reversible binding between calcein and Ce as a signaling element. In this immunoassay, fluorescent calcein is
3+
readily quenched by Ce via a coordination process. The ALPꢀcatalyzed hydrolysis of pꢀnitrophenyl phosphate leads to the forꢀ
3+
mation of pꢀnitrophenol and inorganic orthophosphate. And the newly formed orthophosphate could potently combine with Ce
due to the higher affinity, thus recovering the fluorescence of calcein. The corresponding fluorescence signal triggered by phosꢀ
phate is related to ALP activities labeled on antibody, and thus could be applied to detect target antigen in an enzymeꢀlinked imꢀ
munosorbent assay (ELISA) platform. The fluorescence intensity correlated well to the AFP concentration ranges of 0.2−1.0 ng/mL
and 1.0−4.0 ng/mL, with a detection limit of 0.041 ng/mL. The proposed fluorescence ELISA possesses convincing recognition
mechanism and exhibits excellent assay performance in the evaluation of the AFP level in serologic test, which unambiguously
reveals great application potential in the clinic diagnosis of disease biomarkers.
nzymeꢀlinked immunosorbent assay (ELISA) is mainly
based on the highly specific antigenꢀantibody recognition
cation degree of detection signal. With the development of
nanotechnology, several nanomaterialꢀrelated signal amplificaꢀ
tion strategies based on inꢀsitu synthesis or aggregations of
metal nanoparticles have been extended into the ALPꢀbased
E
1ꢀ2
and the efficient biocatalytic property of an enzyme. By virꢀ
tue of the excellent specificity, low cost and straightforward
readout, ELISA has already become the most widely used forꢀ
8, 31ꢀ32
plasmonic or colorimetric immunoassays.
Though effecꢀ
2
ꢀ4
mat of immunoassay in clinical diagnosis, foods quality conꢀ
tive improvement in sensitivity has been achieved, the syntheꢀ
sis or surface modification of nanomaterials is ineluctably
complicated, laborious and timeꢀconsuming. As a substitute of
conventional colorimetric immunoassays, the fluorescent one
is particularly appealing because of the intrinsic superior sensiꢀ
tivity and the foregoing advantages of fluorescence methods
over traditionally used UV−vis spectrophotometry. However,
currently reported ALPꢀbased fluorescence immunoassays are
quite limited and almost all rely on the ALPꢀenabled formation
5ꢀ6
7
8
trol, environmental monitoring, and in laboratory research.
Alkaline phosphatase (ALP) is extensively used as a labeling
enzyme in ELISA to produce detectable signals because of its
high catalytic activity, broad substrate specificity, easy conjuꢀ
gation to antibodies, mild reaction conditions and good stabilꢀ
9
ity. Moreover, ALP is a significant biomarker for diagnostics
as well, and the abnormal level of ALP in serum is closely
associated with several diseases, including prostatic cancer,
1
0
33ꢀ35
bone disease, liver dysfunction and diabetes. Therefore, highꢀ
ly sensitive and selective sensing of ALP activity is of great
significance for both the ALPꢀrelated diseases diagnosis and
the development of ALPꢀbased ELISA platforms.
or fluorescence change of the fluorescent nanomaterials.
Therefore, despite many advances in the fields of ALP activity
sensing and ELISA, it is still highly desirable to develop simꢀ
ple, rapid responsive, inexpensive and ultrasensitive ALPꢀ
based fluorescence ELISA protocols.
Until now, a large number of analytical methods for ALP
activity determination have been developed, including colorꢀ
Fluorochrome calcein, as a cheap and commercially availaꢀ
ble fluorescent dye, possesses admirable yellow green fluoresꢀ
cence and excellent stability. The intense fluorescence of calꢀ
cein could be quenched by several metal ions, and calcein has
been devoted to fluorometric determination of certain metal
1
1ꢀ14
15ꢀ20
21
imetry,
fluorometry,
chemiluminescence, surface enꢀ
22 23ꢀ27
hancement Raman scattering (SERS), electrochemistry,
28
and chromatography. Among these methods, fluorescence
analysis methods have sparked significant excitement owing to
their high sensitivity, cost effectiveness and convenience. The
majority of the previous fluorescence approaches merely deꢀ
pend on the discrimination of pyrophosphate and phosphate by
3
6ꢀ39
ions and biomolecules.
In this regard, the specific reaction
3+
between calcein and Ce would be particularly attractive and
useful in the area of fluorescence sensing because the fluoresꢀ
2
+ 16, 29ꢀ30
3+ 40
Cu .
As for ALPꢀbased immunoassays, the routine proꢀ
cence of calcein can be dramatically quenched by Ce . On
cedures directly estimate the colored products of enzymatic
hydrolysis by absorption spectroscopy or nakedꢀeye, which
usually suffer from poor sensitivity due to the limited amplifiꢀ
the other hand, phosphate, a universal hydrolysis product of
3+ 41
phosphatase, could chelate with Ce . We can envision that
3+
the nonꢀfluorescent calceinꢀCe complex would be applied to
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assay phosphateꢀrelease reaction in a fluorescence enhanceꢀ
Japan). UV−vis absorption spectra were obtained with a Cary
500 UV−vis spectrophotometer (Varian).
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ment manner based on the exchange of calcein by phosphate.
As far as we know, there is scarcely any ALP sensing or/and
ELISA system using calcein as an indicator up to now.
Pꢀnitrophenyl phosphate (pNPP) is a frequentlyꢀused subꢀ
strate of ALP, and the enzymatic hydrolysis of pNPP by ALP
yields two products, pꢀnitrophenol (pNP) and phosphate ion.
Conventionally, ALP activity is routinely estimated by measurꢀ
Detection of ALP Activity. 100 µL of pNPP (100 µM), 100
3+
µL of ALP with various activities, 50 µL of Ce (100 µM) and
500 µL of ultrapure water were subsequently added into 200
µL of TrisꢀHCl (50 mM, pH 9.0) buffer solution, followed by
incubation at 37°C for 30 min. Afterward, 50 µL of calcein
(100 µM) was added into the above reaction solution, and the
fluorescence intensities of resultant solutions were recorded
directly.
ing colorimetric or fluorescent signal difference between or
15, 19
induced by the substrate pNPP and product pNP.
However,
phosphate, as the other obvious enzymatic hydrolysate, is usuꢀ
ally overlooked, which also has great potential to be employed
to quantify ALP activity. Inspired by this, we have successfulꢀ
ly designed a universal ALPꢀbased fluorescence ELISA system
based on phosphateꢀtriggered fluorescence turnꢀon of the calꢀ
Fluorescence Immunoassay for AFP. First, 100 ꢂL of diꢀ
luted mouse monoclonal antibody (1:200) in coating solution
was added into the wells of a 96ꢀwell polystyrene plate and
incubated at 4°C overnight. After discarding the solutions, the
plate was washed three times with 300 ꢂL of wash buffer
(TBST), and blocked with 1% BSA at 37°C for 1 h to block
nonspecific binding sites. After washing again, 100 ꢂL of AFP
standard solutions with various concentrations (0, 0.2, 0.5, 0.8,
1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10, 20 ng/mL) were added into
each well to incubate at 37°C for 1 h, followed by washing
steps and the addition of rabbit antiꢀAFP (1:500, 100 ꢂL). Afꢀ
ter incubation at 37°C for 1 h, the wells were washed three
times, 100 ꢂL of goat antiꢀrabbit secondary antibody labeled
with ALP (1:3000) was added, and subsequently incubated at
37°C for 1 h. After washing the plate, we performed the enzyꢀ
matic reaction as follows: 100 µL of pNPP (500 µM), 50 µL of
0
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3+
ceinꢀCe complex, with pNPP as the enzyme substrate. The
extent of fluorescence enhancement directly hinges on the
phosphate concentration generated from ALP reaction (i.e., the
amount of ALPꢀsecondary antibody conjugates), which is
thereby indirectly related to the target antigen αꢀfetoprotein
(AFP) concentration. Compared to the previous methods, this
novel strategy shows several advantages. First, phosphate is
the universal hydrolysis product of all the ALP substrates,
therefore this protocol could be extended to other types of
ALPꢀsubstrate systems and ALPꢀbased ELISA. Second, beꢀ
sides the indispensable reagents for ELISA construction, no
special and expensive reagents (only commercially available
3+
Ce (500 µM) and 50 µL of TrisꢀHCl (50 mM, pH 9.0) buffer
solution were subsequently added into each well, and then
incubated at 37°C for 30 min. Afterward, 50 µL of the above
reaction solution was added into 950 µL of TrisꢀHCl (50 mM,
pH 9.0) buffer solution containing calcein (62.5 µL, 100 µM),
and the fluorescence intensities of resultant solutions were
directly recorded.
3+
Ce and calcein) are needed. Third, the fluorescent signal genꢀ
eration procedure could be performed in a facile mixꢀandꢀ
readout manner by simply mixing the enzyme reaction system
3+
with the Ce and calcein, effectively avoiding complicated or
timeꢀconsuming procedures. Finally, the assay is straightforꢀ
ward, selective and sensitive enough for practical application
by means of the intrinsic high specificity of enzyme reaction,
antigenꢀantibody recognition, and the intrinsic high sensitivity
of fluorescent detection.
Fluorescence Immunoassay for the Real Clinical Sam-
ples. Serum samples from two normal people and four patients
who suffered from hepatocellular carcinoma (HCC) were kindꢀ
ly provided by the Second Hospital of Jilin University. The
detailed procedures of the fluorescence ELISA referred to that
for the model protein by just adding the diluted human serum
instead of the AFP standard solution.
EXPERIMENTAL SECTION
Chemicals and Materials. Calcein was obtained from
Aladdin Industrial Corporation (Shanghai, China). Alkaline
phosphatase (EC 3.1.3.1) from bovine intestinal mucosa, 4ꢀ
nitrophenyl phosphate disodium salt hexahydrate (pNPP),
adenosine monophosphate (AMP), adenosine diphosphate
RESULT AND DISCUSSIONS
The Design and Establishment of the Sensing System for
(
(
(
ADP), adenosine triphosphate (ATP), sodium pyrophosphate
ALP Activity. Fluorescent calcein, could be efficiently
3+
Na PPi), bovine serum albumin (BSA), human serum albumin
quenched by combining with Ce ion with 1:1 stoichiometry
4
4
0, 42
HSA), lysozyme, peroxidase from horseradish (HRP), human
probably via an energy or charge transfer mechanism.
The
immunoglobulin G (IgG), casein and trypsin were purchased
from SigmaꢀAldrich (St. Louis, MO). Guanosine monophosꢀ
phate (GMP), guanosine diphosphate (GDP), guanosine triꢀ
phosphate (GTP), uridine monophosphate (UMP), uridine diꢀ
phosphate (UDP), uridine triphosphate (UTP), 2'ꢀ
deoxythymidineꢀ5'ꢀmonophosphate (dTMP), were obtained
from Sangon Biotechnology Co. Ltd. (Shanghai, China). αꢀ
fetoprotein (AFP), rabbit antiꢀAFP and mouse monoclonal
antibody were bought from ProSpec (Ness Ziona, Israel). ALPꢀ
conjugated goat antiꢀrabbit secondary antibody was purchased
from Abcam (Cambridge, MA). Both the wash buffer and anꢀ
tibody dilution buffer for ELISA were purchased from Boster
(Wuhan, China). All reagents were analytical grade and used
as received without any further purification. Ultrapure water
from a Millipore system (resistivity 18.25 Mꢁ cm, Millipore,
USA) was used throughout all experimental procedures.
reaction could be reversed, thus offering the possibility of fluoꢀ
rescence turnꢀon assay of phosphate, which could form therꢀ
3+
modynamically more stable complex with Ce than calcein.
3+
Consequently, the calceinꢀCe system could be extended for
ALP detection by means of the ALPꢀcatalyzed generation of
phosphate.
For an enzymatic reactionꢀbased assay, high discrimination
ability towards enzyme substrate and product is the necessary
prerequisite to successfully detecting the corresponding enꢀ
zyme activity. Therefore, we first evaluate the response signal
3+
of the asꢀproposed calceinꢀCe system toward various phosꢀ
phateꢀcontaining molecules or ions, including phosphate,
pNPP, AMP, ADP, ATP, GMP, GDP, GTP, UMP, UDP, UTP,
dTMP and PPi (10 ꢀM). The results in Figure 1A clearly imply
that only phosphate could evoke significant fluorescence enꢀ
hancement. This is ascribed to the more robust and stable
3+
3+
Apparatus and Characterization. Fluorescence spectra
were recorded on a Hitachi Fꢀ4600 spectrofluorometer (Tokyo,
complex of phosphateꢀCe than calceinꢀCe and the concomiꢀ
tant release of free fluorescent calcein from nonꢀfluorescent
3+
calceinꢀCe complex. The response of other phosphateꢀ
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+
Figure 1. (A) Fluorescence spectra of the calceinꢀCe sensing system in the presence of various phosphateꢀcontaining targets (10 ꢀM).
3+
(B) Relative responses of the phosphateꢀcontaining compounds compared to phosphate. Calcein, 5 ꢀM; Ce , 5 ꢀM; pH 9.0 (10 mM Trisꢀ
HCl). λex = 480 nm.
3
+
Figure 2. (A) Schematic illustration of ALP activity detection based on phosphate induced fluorescence turnꢀon of calceinꢀCe complex.
3+
3+
(
B) Fluorescence spectra of the calceinꢀCe ꢀbased sensing system in the presence of different reaction components. Calcein, 5 ꢀM; Ce , 5
ꢀM; pNPP, 10 ꢀM; ALP, 1 mU/mL; pH 9.0 (10 mM TrisꢀHCl); incubation at 37°C for 30 min. λex = 480 nm.
containing compounds investigated could be ignored compared
ment is related to ALP amount, which lays a foundation for
assessing ALP activity.
3ꢀ
with that of PO , although they possess similar phosphate
4
group. Apparently, the aforementioned pNPP, AMP, ADP,
ATP, GMP, GDP, GTP, UMP, UDP, UTP, dTMP and PPi
As shown in Figure 2B, several tests are conducted to verify
the feasibility of the proposed fluorescence turnꢀon assay for
the sensitive quantitation of ALP activity. In this regard, calceꢀ
in (5 ꢀM) has an intense fluorescence emission peak centered
at around 512 nm (line a, λex = 480 nm), and the coꢀexistence
of ALP (1 mU/mL) and pNPP (10 ꢀM) could not depress the
fluorescence signal of calcein (line b), indicating the ALPꢀ
catalyzed dephosphorylation reaction system has a negligible
effect on the fluorescence emission of calcein. As expected,
3+ 43ꢀ45
molecules do not permit for valid interaction with Ce .
As
3+
a result, the quenched fluorescence of calceinꢀCe complex
could not be recovered by them. The high discrimination abilꢀ
ity towards free phosphate and other phosphateꢀcontaining
molecules or ions provides a sound basis for the fabrication of
a versatile ALP assay, as well as the ALPꢀbased fluorescence
ELISA in the subsequent steps. In this study, pNPP is selected
as a model ALP substrate for the sake of low background sigꢀ
nal and ease of comparison with standard pNPPꢀbased chroꢀ
mogenic method.
3+
when Ce (5 ꢀM) is introduced, the fluorescence signal of
3+
calcein is effectively quenched (line c). However, when Ce is
premixed with ALPꢀpNPP reaction solution, the fluorescence
3+
Figure 2A schematically illustrates the fluorescence strategy
for ALP activity sensing based on the phosphateꢀinduced fluoꢀ
of calceinꢀCe is greatly enhanced (line f) due to the coordinaꢀ
3+
tion of the newly formed phosphate with Ce . In several other
3
+
3+
rescence turnꢀon of calceinꢀCe complex. The organic phosꢀ
experiments, the preꢀincubation of pNPP and Ce could not
3+
3+
phate group on the pNPP is unable to coordinate with Ce , so
influence the coordination of calcein with Ce and the fluoresꢀ
cence quenching of calcein, which overwhelmingly confirms
that the phosphate group on pNPP is incapable of coordinating
3
+
the fluorescence of calceinꢀCe cannot be recovered in the
presence of pNPP. However, the presence of ALP can catalyze
3+
3+
the dephosphorylation of the pNPP to produce phosphate,
with Ce (line d). Moreover, the mixture of ALP with Ce
3+
which could combine with Ce to form more stable CePO
4
also invokes no fluorescence enhancement (line e). All these
results undoubtedly reveal that, it is the ALPꢀcatalyzed release
of phosphate from the pNPP actuates the fluorescence enꢀ
hancement.
ꢀ
24
3+
41
(
Ksp ~ 10 ) complex compared to calceinꢀCe complex. In
this case, calcein is deprived of Ce ion by newly generated
phosphate, accompanied by the recovery of the fluorescence of
3+
3+
Ce ꢀquenched calcein. The degree of fluorescence enhanceꢀ
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Figure 3. (A) The fluorescence emission spectra of the sensing system toward ALP with various activities (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0
ꢀ
.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mU/mL). (B) The plots of fluorescence intensities at 512 nm versus the ALP activities. Calcein, 5
3+
M; Ce , 5 ꢀM; pNPP, 10 ꢀM; pH 9.0 (10 mM TrisꢀHCl); incubation at 37°C for 30 min. λ = 480 nm.
ex
Fluorescence Turn-on Sensing of ALP Activity. Prior to
pNP to make a comparison with the fluorescence method. Both
3+
assessing the capability of the proposed calceinꢀCe complex
to detect ALP activity, we have optimized several experiꢀ
mental conditions like enzymatic reaction time, pH, pNPP
the absorption spectra and absorbance intensity at 405 nm inꢀ
crease progressively with the increase of ALP activities from
0.1 to 2.0 mU/mL (Figure S5). The detection limit is 0.13
mU/mL, which is 1 order of magnitude higher than that of the
proposed fluorescence method. As the normal range of serum
ALP in adults is reported to be about 40–190 mU/mL, the asꢀ
established fluorescence assay is sensitive enough for practical
detection of ALP activity in biological samples. Besides high
sensitivity, the intrinsic high specificity of enzyme reaction
would confirm an ideal selectivity for ALP sensing. The deꢀ
veloped fluorescence assay is simple, sensitive, selective and
costꢀeffective to monitor ALP activity, which holds great
promise for fluorescence immunoassays.
3
+
concentrations and Ce concentrations. Fascinatingly, premixꢀ
3+
ing of Ce with ALPꢀpNPP reaction solution would not imꢀ
pede the ALPꢀcatalyzed dephosphorylation reaction (Figure
S1). Thus it is possible to simultaneously conduct ALPꢀ
catalyzed dephosphorylation and CePO formation reactions in
4
one step, which makes the ALP analysis more simple and
quick. And as a side benefit, since phosphate is a competitive
inhibitor for the enzymatic reaction via occupying active site in
16, 46
ALP,
the immediate consumption of generated phosphate
3+
by Ce will accelerate the enzymatic reaction due to the reꢀ
straint of phosphate inhibition. In this work, the fluorescence
intensity of the analytical solution is increased gradually with
increasing enzymatic reaction time, and levels off after 30 min,
while the background hydrolysis reaction cannot proceed withꢀ
in as least 50 min (Figure S2). Thus, we choose 30 min as the
optimum enzymatic reaction time. As is wellꢀknown, alkaline
condition is propitious to the ALPꢀparticipated catalytic reacꢀ
tion, hence pH within the range from 7.5 to 9.5 is performed,
and pH 9.0 is the optimal value in following experiments (Figꢀ
ure S3). The fluorescence intensity of calcein is directly moduꢀ
Investigations on the inhibitor of enzyme are of great imꢀ
portance in drug design. Therefore, besides the ability of ALP
activity detection, this proposed fluorescence assay has been
also utilized to evaluate the enzyme inhibition efficiency. As
an acknowledged ALP inhibitor, Na VO is employed for inꢀ
3
4
hibiting assays. With the addition of Na VO into the assay
3
4
solution, the release of phosphate (i.e., hydrolysis of pNPP) is
restricted due to inhibition of ALP activity (1.2 mU/mL), and
the fluorescence recovery would be depressed accordingly
(Figure S6A). Figure S6B manifests that the fluorescence inꢀ
tensity at 512 nm decreases progressively with the increase of
Na VO concentration from 0 to 1000 ꢀM. The IC (the inhibꢀ
3+
lated by Ce level in this assay. To ensure a low background
3+
signal in the sensing system, 5 ꢂM of Ce (identical to the
3
4
50
concentration of calcein) is used in further experiments. Beꢀ
cause the detection range and sensitivity of an enzyme greatly
depend on the substrate concentration, pNPP concentration is
evaluated and optimized to be 10 ꢂM (Figure S4).
itor concentration required for 50% inhibition of the enzyme
activity) is calculated to be approximately 61.2 µM, which is
in accord with the reported values determined by other
19, 47
works.
These results clearly prove that our method is proꢀ
Having ascertained these experimental conditions, the fluoꢀ
rescence response of the proposed sensing system toward ALP
with various activities is studied and depicted in Figure 3.
When ALP activity increases from 0 to 2.0 mU/mL, the fluoꢀ
rescence emission spectra centered at around 512 nm enhance
gradually (Figure 3A) due to the increase of produced phosꢀ
phate concentrations. Figure 3B shows the plots of fluoresꢀ
cence intensities at 512 nm versus the ALP activities. The fluoꢀ
rescence recovery correlates well to the ALP activities in the
spective for the screening of ALP inhibitors in drug discovery.
Fluorescence ELISA for Detection of AFP. Inspired by
the successful and extensive use of ALP in ELISA, we proceed
to investigate the potential application of the fluorescence turnꢀ
on ALP activity sensing system to ALPꢀlabeled immunoassay
for target antigen. AFP is taken as our model antigen considerꢀ
ing its importance in hepatocellular carcinoma (HCC). In this
case, the capture antibody and the primary antibody are mouse
antiꢀAFP monoclonal antibody and rabbit antiꢀAFP, respecꢀ
tively, and the secondary antibody is goat antiꢀrabbit IgG laꢀ
beled with ALP. The procedures of this fluorescence ELISA
2
ranges of 0.1–0.4 mU/mL (R = 0.995) and 0.4–1.2 mU/mL
2
(R = 0.996). The calculated detection limit is as low as 0.023
48ꢀ49
mU/mL based on 3σ/S, which is 1~2 order of magnitude better
are nearly the same as the conventional ones.
Figure 4A
34
than the pNPPꢀbased standard chromogenic method. The
sensitivity is also comparable with or superior to most previꢀ
ously reported methods (Table S1). In this work, we also have
detected the ALP activity via estimating the colored product
schematically represents the ALPꢀlabeled immunoassay for the
detection of AFP. In this approach, the target AFP is first capꢀ
tured with specific antibodies preꢀimmobilized on a 96ꢀwell
plate. Subsequently, rabbit antiꢀAFP and second antibody label
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Figure 4. (A) Schematic representation of the fluorescence ELISA strategy via phosphate triggered fluorescence turnꢀon of the calceinꢀ
3+
Ce complex. (B) The fluorescence emission spectra and (insert) fluorescence intensities at 512 nm of the sensing system toward AFP
with various concentrations. (C) The linear calibration plots for AFP detection. (D) Fluorescence responses of the developed ELISA
against AFP (4 ng/mL) or other control enzymes/proteins (40 ng/mL). Experimental conditions for the enzymatic reaction: Calcein, 6.25
3+
ꢀM; Ce , 6.25 ꢀM; pNPP, 12.5 ꢀM; pH 9.0 (10 mM TrisꢀHCl); incubation at 37°C for 30 min. λ = 480 nm.
ex
with ALP are successively immobilized on the plate via antiꢀ
genꢀantibody specific recognition. After immobilization of
these molecules on a 96ꢀwell plate following the procedures of
fluorescence signal (Figure 4D), proving that our fluorescence
immunoassay system is sufficiently selective for the detection
of target AFP.
3
+
conventional ELISA, pNPP and Ce are added to generate
Encouraged by the high sensitivity and selectivity of this
fluorescence ELISA, we apply it to the detection of target AFP
in real samples. For this purpose, six different human serum
samples from two normal adults and four patients with HCC
are analyzed. The experiment is performed on the six unrelated
human serum samples, and the results are shown in Table S3.
As the AFP levels in serums of both normal adults (typically <
20 ng/mL) and patients with HCC (typically > 400 ng/mL) are
outside of the linear range of our developed assay, the serums
samples are diluted 10 and 200 times respectively to reduce
AFP to an appropriate level. The results obtained from this
assay are consistent well with the results from the pNPPꢀbased
standard ELISA method (Table S3), which verifies that our
assay has a high accuracy and excellent performance in real
sample analysis.
orthophosphate and form CePO simultaneously, then calcein
4
is introduced, giving a fluorescent readout signal. To demonꢀ
strate the viability of our design, quantitative AFP detection
experiment is implemented through our ALPꢀbased ELISA
system. The more AFP is introduced, the more ALP labeled on
antibody would be banded. Therefore, the fluorescence intensiꢀ
ties at 512 nm gradually increase when the AFP concentrations
increase from 0 to 20 ng/mL (Figure 4B), exhibiting good lineꢀ
2
arity with AFP from 0.2 to 1.0 ng/mL (R = 0.971) and from
2
1
.0 to 4.0 ng/mL (R = 0.995) (Figure 4C). The detection limit
is 0.041 ng/mL calculated from 3σ/S, which is comparable
with or better than other previously reported immunoassays for
the detection of AFP (Table S2). The AFP concentration in
normal subjects is less than 20 ng/mL, and abnormally elevatꢀ
ed AFP (typically exceeds 400 ng/mL) is usually observed in
the serum of patients with HCC. Obviously, this method could
be available for the sensitive detection of disease biomarkers
CONCLUSIONS
A versatile fluorescence biosensor for ALP that can be diꢀ
rectly imposed on the traditional ELISA platform is established
with improved performance. The proposed method is based on
the phosphateꢀtriggered fluorescence turnꢀon of the calceinꢀ
(AFP) in clinical applications by simply diluting real samples.
To verify the sensing ability of the assay in real samples,
high specificity toward target antigen AFP is required. Thereꢀ
fore we challenge the ELISA platform with other nonspecifiꢀ
proteins, such as HSA, lysozyme, HRP, human IgG, casein,
and trypsin. Because this proposed ELISA employs a particuꢀ
lar antibody with specificity for AFP, it almost certainly has a
splendid capability of selective recognizing AFP. Unsurprisꢀ
ingly, other examined proteins induce negligible change in
3+
Ce complex. It has proven to be straightforward, facile and
costꢀeffective, and exhibits a much higher sensitivity compared
with the traditional chromogenic assay for ALP. It also proꢀ
vides a potential platform for trace ALP inhibitor screening in
drug discovery. Furthermore, using ALP as the labeling tracer
and calcein as the signaling element, an ALPꢀbased fluoresꢀ
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cence turnꢀon ELISA platform for AFP detection is thereby
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developed. The detection limit is as low as 0.041 ng/mL, and
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AUTHOR INFORMATION
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13, 5458ꢀ5479.
Corresponding Author
(26) Goggins, S.; Naz, C.; Marsh, B. J.; Frost, C. G., Chem.
Commun. 2015, 51, 561ꢀ564.
*
Eꢀmail: mse_luyz@ujn.edu.cn
Eꢀmail: jiansun@ciac.ac.cn
(
(
27) Jiang, H.; Wang, X., Anal. Chem. 2012, 84, 6986ꢀ6993.
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*
Umemura, T.; Haraguchi, H., Bull. Chem. Soc. Jpn. 2006, 79, 1211ꢀ
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Author Contributions
(
29) Zhang, L.; Zhao, J.; Duan, M.; Zhang, H.; Jiang, J.; Yu, R.,
Anal. Chem. 2013, 85, 3797ꢀ3801.
30) Sun, J.; Yang, F.; Zhao, D.; Yang, X., Anal. Chem. 2014, 86,
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
(
7
883ꢀ7889.
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267ꢀ273.
ACKNOWLEDGMENT
(
32) Jin, L. Y.; Dong, Y. M.; Wu, X. M.; Cao, G. X.; Wang, G. L.,
This work was supported by the National Natural Science Foundaꢀ
tion of China (No. 21605139, 21435005, 21705056), the Natural
Science Foundation of Shandong Province (ZR2017MB022) and
the startꢀup funding from University of Jinan (511ꢀ1009408).
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Chem. 2016, 88, 9789ꢀ9795.
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