Fluorescent and Colorimetric Dual-Readout Assay for Inorganic Pyrophosphatase with Cu2+-Triggered Oxidation of o-Phenylenediamine

Article abstract of DOI:10.1021/acs.analchem.5b03848

We demonstrate a rationally designed fluorescent and colorimetric dual-readout strategy for the highly sensitive, quantitative determination of inorganic pyrophosphatase (PPase) activity, a key hydrolytic enzyme involved in a variety of metabolic processes. Inspired by the selective oxidative and chromogenic reaction of o-phenylenediamine (OPD) with Cu²⁺, the special inhibitory effects of pyrophosphate (PPi) on the oxidative ability of Cu²⁺, and the specific hydrolysis of PPi into orthophosphate by PPase, a convenient small molecule OPD-based analytical system was developed for Cu²⁺/PPi recognition and PPase activity assay. We have confirmed that Cu²⁺ acts as the oxidant in the reaction and the main chromogenic product of OPD is 2,3-diaminophenazine (usually called OPDox) in the assay by combining the ESI-MS, ¹H NMR, and XPS spectra analysis. Direct electrochemical insights into the Cu²⁺-triggered and PPi-inhibited mechanism were performed by cyclic voltammetry characterizations of the Cu²⁺ in the absence and presence of PPi for the first time. Furthermore, the proposed analytical system with clear response mechanism exhibits a promising outlook for the PPase activity assay in real biological samples and inhibitor screening.

Full text of DOI:10.1021/acs.analchem.5b03848

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Article
Fluorescent and Colorimetric Dual-Readout Assay for Inorganic
2+
Pyrophosphatase with Cu-Triggered Oxidation of o-Phenylenediamine
Jian Sun, Bin Wang, Xue Zhao, Zong-Jun Li, and Xiurong Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03848 • Publication Date (Web): 24 Dec 2015
Downloaded from http://pubs.acs.org on December 24, 2015
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Analytical Chemistry
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Fluorescent and Colorimetric Dual-Readout Assay for Inor-
ganic Pyrophosphatase with Cu -Triggered Oxidation of o-
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Phenylenediamine
†
‡
†,§
†
,†
Jian Sun , Bin Wang , Xue Zhao , ZongꢀJun Li , and Xiurong Yang*
†
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciꢀ
ences, Changchun, Jilin 130022, China
‡
School of Material Science and Engineering, Tianjin University, Tianjin 300072, China
§
University of Chinese Academy of Sciences, Beijing 100049, China
*Fax: +86 431 85689278. Eꢀmail: xryang@ciac.ac.cn
ABSTRACT: We demonstrated a rationally designed fluorescent and colorimetric dualꢀreadout strategy for the highly sensitive,
quantitative determination of inorganic pyrophosphatase (PPase) activity, a key hydrolytic enzyme involved in a variety of metabolꢀ
2+
ic processes. Inspired by the selective oxidative and chromogenic reaction of oꢀphenylenediamine (OPD) with Cu , the special
2+
inhibitory effects of pyrophosphate (PPi) on the oxidative ability of Cu , and the specific hydrolysis of PPi into orthophosphate by
2+
PPase, a convenient small molecule (OPD)ꢀbased analytical system was developed for Cu /PPi recognition and PPase activity asꢀ
2
+
say. We have confirmed that Cu acts as the oxidant in the reaction and the main chromogenic product of OPD is 2,3ꢀ
1
diaminophenazine (usually called OPDox) in the assay by combining the ESIꢀMS, H NMR and XPS spectra analysis. Direct elecꢀ
2
+
trochemical insights into the Cu ꢀtriggered and PPiꢀinhibited mechanism were performed by cyclic voltammetry characterizations
2+
of the Cu in the absence and presence of PPi for the first time. Furthermore, the proposed analytical system with clear response
mechanism exhibits a promising outlook for the PPase activity assay in real biological samples and inhibitor screening.
posed a AuNPs@cysteineꢀbased colorimetric assay and a
AuNCs@11ꢀmercaptoundecanoic acidꢀbased fluorescent assay
INTRODUCTION
As a ubiquitous hydrolytic enzyme, inorganic pyrophosphaꢀ
tase (PPase, EC 3.6.1.1) has been demonstrated to be directly
involved into the carbohydrate and phosphours metabolism,
lipid synthesis and degradation, bone formation, DNA syntheꢀ
sis, as well as many other biological processes and biochemiꢀ
for monitoring PPase activity. However, such functionalized
Auꢀbased nanomaterials also require a timeꢀconsuming prepaꢀ
ration procedure and it is still highly desirable to establish
more convenient and sensitive PPase activity assays.
To address the aforementioned drawbacks, we have concenꢀ
trated on the discovery and utilization of conventional yet
promising organic chromophore or fluorophore. Phenylenediꢀ
amine, especially oꢀphenylenediamine (OPD), is an important
1ꢀ3
cal events. When activated by certain metal ions (typically
2
+ 4
Mg ), PPase plays a critical role in regulating the cellular
concentration of inorganic pyrophosphate (PPi) owing to its
catalysis during the conversion of one molecule of PPi into
precursor for fabricating numerous heterocyclic compounds or
5
two orthophosphate ions (Pi). More significantly, this PPi
12ꢀ15
polymers with unique properties.
OPD could be oxidized
hydrolytic process is an extremely exergonic reaction, and the
released energy can provide a thermodynamic push for several
energetically unfavorable biosynthetic reactions and biochemꢀ
by certain metal ions or hydrogen peroxide, and the resultant
16
2
,3ꢀdiaminophenazine (usually called OPDox) solution exꢀ
hibit a visible pale yellow color as well as an orangeꢀyellow
fluorescence when irradiated by ultraviolet light. In fact, this
chromogenic response has already been utilized to develop a
6
ical transformations to completion. Accordingly, the abnorꢀ
mal level of PPase is associated with certain diseases, and thus
developing a powerful and convenient PPase assay is necesꢀ
sary to elucidate the molecular mechanism of phosphateꢀ
related hydrolysis and biological processes.
series of assays for relevant reactants in the previous literaꢀ
17ꢀ19
tures.
For instance, Wang et al. have already reported a
+
novel nanoparticle autocatalytic assay for directly sensing Ag
2
+
+
2+
By determining the change in the concentration of PPi subꢀ
strate or Pi product, several conventional PPase activity assays
based on chromatographic, enzymatic, and spectral methods
have been developed for decades. Unfortunately, most existing
assays are limited by the complicated, laborious operations,
and Cu based on the oxidative capability of Ag and Cu
20
toward OPD.
As expected, we have found that the oxidative and chromoꢀ
genic reaction of OPD could be selectively triggered by Cu
2+
over other common metal ions and inorganic anions in Trisꢀ
HCl buffer at pH 7.4 in the current study (Scheme 1A). More
7
ꢀ9
expensive precursors and specialized equipments. Recently,
with the development of nanomaterialsꢀbased sensors and in
2+
interestingly, the redox reaction between OPD and Cu can be
2
+
view of the different affinity for copper ions (Cu ) between Pi
inhibited by introducing the PPi, revealing remarkable coordiꢀ
1
0
11
and PPi, Mao et al. and our group have respectively proꢀ
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+
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nation ability between Cu and PPi.
Inspired by the fact tris(hydroxymethyl)aminomethane into the ultrapure water
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that PPase can specifically hydrolyze the PPi into Pi and the
resultant Pi can hardly inhibit the redox reaction, a novel fluoꢀ
rescent and colorimetric dualꢀreadout assay for PPase with
and adjusting the pH to 7.4 with 1 M HCl.
Apparatus and Characterization. The absorption and fluꢀ
orescence intensity spectra were recorded on a CARY 500
UV−Vis−NIR Varian spectrophotometer (CA, USA) and a
Hitachi Fꢀ4600 spectrofluorometer (Tokyo, Japan), respectiveꢀ
ly. The Xꢀray photoelectron spectroscopy (XPS) were surꢀ
veyed on a Thermo ESCALAB VG Scientific 250 (West Susꢀ
sex, UK) equipped with monochromatized Al Kα excitation as
the Xꢀray source and the binding energies were calibrated with
C 1s (284.6 eV). Nuclear magnetic resonance (NMR) measꢀ
urements were preformed on a BRUKER AVANCEꢀ400
2+
Cu ꢀregulated and PPiꢀinhibited oxidation of OPD has been
innovatively developed for the first time (Scheme 1B). In case
2
+
of adding constant concentration of OPD, Cu and PPi as well
as varied activities of PPase into the analytical system, the
absorbance and fluorescence intensity of the resultant solution
2
+
are directly related to the concentration of free Cu (i.e., the
remaining concentration of PPi), whereas are indirectly related
to the level of PPase activity (i.e., the consumption of PPi). In
fact, such spectral sensing assay with fluorescent and coloriꢀ
metric dual modes had already sparked significant excitement
due to the fact that they can present more than one kind of
output signal conveniently and rapidly, thus making the anaꢀ
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NMR spectrometer, with CDCl as the solvent. Electrospray
3
ionization mass spectrometry (ESIꢀMS) was carried out on a
Finnigan LCQ ion trap mass spectrometer (Finnigan, San Jose,
CA). Cyclic voltammetric experiments were performed with a
CHI 660B electrochemical workstation by using a conventionꢀ
al threeꢀelectrode cell, including a Ag/AgCl (saturated KCl)
reference electrode, a platinum spiral as the counter electrode
and a glassy carbon (GC) electrode as the working electrode.
2
4ꢀ25
lytical results more convincing.
Furthermore, it is also the
first time for using physicalꢀchemical methods (cyclic voltꢀ
ammetric experiment and so on) to conclude the ambiguous
26
OPDꢀbased reaction mechanism and sensing principle. The
unequivocal recognition mechanism would be significantly
beneficial for the further sensing application on the basis of
Fluorescent and Colorimetric Assay for PPase. For evalꢀ
2+
2
+
uation the effects of Cu ions on the oxidation of OPD, a 100
the oxidative reaction of OPD employing Cu or relevant
coordinator as a signal transducer medium.
2+
ꢁ
L volume of Cu solution (prepared from CuSO ·5H O)
4 2
with varied concentrations were added respectively into the
aqueous solution consisting of freshly prepared OPD (200 ꢁL,
10 mM), TrisꢀHCl buffer (1 mL, 20 mM, pH 7.4) and H O
(700 ꢁL). The mixture solutions were incubated at 37 °C for 2
hours, then were taken out and cooled to room temperature for
5 min for the following absorption and fluorescence measureꢀ
ments. To assess other metal ions and inorganic anion, the
2+
Scheme 1. Schematic representations of the Cu -regulated
2
oxidation of OPD (A) and fluorescent/colorimetric dual-
readout assay strategy for sensing PPase (B).
2
+
experimental procedures are similar to that for Cu ions, exꢀ
2+
cept that Cu was replaced with others.
For PPi recognition, 100 ꢁL of varied concentrations of PPi
2+
were respectively mixed with Cu solution (100 ꢁL, 200 ꢁM),
and then the mixtures were added into the aqueous solution
consisting of OPD (200 ꢁL, 10 mM), TrisꢀHCl buffer (1 mL,
2
0 mM, pH 7.4) and H O (600 ꢁL). The above solutions were
2
incubated at 37 °C for 2 hour, and cooled to room temperature
for 5 min before the spectra measurements. The selectivity of
this recognition system for PPi was evaluated by using other
common anions or biomolecules instead of PPi.
A fluorescent and colorimetric dualꢀreadout assay for PPase
activity was performed under the following procedures. A 200
EXPERIMENTAL SECTION
Chemicals and Materials. Copper(II) sulfate pentahydrate
ꢁ
L volume of freshly prepared PPase with different activities
ranging from 0 to 500 mU/mL were firstly added into the mixꢀ
(
CuSO ·5H O), tris(hydroxymethyl)aminomethane, pyrophosꢀ
4 2
2+
2+
ture solution consisting of Mg (20 ꢁL, 1 mM), Cu (100 ꢁL,
00 ꢁM), PPi (200 ꢁL, 200 ꢁM) , TrisꢀHCl buffer (1 mL, 20
mM, pH 7.4) and H O (280 ꢁL). After incubation at 37 °C for
phatase (EC 3.6.1.1), inorganic from baker’s yeast (Saccharoꢀ
myces cerevisiae), acetylcholinesterase, bovine serum albumin,
glucose oxidase, lysozyme and trypsin were purchased from
SigmaꢀAldrich (St. Louis, MO, USA). Sodium pyrophosphate,
adenosine 5′ꢀtriphosphate disodium salt hydrate (ATP), adenoꢀ
sine 5′ꢀdiphosphate sodium salt (ADP), adenosine 5′ꢀ
monophosphate disodium salt (AMP), sodium fluoride (NaF)
and oꢀPhenylenediamine were purchased from Aladdin Indusꢀ
trial Corporation (Shanghai, China). Fish sperm DNA was
purchased from DingGuo Biotech. Ltd. (Beijing, China). Othꢀ
er metal ions and inorganic anions were purchased from Beiꢀ
jing Chemical Works (Beijing, China) or Aladdin Industrial
Corporation. All reagents were analytical grade and used as
received without any further purification. The ultrapure water
from a Millipore system was used in all aqueous solution. The
2
2
2+
3
0 min, these PPaseꢀtreated Cu −PPi solutions were mixed
with the OPD solution (200 ꢁL, 10 mM), and then the above
mixtures were incubated at 37 °C for 2 hour, cooled to room
temperature for 5 min before the spectra measurements.
PPase Inhibition Assay. We initially added 100 ꢁL of varꢀ
ied concentrations of NaF into the freshly prepared PPase soꢀ
lution (100 ꢁL, 200 mU/mL). After a brief mixing, these NaFꢀ
treated PPase solutions were added into the mixture solution
2+
2+
consisting of Mg (20 ꢁL, 1 mM), Cu (100 ꢁL, 200 ꢁM),
PPi (200 ꢁL, 200 ꢁM) , TrisꢀHCl buffer (1 mL, 20 mM, pH
7
.4) and H O (280 ꢁL). The resulting mixtures were incubated
2
at 37 °C for 30 min, mixed with the OPD solution (200 ꢁL, 10
mM), further incubated at 37 °C for 2 hour, cooled to room
TrisꢀHCl
buffer
was
prepared
by
dissolving
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could be monitored by absorption and fluorescence intensity
temperature for 5 min and then were used for the spectra
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measurements.
spectra, and thus a convenient dualꢀreadout assay could be
designed and established for sensing PPase sensitively and
selectively.
RESULTS AND DISCUSSION
2+
Design and Establishment of PPase Sensing System. As
shown in Figure 1, the aqueous solution of pure OPD has alꢀ
ways remained colorless and exhibited negligible fluorescence
under the incubation at 37 °C for 2 hours (curve and vial 1). In
Sensitive and Selective Recognition of Cu and PPi. In
previous literatures, the oxidative capability of certain metal
ions toward OPD has already been demonstrated and utilized
+
2+ 19ꢀ20
in the detection of Ag and Cu .
Herein, we have firstly
2
+
2+
contrast, the preꢀintroduction of Cu essentially triggers the
oxidative reaction of OPD under the same incubation condiꢀ
tion, during which the original colorless and nonꢀfluorescent
solution has gradually turned to pale yellow in color and
showed intense orangeꢀyellow fluorescence under ultraviolet
light (curve and vial 2). As shown in Figure 1A, the
investigated the effect of Cu concentration on the oxidation
2+
of OPD by adding different concentrations of Cu respectiveꢀ
ly into the aqueous solution of OPD (1 mM) in 10 mM Trisꢀ
HCl buffer at pH 7.4. As shown in Figure 2A and 2B, the fluoꢀ
rescence intensity of the resultant mixture solution results in a
continual and drastic fluorescence enhancement with increasꢀ
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2+
Cu −OPD mixed solution has an obvious absorption peak at
ing concentrations of Cu from 0 to 50 ꢁM. Meanwhile, along
2+
about 417 nm, which belongs to the characteristic absorption
spectrum of OPDox, the main oxidation product of OPD.
Meanwhile, the corresponding fluorescence emission spectrum
in Figure 1B exhibited a peak at about 560 nm, which is simiꢀ
lar to that of OPDox in previous work as well.
with the Cu concentrations increasing from 0.25 to 10 ꢁM,
18
the fluorescence intensity values at 560 nm display a quasiꢀ
2+
linear relationship. Thus, the final concentrations of Cu in
the following assays was set to be 10 ꢁM because this concenꢀ
tration would be beneficial for the following quantitative deꢀ
tection and could induce sufficient enhancement (more than
120 times). Meanwhile, the concentrationꢀdependent oxidative
reaction of OPD could also be monitored by UV−vis absorpꢀ
2
0,26
The further
confirmation of reaction product and the investigation on releꢀ
vant mechanism will be described in the later section.
2
+
tion spectrum. With increasing concentrations of Cu , the
absorption band gradually increased, and the absorbance valꢀ
2
+
ues at 417 nm versus varied concentrations of Cu reflected
the quasiꢀlinearly concentrationꢀdependent process as well
(
Figure S1).
Figure 1. Absorbance (A) and fluorescence emission spectra (B)
2
+
2+
of the OPD (1), OPD + Cu (2), OPD + Cu + PPi (3), and OPD
2+
+ Cu + PPi + PPase (4) in 10 mM TrisꢀHCl buffer at pH 7.4.
The corresponding photos under visible (C) and ultraviolet light
2+
(D). The final concentrations or activity of the OPD, Cu , PPi,
and PPase are 1 mM, 10 ꢁM, 20 ꢁM, and 50 mU/mL, respectively.
Figure 2. (A) Fluorescence spectra of the analytical solution preꢀ
2+
pared by adding varied concentrations of Cu (0–50 ꢁM) into the
OPD solution. (B) Fluorescence intensity values at 560 nm for the
Next, we have found the significant enhancement in the colꢀ
or and fluorescence along with the oxidative reaction of OPD
could be partially inhibited in the presence of PPi (curve and
vial 3), because of the preferential complexation affinity of
2+
analytical solution versus the Cu concentrations. Inset: linear
curve. The fluorescence intensities of the OPD solution in the
presence of other control metal ions (10 ꢁM) only (C) and coexꢀ
2+
PPi to Cu . On the other hand, the addition of PPase into the
2+
2+
istence of Cu ions (10 ꢁM) (D). All measurements were
performed by using OPD (1 mM) solution in TrisꢀHCl buffer (10
mM, pH 7.4).
preꢀmixed Cu −PPi solution could catalyze the hydrolysis of
2
+
PPi towards Pi and regain the oxidative ability of Cu by disꢀ
abling the PPiꢀbased complexation. In this case, the conseꢀ
quent solution mixed with PPaseꢀtreated Cu −PPi system and
OPD (curve and vial 4) possesses similar optical and spectral
properties to the initial aqueous mixtures of Cu and OPD,
but is obviously different from the OPD–Cu –PPiꢀbased soluꢀ
tion (curve and vial 3). In addition, PPi and PPase have negliꢀ
gible interference on the absorbance or fluorescence of the
initial OPD solution (data not shown). Thus, the above pheꢀ
nomena illustrate that PPi could restrain the OPD–Cu oxidaꢀ
tive reaction, and yet PPase could indeed relieve the PPiꢀ
induced confinement. Most significantly, these processes
2
+
2+
Besides Cu ions, we further investigated the effects of othꢀ
er common metal ions (Figure 2C) and anion (Figure S2), all
of which could not induce obvious fluorescence changes at the
concentration of 10 ꢁM. And the coexistence of other relevant
2
+
2+
2+
metal ions had negligible influence when recognising Cu in
our expremental condition (Figure 2D). The corresponding
absorbance changes induced by these potential interfering ions
are similar to the case of fluorescence, and the data were not
2
+
+
shown here. It is noteworthy to mention that Ag could also
trigger the oxidative reaction of OPD owing to its stronger
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+
19ꢀ20
oxidative ability than Cu in the previous studies.
Howevꢀ
Investigation into the Response Mechanism for PPi. It is
ꢀ
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er, the large amounts of Cl ions in our analytical system (Trisꢀ
HCl buffer) would easily prevent the interference from Ag ,
noteworthy to mention that our proposed sensing system could
+
2+
be utilized in the Cu and PPi detection, based on the aforeꢀ
based on the recognized masking effect of halide ions to
Ag .
mentioned exquisite sensing behaviour. The excellent selectivꢀ
ity, high sensitivity, and even determined spectral signalꢀ
concentration quasiꢀlinear relationship for the relative subꢀ
strate recognition are beneficial and necessary to the subseꢀ
quent enzyme activity assay. On the other hand, further inꢀ
sights into the unambiguous response mechanism of the subꢀ
strate also play an important role in the construction of the
analytical system.
+
27ꢀ28
Up to now, using a certain metal ion as a medium or binding
site has been considered as the most successful strategy in the
32
2
9ꢀ30
PPi sensing methods.
As shown in Figure 3 and Figure S3,
2+
the preꢀmixing of PPi and Cu could inhibit the oxidative
reaction of OPD and reduce the absorption/fluorescence specꢀ
tra enhancement with PPi concentrations over the range from
0
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to 500 ꢁM in our assay system. Typically, with increasing
Taking these considerations into accounts, we have firstly
2
+
concentrations of PPi from 2 to 20 ꢁM, the fluorescence intenꢀ
sity values at 560 nm were plotted, which displayed a quasiꢀ
linear relationship. Meanwhile, the final concentrations of PPi
in the following assays was set to be 20 ꢁM under current
experimental conditions based on the similar reason for the
investigated the selective reaction process between Cu and
OPD, during which OPDox have been considered as the main
chromogenic product of OPD in the previous reports. To conꢀ
firm the formation of OPDox, we further purified the incubatꢀ
2+
ed mixture solution of Cu and OPD by thin layer
2+
optimum concentration of Cu . Furthermore, we measured the
chromatography (TLC) and the products were anyalyzed by
2+
1
recognition response of this OPD−Cu mixed system with
ESIꢀMS and H NMR. The molecular ion peak at 211.1 (m/z,
+
sixteen different common anions (also 20 ꢁM) in the same
condition. As expected, only PPi could significantly reduce the
fluorescence intensity, whereas, the coexistence of these
relevant anions (including the phosphateꢀrelated anions,
[M+H] ) assigned to OPDox can be obviously observed in
ESIꢀMS spectrum (Figure S5). Additionally, the resonance
peaks assigned to –NH group shifted to lowꢀfield. The peak
2
area ratio is a:b:c:d=4:2:2:2 and the peak integrals are
consistent with the total numbers of the hydrogen atoms in
OPDox (Figure S6). Combining NMR analysis and ESIꢀMS
data, we have confirmed that the OPDox is an exclusive
ꢀ
2ꢀ
3ꢀ
H PO , HPO and PO ) has minimal interference on the PPi
2
4
4
4
recognition (Figure 3C and 3D).
2+
product in this Cu ꢀmidiated oxidation reaction.
Figure 4. (A) XPS spectra of Cu 2p for the CuSO4 (black),
CuSO after adding OPD (red), and CuSO after adding PPi and
4
4
OPD (blue). (B) Cyclic voltammograms of the CuSO (black),
4
CuSO in the presence of PPi (red), and CuSO in the presence of
4
4
Figure 3. (A) Fluorescence spectra of the analytical solution preꢀ
Pi (blue) in KCl solution.
2
+
pared by adding the mixture of Cu and varied concentrations of
PPi (0–500 ꢁM) into OPD solution. (B) Fluorescence intensity
values for the analytical solution versus the PPi concentrations.
2+
For more inꢀdepth investigation on the role of Cu in the
oxidative and chromogenic reaction, the XPS analysis was
2+
2+
performed and applied to illustrate the oxidation states of Cu
Inset: linear curve. The fluorescence intensities of the OPD–Cu
in the absence and presence of OPD/PPi. The asꢀprepared mixꢀ
system in the presence of other control anions (20 ꢁM) only (C)
and coexistence of PPi ions (20 ꢁM) (D). All measurements were
ture solutions (CuSO , CuSO with OPD, and CuSO with
4
4
4
2+
PPi/OPD) were respectively deposited on the silicon wafer for
the XPS measurement. As shown in Fig. 4A, a peak around
935.0 eV of the black line is assigned to the Cu 2p , indicatꢀ
performed by using OPD (1 mM) and Cu (10 ꢁM) solution in
TrisꢀHCl buffer (10 mM, pH 7.4).
3/2
In addition, we further investigated the recognition response
2+
ing the predominant oxidation state of the Cu is bivalent (Cu )
2+
of the OPD−Cu system towards several phosphateꢀrelated
biomolecules (50 ꢁM of ATP, ADP, AMP and 50 ꢁg/mL of
fish sperm DNA). The results in Figure S4 suggested that the
presence of these biomolecules (mainly ATP and ADP) had a
33ꢀ34
in the original CuSO4.
consequent formation of OPDox, lower bind energy of Cu
p3/2 and another peak around 932.5 eV can be observed (red
After the addition of OPD and the
2
+
0
line), revealing the existence of Cu or Cu in the resultant
2+
little influence on the OPD−Cu system either in the absence
33,35
2+
mixture.
In contrast, adding OPD into the PPiꢀtreated Cu
or presence of PPi (20 ꢁM). This is mostly because the ATP
solution does not have a significant influence on the bind enꢀ
(
or ADP) molecules with the pyrophosphate group also exhibit
ergy of Cu 2p3/2 (blue line), which is quite similar to that of
2+ 31
rather strong binding affinity to Cu , although this affinity is
2+
original CuSO . Thus, we believe that Cu , served as the
2
+
4
much weaker than that between PPi and Cu . Therefore, our
following proposed bioassay would have certain limits in the
presence of high levels of these potential interfering
biomolecules.
+
0
2+
oxidant, could be reduced to Cu or Cu in the OPD–Cu reꢀ
dox reaction, on which the PPi exhibits an inhibitory effect, on
2+
the basis of the strong affinity of PPi to Cu .
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Figure 5. Sensitivity of the proposed assay for PPase by fluorescence (A) and absorbance (C) spectra enhancement. Plots of the correꢀ
sponding fluorescence intensity values at 560 nm (B) and absorbance values at 417 nm (D) versus different activities of PPase. (E) Fluoꢀ
rescence intensities of the analytical solution in addition of the control proteins or enzymes (10 ꢁg/mL) without and with the coexistence of
2+
PPase (10 mU/mL). All measurements were performed by using Cu (10 ꢁM), PPi (20 ꢁM) and OPD (1 mM) solution in TrisꢀHCl buffer
2+
2+
2+
(
10 mM, pH 7.4). (F) Fluorescence spectra of the OPD, OPD + Cu , OPD + Cu + PPi, and OPD + Cu + PPi + PPase in 2% fetal boꢀ
vine serum.
Nevertheless, the underlying reasons or mechanisms of why
vate the PPase activity while does not interfere in any other
recognitions. Figure 5A depicts the fluorescence spectra of the
obtained OPDox after incubating OPD–Cu –PPi sensing sysꢀ
2
+
the coordination of Cu with PPi could inhibit the redox reacꢀ
2+
2+
tion between Cu and OPD are still not yet clear as far. To
further ascertain the possible response mechanism for PPi
recognition, we have conducted the cyclic voltammetric experꢀ
iments, in which the electrochemical redox behaviors of the
tem with PPase at different activities from 0 to 50 mU/mL. It
is clearly observed that the fluorescence intensity enhanced
gradually with increasing PPase activity. Meanwhile, the fluoꢀ
rescence intensity varied more sensitively with PPase activity
at the low activity range (typically <10 mU/mL). In contrast,
the fluorescence intensity enhanced slower and seemed to
reach a plateau at the higher activity domain. It has been reꢀ
ported that the binding of enough PPi could increase the hyꢀ
2
+
solution of Cu in the absence and presence of PPi were inꢀ
vestigated for the first time. We have used a conventional
threeꢀelectrode cell, including a Ag/AgCl (saturated KCl) refꢀ
erence electrode, a platinum spiral as the counter electrode and
a glassy carbon (GC) electrode as the working electrode.
Meanwhile, a solution of KCl (0.5 M) was used as an electroꢀ
lyte and the related test samples are added and mixed into the
KCl solution. Figure 4B shows the representative cyclic voltꢀ
ammograms (CVs) obtained at a sweeping rate of 50 mV/s
from –0.5 to 0.5 V versus Ag/AgCl for the related samples in
38
drolysis rate of PPase. Therefore, it is speculated that the
addition of higher level of PPase could induce more consumpꢀ
tion of PPi in the sensing system, which was not beneficial for
the subsequent activation of PPase (due to a lack of PPi), and
could hardly have the same rate in the subsequent hydrolysis
of residual PPi. Furthermore, a quasiꢀlinear relationship of the
logarithm of the fluorescence intensity values at 560 nm with
the logarithm of PPase activities was obtained over the range
of 0.2−50 mU/mL (Figure 5B). The fitted linear data could be
2+
0
.5 M KCl solution. The CV curve of the pure Cu solution
shows an obvious oxide reduction peak around 0.1 V, and the
addition of Pi has negligible influence on the CV behaviours.
Very impressively, the copper oxidation–reduction potentials
2+
2
of the mixture of Cu and PPi descreas significantly, with the
expressed as Log I = 1.474 + 0.090 Log (CPPase, mU/mL), (R
oxide reduction peak shifted from around 0.1 V to below –0.2
= 0.993). Such fluorescence assay is quite sensitive, during
which 0.2 mU/mL PPase could be easily detected in current
experimental conditions.
2
+
V. It is therefore suggested that the Cu could be reduced
more difficultly in the presence of PPi, and OPD could be
2
+
oxidized more easily and simply by free Cu than the PPiꢀ
Furthermore, the assay of PPase activity could also be moniꢀ
tored by the absorption spectra of obtained OPDox (Figure 5C
and 5D). Likewise, with increasing activities of PPase, the
absorption band at 417 nm increased gradually, and the logaꢀ
rithm of the absorbance values were quasiꢀlinear with the
logarithm of PPase activities over the range of 0.2−50 mU/mL
36ꢀ37
bound one.
In addition, Figure S7 shows the representative
CVs of the pure KCl solution, PPi, Pi and OPD under the same
experimental conditions. A series of oxide reduction peak of
2+
2
+
OPD between that of free Cu (around 0.1 V) and PPi–Cu
complexation ( –0.2 V) might be the reason why the presence
<
2
+
2
of PPi could inhibit the redox reaction between Cu and OPD.
(Log A = − 0.613 + 0.115 Log (CPPase, mU/mL), R = 0.993).
Fluorescent and Colorimetric Dual-Readout Assays for
In this regard, the results for absorbance measurement were
consistent with those for fluorescence measurement, and these
identical phenomena make the results more convincing. Noꢀ
ticeably, we had used rather low concentration of components
PPase. The assay of PPase activity was carried out by using
2
+
OPD–Cu –PPi sensing system in 10 mM TrisꢀHCl buffer at
2
+
2+
pH 7.4 containing 10 ꢁM of Mg . The adopted Mg can actiꢀ
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2
+
(
typically 10 ꢁM for Cu and 20 ꢁM for PPi) in the sensing Moreover, the corresponding absorbance changes of our proꢀ
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system for the sake of the higher sensitivity, because it is not
beneficial for sensing analyte under high concentration. Thereꢀ
fore, our bioassay has certain limits in the precise determinaꢀ
tions of high level of PPase and dilute sample seems to be
necessary, in spite of the quasiꢀlinear relationship between the
double logarithmic plots over the range of 0.2−50 mU/mL of
PPase activity.
posed bioassay system were similar to the case of fluorescence
intensity and the IC50 calculated by absorbance data was apꢀ
proximate 53.7 ꢁM (Figure 6C and 6D), close to the value
from the fluorescence result. These values are similar to those
previously reported by other PPase activity assays and the
experiment results have clearly demonstrated that our bioassay
2
2,41
system could be used to screen potential PPase inhibitors.
To investigate the specificity of such bioassay toward PPase,
a number of proteins/enzymes including bovine serum albuꢀ
min (BSA), acetylcholinesterase (AChE), glucose oxidase
CONCLUSION
In summary, we have rationally designed and successfully
developed a fluorescent and colorimetric dualꢀreadout analytiꢀ
cal system for highly sensitive, selective, convenient, efficaꢀ
0
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(GOx), lysozyme and trypsin were performed as control. The
results in Figure 5E have conceivably demonstrated that none
of these control samples (10 ꢁg/mL) could induce remarkable
fluorescence response in the bioassay system and interfere in
the detection of PPase activity. To further evaluate the potenꢀ
tial applications of the OPDꢀbased bioassay, the recognition of
2+
cious recognition of Cu /PPi and determination of PPase acꢀ
tivity. To the best of our knowledge, such OPDꢀbased bioasꢀ
say owns several advantages as follow. (1) The commercially
2+
available materials containing OPD, Cu as well as PPi made
2+
Cu /PPi and sensing of PPase in the diluted fetal bovine seꢀ
the bioanalytical system pretty convenient, timeꢀsaving, and
inexpensive. (2) The sensing assay with fluorescent and colorꢀ
imetric dual modes could present more than one kind of output
signal, and make the analytical results more convincing. (3)
This is the first elaborate investigation by combining the ESIꢀ
rum (2%) and diluted 3T3L1 cell lysate (2%) was carried out.
As shown in Figure 5F and S8, the fluorescence response reꢀ
sults in the diluted biological samples were similar to that in
the standard assay system, unambiguously suggesting our
OPDꢀbased analytical system is promising to detect PPase
activity in real biological samples.
1
MS, H NMR, XPS spectra and especially cyclic voltammetric
2
+
analysis on not only the reaction mechanism of Cu ꢀtriggered
oxidation of OPD but also the inhibition mechanism of PPi on
2+
the oxidative ability of Cu . Based on the clear response prinꢀ
2+
ciple, several other potential OPDꢀ and/or Cu ꢀrelated bioasꢀ
says could be designed and such a kind of promising analytical
system might be used for the real biological sample analysis as
well as inhibitor screening in the near future.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: xryang@ciac.ac.cn; Fax: +86 431 85689278
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Financial supports by the National Natural Science Foundation of
China (Nos. 21175124, 21435005), and the Development Project
of Science and Technology of Jilin Province (No.
20150520011JH) are gratefully acknowledged.
Figure 6. Fluorescent (A) and absorbance (C) spectra of the anaꢀ
ASSOCIATED CONTENT
Supporting Information
lytical solution prepared by adding different concentrations of
2+
NaFꢀtreated PPase (10 mU/mL) into the mixtures containing Cu
10 ꢁM), PPi (20 ꢁM) and OPD (1 mM) in TrisꢀHCl buffer (10
(
The Supporting Information is available free of charge on the
ACS Publications website.
mM, pH 7.4). (B) Kinetic plots of the corresponding fluorescence
intensity (B) and absorbance values (D) against the logarithm of
the NaF concentration.
Absorption spectra and absorbance values for OPD versus
2+
2+
the Cu concentrations and OPD–Cu versus the PPi conꢀ
centrations, the effect of inorganic anions on the OPD, the
effect of phosphateꢀrelated biomolecules on the recognition
2
+
PPase Inhibition Assay. The OPD–Cu –PPiꢀbased PPase
activity bioassay system proposed herein could be extended to
evaluate PPase inhibition, by using a typical PPase inhibitor
1
of PPi, ESIꢀMS spectrometry of OPD, H NMR spectromeꢀ
3
9ꢀ40
try of OPD and OPDox, additional cyclic voltammograms,
fluorescence spectra and intensities in 2% 3T3L1 cell lyꢀ
sate. (PDF)
(sodium fluoride, NaF) as a model.
With the NaF concenꢀ
trations increasing from 0 to 10 mM in the analytical system,
the PPase activity would be inhibited and the fluorescence of
the resultant solution exhibited a gradual quenching (Figure
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