Research ArticleDesign and Synthesis of a Dinuclear Copper(II) Probe for SelectiveFluorescence Sensing of Pyrophosphate

Jinhe Xu,1 Jing Li,1 Chenxi Liu,1 Linlin Yang,1 Guangjie He ,1 Tianjun Ni ,2 Aiying Fan,1

Songjun Wang,3 and Qingzhi Wang 1

1Xinxiang Key Laboratory of Forensic Science Evidence, School of Forensic Medicine, Xinxiang Medical University, Jinsui RoadNo. 601, Xinxiang, 453003 Henan Province, China2School of Basic Medical Science, Xinxiang Medical University, Jinsui Road No. 601, Xinxiang, 453003 Henan Province, China3Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, East Zhongshan Road No. 361, Shijiazhuang,050017 Hebei Province, China

Correspondence should be addressed to Guangjie He; guangjiehe@163.com, Tianjun Ni; tjni@xxmu.edu.cn,and Qingzhi Wang; wqzmgl@126.com

Received 7 November 2018; Accepted 9 January 2019; Published 28 February 2019

Academic Editor: Roberto Paolesse

Copyright © 2019 Jinhe Xu et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A novel coumarin-based compound DPAC with two dipicolylamine (DPA) arms as the chelator sites was designed andsynthesized. The compound DPAC exhibits a highly selective response to Cu2+ ions with a distinctly emission-quenchingphenomenon. Moreover, the in situ formed complex DPAC-Cu2+ was used for the detection of pyrophosphate (PPi). Thebinding manner of probe DPAC-Cu2+ with PPi in 1 : 1 stoichiometry was supported by the Benesi-Hildebrand fitting, ESI-MSand HPLC analysis. The linear range of PPi concentration was 1-4 μM, and the detection limit was 0.53 μM. The competingexperiments illustrated that the probe DPAC-Cu2+ had good sensitivity and selectivity for PPi than other anions, includingATP, ADP, AMP, and Pi in CH3CN :HEPES (3 : 2, v/v, pH = 7 20) buffer. Further, cell fluorescence imaging experimentsindicated that the probe DPAC-Cu2+ had a potential to be used to detect PPi in vivo.

1. Introduction

Anions, widely spread in nature, play a fundamental role inbiological, environmental, and chemical fields [1, 2]. Anionsrecognition and sensing have become one of the extremelypromising topics in current research [3, 4]. Recently, consid-erable effort has been devoted to the design of probes thathave the ability to selectively bind and recognize biologicallyessential anions with the output of spectra signals [5–8].Biological phosphate anions, such as adenosine triphosphate(ATP), adenosine diphosphate (ADP), and adenosine mono-phosphate (AMP), were essential to control the cellularmovements and metabolic processes in the living organisms[9, 10]. The selective recognition of adenine-based nucleo-tides was considered to be meaningful as they were associatedwith the health status of biological cells. In particular, pyro-phosphate (PPi), as the hydrolysis product of ATP, was

formed by the combination of two phosphate units undercellular conditions [11]. The detection of PPi has successfullybeen used in a real-time DNA-sequencing method [12]. Andthe alteration of PPi concentrations in cells could also beemployed to detect or diagnose many diseases. For instance,the high intracellular level of PPi in knee synovial fluidinhibited apatite deposition, which caused the calcium pyro-phosphate dehydrate (CPPD) crystal deposition disease [13].Recently, intracellular PPi in low concentration has becomean important indicator for early cancer research [14, 15].Therefore, the development of high-efficient probes for thedetection and discrimination of PPi has become an attractiveand challenging task in recent years [16–20].

Among the variety of methods for detecting PPi, fluo-rescent chemosensors have attracted considerable attentionbecause of their superiority in high sensitivity and selectiv-ity, low cost, and fast detection [21]. The sensors, which

HindawiJournal of SensorsVolume 2019, Article ID 5349124, 8 pageshttps://doi.org/10.1155/2019/5349124

selectively recognize the PPi anions, often rely on the interac-tion of hydrogen bonding, electrostatic, and coordinationbonds (or combinations of these). In particular, the metalcomplexes based on strong coordination ability with anionswere considered to be ideal candidates for PPi recognition[22]. Many of previously reported Cu(II)-based PPi sensorsdetected the PPi anions by releasing quenching copper(II)ion [23–27]; the probes based on PPi binding recognitionmechanism were still rare [28–31].

In recent years, a number of highly effective fluorescenceprobes based on coumarin derivatives have been developeddue to their high fluorescence quantum yield and large stokesshift [32, 33]. And the spectra of coumarin derivatives couldbe easily modified to fall well within the visible range. Mean-while, metal complexes based on dipicolylamine (DPA) unitswere known as good affinity groups to selectively bind multi-anionic phosphorylated biomolecules in the outer surfaces ofcell membranes [34–36]. The configuration of dinuclear metalcomplexes is propitious to the binding action and display highaffinity toward PPi, which could be a good candidate for PPisensing [37–40]. Herein, we reported a new fluorescenceprobe (complex DPAC-Cu2+), a dinuclear copper ions com-plex for PPi, which was synthesized based on the coumarinand DPAmoieties. This complex exhibited weak fluorescence,but the addition of 1 equiv. of PPi caused a significant recoveryof the fluorescence intensity. The probe showed a high sensi-tivity and selectivity for PPi via fluorescence “turn-on” man-ner over other anions, including Pi, ATP, ADP, and AMP,and the recognition mechanism of DPAC-Cu2+ to PPi wasalso demonstrated. Furthermore, cell fluorescence imagingexperiments indicated that the probe DPAC-Cu2+ had apotential to detect PPi in real-time imaging in living cells.

2. Experimental

2.1. Apparatus. 1H NMR and 13C NMR spectra were per-formed on Bruker Ascend™ 400 spectrometer with chemical

shifts reported as ppm with TMS as internal standard. Massspectrometric data were carried on Bruker Microtof-QIIIspectrometry. UV-vis absorption spectra were measured onShimadzu UV2600 spectrophotometer. Fluorescence spectrawere measured with Edinburgh FS-5 fluorescence spectro-photometer. Biological cell imaging was recorded with LeicaDMI8 inverted fluorescence microscope.

2.2. Reagents and Chemicals. All the chemicals of analyticalgrade were obtained from commercial sources and used assupplied. Perchlorate solutions (2 0 × 10−2 M) of variousmetal ions (Al3+, K+, Na+, Mg2+, Ca2+, Cr3+, Mn2+, Fe2+,Fe3+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+, Ag+, Pb2+, and Cu2+)were prepared in aqueous solutions. The anions (PPi, Pi,ATP, ADP, AMP, S2-, F-, Cl-, Br-, I-, HSO3

-, HCO3-, ClO4

-,SO4

2-, CH3COO-, and CN-) were dissolved in aqueous solu-

tions to prepare stock solutions (2 0 × 10−2 M).

2.3. Synthetic Procedures. As shown in Scheme 1, 7-(diet-hylamino)-2-oxo-2H-chromene-3-succinimidyl ester (com-pound D) and 4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl) phenoxy)-butan-1-amine (compound E) weresynthesized according to the literatures, respectively. Thefluorescent sensing probe DPAC was synthesized by the con-densation of compound D and E and characterized by 1HNMR, 13C NMR, and ESI-MS spectra (Figure S1, S2, and S3).

2.3.1. Synthesis of Compound C (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxylic Acid) [41]. Diethyl malonate(6.40 g, 0.04mol), 4-N, N-diethyl amino salicylic aldehyde(3.86 g, 0.02mol), and 4mL piperidine were dissolved in60mL dry ethanol and refluxed for 6 hours. After the solu-tion was cooled to room temperature, 120mL 10% NaOHsolution was added and refluxed for 15 minutes to hydrolyzethe product. The mixture was cooled to room temperature,and the solvent was removed by rotary evaporation. Theresidual product was acidified to pH = 2 with concentrated

(Et)2N

(Et)2N

(Et)2N

H2N

(Et)2N (Et)2NCH2 (COOEt)2

COOEtNaOH

COOH

HCIO

O

O

N

O

N

N

N N

N

O O

O

OC N

O

O O O

C N

N

N

N

NDPAC N

N

H

O O OPiperidine

CHO

OH

A B

D

D

E

C

EDCNHS

Scheme 1: Synthesis procedures of compound DPAC.

2 Journal of Sensors

hydrochloric acid under ice cooling. The precipitate wascollected by filtration, washed with cold ethanol, and driedin vacuo. Compound C was obtained as an orange solid in79% yield: 1H NMR (400MHz, CDCl3) δ 8.69 (s, 1H),7.48 (d, J = 9 0Hz, 1H), 6.74 (dd, J = 9 0, 2.4Hz, 1H),6.56 (d, J = 2 3Hz, 1H), 3.65-3.43 (m, 4H), and 1.29 (t, J =7 1Hz, 6H); 13C NMR (100MHz, CDCl3) δ 165.65 (s),164.64 (s), 158.21 (s), 154.05 (s), 150.22 (s), 131.98 (s),110.98 (s), 108.96 (s), 105.39 (s), 96.74(s), 45.38 (s), and12.40 (s); HRMS: m/z: 284.0891, [compound C+Na]+.

2.3.2. Synthesis of Compound D (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Succinimidyl Ester) [42]. 1-(3-Dimethylami-nopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12mmol)and N-hydroxysuccinimide (1.29 g, 11.2mmol) were dis-solved in 32mL anhydrous DMF. Compound C (2.04 g,7.84mmol) in 32mL anhydrous DMF was added dropwiseto the above solution. The mixture was stirred at room tem-perature for 48 hours and poured into 500mL ice water. Theresulting precipitate was filtered to give compound D as ayellow solid that was taken forward in the synthesis withoutfurther purification: 1H NMR (400MHz, d6-DMSO) δ 8.80(s, 1H), 7.73 (d, J = 9 1Hz, 1H), 6.85 (d, J = 9 0Hz, 1H),6.60 (s, 1H), 3.53 (d, J = 6 9Hz, 4H), 2.87 (s, 4H), and 1.16(t, J = 6 7Hz, 6H); 13C NMR (100MHz, d6-DMSO) δ171.04 (s), 159.52 (s), 156.94 (s), 154.67 (s), 151.86 (s),133.35 (s), 111.03 (s), 107.88 (s), 100.98 (s), 96.40 (s),45.25 (s), 25.95 (s), and 12.97 (s); HRMS: m/z: 381.1073,[compound D+Na]+.

2.3.3. Synthesis of Compound DPAC. Compound D (0.18 g,0.50mmol) and compound E (4-(3,5-bis((bis(pyridine-2-yl-methyl)amino)methyl)phenoxy)-butan-1-amine) [43] (0.27 g,0.46mmol) were dissolved in 10mL anhydrous DMF. Themixture was stirred for 24h at room temperature. The prod-uct was poured into ice water and centrifuged. The obtainedprecipitate was dried and purified by flash column chroma-tography (silica gel, CH2Cl2 CH3OH = 20 1) to affordcompound DPAC (60% yield and 95% purity): 1H NMR(400MHz, CDCl3) δ 8.89 (t, J = 5 6Hz, 1H), 8.71 (s, 1H),8.50 (d, J = 4 8Hz, 4H), 7.67-7.60 (m, 8H), 7.43 (d, J = 9 0Hz, 1H), 7.15-7.12 (m, 4H), 7.04 (s, 1H), 6.89 (s, 2H), 6.65(dd, J = 8 9, 2.4Hz, 1H), 6.50 (d, J = 2 2Hz, 1H), 3.99(t, J = 5 9Hz, 2H), 3.83 (s, 8H), 3.66 (s, 4H), 3.55-3.43(m, 6H), 1.91-1.87 (m, 2H), 1.83-1.80 (m, 2H), and 1.24(t, J = 7 1Hz, 6H); 13C NMR (100MHz, CDCl3) δ 163.21(s), 162.83 (s), 159.73 (s), 159.24 (s), 157.63 (s), 152.53 (s),148.86 (s), 148.07 (s), 140.56 (s), 138.00 (s), 136.56 (s),131.13 (s), 127.39 (s), 122.81 (s), 121.99 (s), 121.41 (s),113.46 (s), 110.34 (s), 109.96 (s), 108.40 (s), 96.57 (s), 67.40(s), 60.05 (s), 58.62 (s), 45.09 (s), 39.30 (s), 26.87 (s), 26.43(s), and 12.44 (s); HRMS: m/z: 831.4341, [compoundDPAC+H]+; 853.4159, [compound DPAC+Na]+.

2.3.4. Synthesis of Complex DPAC-Cu2+. The correspondingDPAC-Cu2+ complex was prepared by mixing compoundDPAC and Cu(ClO4)2·6H2O with the ratio of 1 : 2 in solutionin situ, in which the DPA unit coordinated with Cu2+ ions toform the dinuclear complex DPAC-Cu2+.

2.4. General UV-Vis and Fluorescence Spectra Measurements.The stock solution of compound DPAC (1mM) was pre-pared in DMSO. A portion of the stock solution (1mL) ofthe probe DPAC was added to the CH3CN solution andadjusted to 100mL CH3CN :HEPES (3 : 2, v/v, pH = 7 20)solution with 0.1M HEPES buffer. The test solution (10μM)was formed and well-mixed.

Each time a 3mL test solution of compound DPAC orcomplex DPAC-Cu2+ was filled in a quartz cell of 1 cm opti-cal path length, and stock solutions of different metal ions oranions were added into the quartz cell gradually by using amicrosyringe. Excitation wavelength for probe DPAC andDPAC-Cu2+ was 423nm with slit width as 1.2 nm.

2.5. Calculation Methods. The quenching constant (Ksv) wascalculated by using the Stern-Volmer method [44]:

F0F

= 1 + Ksv C , 1

where F is the fluorescence intensity of compound DPAC inthe presence of Cu2+, F0 is the fluorescence intensity of probeDPAC, Ksv is the quenching constant, and C is the concen-tration of Cu2+.

The Benesi-Hildebrand equation was used as shownbelow [45]:

log F − F0FL − F

= log Ka + n log C , 2

where Ka is the association constant; F and F0 represent thefluorescence intensity of DPAC-Cu2+ in the presence andabsence of PPi, respectively. FL is the saturated fluorescenceintensity, C is the concentration of PPi, and n is the stoichi-ometry for the binding of PPi.

The detection limit is then calculated with the followingequation [46]:

LOD = K × δ

S, 3

where δ is the standard deviation of blank measurements; S isthe slope between intensity versus sample concentration.

3. Results and Discussion

3.1. The Absorption and Fluorescence Spectra Responses ofCompound DPAC to Cu2+ Ions. The UV-vis absorption spec-tra of compound DPAC (10μM) upon the addition of vari-ous metal ions (2 equiv.) in the CH3CN :HEPES (3 : 2, v/v,pH = 7 20) solutions were shown in Figure S4. Themaximum absorption peaks of compound DPAC and thecorresponding metal complexes were all at about 423nm.Almost no obvious changes could be monitored with theaddition of various metal ions. The fluorescence spectra ofcompound DPAC (10μM) in the CH3CN :HEPES (3 : 2, v/v,pH = 7 20) solution showed a strong emission peak at470nm when excited at 423nm. To calculate the fluorescent

3Journal of Sensors

response ability of compound DPAC, the fluorescenceexperiment was monitored by adding copper ions to thesolution of compound DPAC (Figure 1(a)). The fluorescenceintensity gradually decreased with the increasing concen-tration of Cu2+ ions. After the addition of 2 equiv. of Cu2+

ions, the fluorescence intensity nearly reached equilibrium,and the probe DPAC-Cu2+ was formed in situ. The lowfluorescence intensity of the probe might be due to thequenching effect from the paramagnetism of copper ions[47]. The plot of fluorescence intensity showed a good linearrelationship (R2 = 0 996) to the concentration of Cu2+ in therange of 1-10μM (Figure 1(b)). The quenching constant ofCu2+ was calculated as 1 47 × 105 Lmol-1 based on theStern-Volmer fitting of the titration plots. The detectionlimit calculated following the S/N = 3 1 was estimated to beabout 0.36μM, which is lower than the WHO guideline inthe drinking water. The Benesi-Hildebrand fitting methodindicated that the stoichiometric ratio of compound DPACwith Cu2+ was 1 : 2 in complex DPAC-Cu2+(Figure S5). Theapparent association constant of DPAC and Cu2+ wasestimated to be 1 67 × 105 M-2. The fluorescence selectivityof compound DPAC was carried out by additions ofdifferent metal ions to the solution of compound DPAC, andthe results were displayed in Figure S6. However, Co2+ ionsshowed similar quenching phenomenon, whereas the othermetal ions did not cause any detectable changes.

The complexation formation of DPAC with Cu2+ in the1 : 2 ratio was further confirmed by ESI-MS spectra. As shownin Figure S7, upon the addition of 2 equiv. Cu2+metal ions tothe solution of DPAC in CH3CN, the signals at m/z =352 3921, 578.0921, and 1255.1358 were observed. The peakswere assigned to the [Cu2(DPAC)(ClO4)]

3+, [Cu2(DPAC)(ClO4)2]

2+, and [Cu2(DPAC)(ClO4)3]+ species, respectively.

The specific isotopic patterns fitted well with the simulatedpeaks calculated by IsoPro 3.0 program (Figure S8 and S9).These results were consistent with the stable presence ofCu2(DPAC) species in the solution. The coordinated ClO4

-

anion was easy to leave, and the quenching effect could be

inhibited by other coordinated anions. All these phenomenaindicated that the complex DPAC-Cu2+ could be used as apotential candidate chemosensor for biological anions [48].

3.2. The Fluorescence Spectra Response of the Probe (ComplexDPAC-Cu2+) to PPi. Based on the moderate binding ability ofthe DPA group and high charge density of the complexDPAC-Cu2+, it was proposed to be used as an anion-selective fluorescence probe for PPi. The experimental datashowed that the fluorescence intensity was gradually restoredwhen PPi was added to the CH3CN :HEPES (3 : 2, v/v,pH = 7 20) solution of the complex DPAC-Cu2+. To under-stand the detection mode of the complex DPAC-Cu2+ withPPi, the titration experiment was performed. As shown inFigure 2(a), after the addition of about 1 equiv. of PPi, theemission intensity was up to the maximum and reached toequilibrium in spite of further addition. It was deduced thatthe probe DPAC-Cu2+ bound PPi with 1 : 1 stoichiometryby the Benesi-Hildebrand method (Figure 2(b)). The detec-tion limit was calculated as about 0.53μM based on the equa-tion of LOD = 3δ/S (Figure S10).

3.3. Detection Mechanism of DPAC-Cu2+ for PPi. Thedetection mechanism of DPAC-Cu2+ to PPi was furtherinvestigated. ESI-MS spectra analysis indicated that DPAC-Cu2+ and PPi formed a 1 : 1 complex as DPAC-Cu2+-PPi.As shown in Figure S11 and Figure S12, a peak at m/z1133.2058 corresponding to [DPAC+2Cu+PPi+1H]+ wasclearly observed when excess amount of PPi was added tothe solution of DPAC-Cu2+. The ESI-MS spectra results fitwell with the proposition that the probe DPAC-Cu2+

provided a platform to selectively detect PPi [49]. Thefurther evidence of the binding mechanism came fromHPLC analyses (Figure S13). Upon the addition of Cu2+

ions, the peak of DPAC (rt = 7 3 min) gradually disappearedalong with the rise of the complex DPAC-Cu2+ peak(rt = 0 9 min). Interestingly, after adding 1 equiv. PPi, thepeak of DPAC-Cu2+ finally vanished with the appearance

1.0 1.0

0.8 0.8

0.60.6

0.4

0.4

0.0 0.5 1.0 1.5Equiv. of Cu2+

2.0 2.5 3.0

0.2

0.0

450 500 550Wavelength (nm)

F/F

0

F/F

0

600 650 700

(a)

2.4

2.0

1.6

Y = 0.95486 + 1.47152XR

2 = 0.996

1.2

0.8

F/F

0

0.0 0.2 0.4Concentration of Cu2+ (10−5mol/L)

0.6 0.8 1.0

(b)

Figure 1: (a) Fluorescence spectra of compound DPAC (10 μM) upon the continuously addition of Cu2+ ions (0-3 equiv.) in theCH3CN :HEPES (3 : 2, v/v, pH = 7 20) solution. Inset: fluorescence titration profile of DPAC at 470 nm upon the addition of Cu2+ ions.(b) The Stern-Volmer fitting of titration plots with the concentration of Cu2+ ions (1-10μM).

4 Journal of Sensors

of a new peak at 1.3min, which was attributed to theformation of DPAC-Cu2+-PPi. These results confirmedthat the probe DPAC-Cu2+ combined PPi to form thestable sandwiched complex DPAC-Cu2+-PPi as shown inScheme 2 and thus inhibited the PET-quenching processalong with fluorescence intensity enhancement. The specificstructure was ascribed to the strong metal coordinationinteraction between the bis(2-pyridylmethyl)amine (DPA)-Cu2+ complex and PPi [50]. Therefore, this unique bindingfeature could be utilized to discriminatively detect PPi fromother phosphate anions in the fluorescence “off-on” approach.

For further evaluating the selectivity of the probeDPAC-Cu2+for PPi, the fluorescence emission intensitychanges coexisted with the anions including PPi, F-, Cl-,Br-, I-, S2-, ATP, ADP, AMP, Pi, HSO3

-, HCO3-, SO4

2-,CH3COO

-, ClO4-, and CN- in CH3CN :HEPES (3 : 2, v/v,

pH = 7 20) buffer were investigated. As shown in Figure 3,among these anions, in the presence of PPi, the fluorescenceintensity of the complex DPAC-Cu2+ (10μM) increased.Nevertheless, no obvious fluorescence intensity changes wasobserved in the presence of the other anions includingATP, ADP, AMP, and Pi. Moreover, subsequently adding 2equiv. of PPi to the other anion solutions gave rise to similar

enhancement in accordance with the addition of equalamount of PPi alone, indicating that the PPi had specificresponse to the probe. The experiments above demonstratedthat the complex DPAC-Cu2+ had good selectivity for PPi,which was not disturbed by the competitive anions. Theselectivity for PPi over other phosphate anions was possiblydue to the higher anionic charge density of the four O–P oxy-gen atoms involved in the complexation between the PPi andthe two Cu2+ sites. Moreover, the hexacoordination of Cu2+

ions with PPi is clearly reflected in the extremely higher asso-ciation constant than other phosphate anions. And the spa-tial configuration of the two Cu2+-DPA sites was benefitedfor the selective sensing of PPi.

3.4. The Fluorescence Cell Imaging of the Probe (ComplexDPAC-Cu2+) for PPi. The fluorescence imaging of the probeDPAC-Cu2+ for PPi was performed in lung cancer cellsA549. Prior to fluorescence imaging, A549 cells were cul-tured in a 12-well cell plate for 24 hours and incubated withcompound DPAC (1μM) at 37°C, 5% CO2 for 30min andthen washed with PBS solution three times. Under LeicaDMI8 inverted fluorescence microscope, the cell imagingresults of A549 were shown in Figure 4. Excited with blue

(Et)2N (Et)2N

O

O O

NN N

OO

O PPO

O

OO

O

C NN

N N

HO O O PPi

N

NNCu2+

DPAC-Cu2+ DPAC-Cu2+-PPi

Cu2+

Cu2+

Cu2+

N

N

NOC N

H

Scheme 2: Schematic illustration of the proposed sensing mechanism of DPAC-Cu2+ toward PPi.

2.5

2.02.0

1.6

1.2

0.0 0.5Equiv. of PPi

1.0 1.5 2.0

1.5

1.0

0.5

0.0

450 500 550Wavelength (nm)

F/F

0

F/F

0

600 650 700

(a)

0.6

0.4

0.2

Y = 5.953 + 1.062XR

2 = 0.994

0.0

−0.2

−0.4

Ig[(F

-F0)

/(F

L-F

)]

−6.0 −5.8 −5.6

Ig (PPI)−5.4 −5.2 −5.0

(b)

Figure 2: (a) Fluorescence alteration of compound DPAC-Cu2+ (10 μM) upon the addition of PPi with concentrations up to 20μM in theCH3CN :HEPES (3 : 2, v/v, pH = 7 20) solution. Inset: fluorescence titration profile of DPAC-Cu2+ at 470 nm upon the addition of PPi. (b)The Benesi-Hildebrand fitting of titration plots with the concentration of PPi in the range of 1-8 μM.

5Journal of Sensors

light, compound DPAC showed significant intracellulargreen fluorescence in A549 cells, indicating that compoundDPAC had strong cell permeability. After the addition ofexcess 4 equiv. of copper ions, the probe DPAC-Cu2+ wasprepared in situ, and the green fluorescence in A549 cellswas significantly quenched. However, after the excess Cu2+

ions were washed away and 2 equiv. PPi were subsequentlyadded, the fluorescence in the cells was recovered. Theseresults demonstrated that the probe DPAC-Cu2+ could beused for imaging the PPi anions in living cells.

4. Conclusions

In summary, a coumarin-based dinuclear copper complexDPAC-Cu2+ was synthesized and investigated as a fluorescent

probe for PPi. The Benesi-Hildebrand fitting analysis, ESI-MSand HPLC analyses, indicated a 1 : 1 stoichiometry of the com-plex DPAC-Cu2+ with PPi. The detection limit of PPi was esti-mated about 0.53μM. The fluorescence spectra indicated thatthe probe had good selectivity and sensitivity for PPi inCH3CN :HEPES (3 : 2, v/v, pH = 7 20) buffer. Ultimately, cellimaging results indicated that DPAC-Cu2+ could visuallymonitor the PPi anions in A549 cells.

Data Availability

The data used to support the findings of this study areincluded within the article and supplementary informationfile(s).

2.5

2

1.5

1

0.5

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Figure 3: Fluorescence intensity of probe DPAC-Cu2+ (10 μM) upon the addition of various competitive anions (2 equiv.) (blue bars) andsubsequent addition of PPi (2 equiv.) (red bars) in CH3CN :HEPES (3 : 2, v/v, pH = 7 20) buffer. Anions: (1) PPi, (2) F-, (3) Cl-, (4) Br-,(5) I-, (6) S2-, (7) ATP, (8) ADP, (9) AMP, (10) Pi, (11) HSO3

-, (12) HCO3-, (13) SO4

2-, (14) CH3COO-, (15) ClO4

-, and (16) CN-.

(a) (b) (c)

(d) (e) (f)

Figure 4: The fluorescence images of A549 cells incubated with (a) compound DPAC (1 μM), (b) probe DPAC-Cu2+, (c) DPAC-Cu2+-PPi,and (d-f) their corresponding bright-field images.

6 Journal of Sensors

Conflicts of Interest

The authors declare that there is no conflict of interestregarding the publication of this paper.

Authors’ Contributions

Jinhe Xu and Jing Li contributed equally to this work.

Acknowledgments

We gratefully acknowledge the financial support fromthe National Natural Science Foundation of China (Nos.81401470 and 21371148), the Natural Science Foundationof Henan Province (No. 182300410309), the Key Scien-tific and Technological Project of Henan Province (No.182102310648), the Key Research Project of Higher Edu-cation Institutions of Henan Province (No. 18A150044),and the Hebei Key Laboratory of Forensic Medicine (No.KF201601).

Supplementary Materials

Figures S1, S2, and S3: NMR and MS spectra of compoundDPAC; Figures S4 and S6: UV-vis absorption and emissionspectra of compound DPAC upon addition of various metalions; Figure S5: the Benesi-Hildebrand fitting of titrationplots with the titration of Cu2+; Figures S7, S8, and S9:ESI-MS spectra of compound DPAC in the presence of Cu2+;Figure S10: LOD calculation of DPAC-Cu2+ for PPi basedon fluorescence intensity; Figures S11, S12, and S13: ESI-MSand HPLC study of compound DPAC-Cu2+ upon addition ofPPi; Figure S14: fluorescence spectra of compound DPAC indifferent buffer solutions. (Supplementary Materials)

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