PyridineâBiquinolineâMetal Complexes for Sensing Pyrophosphate

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Pyridine-Biquinoline-Metal Complexes for Sensing Pyrophosphate and Hydrogen Sulfide in Aqueous Buffer and in Cells Zijuan Hai, Yajie Bao, Qingqing Miao, Xiaoyi Yi, and Gaolin Liang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504536q • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

Pyridine-Biquinoline-Metal Complexes for Sensing Pyrophosphate and Hydrogen Sulfide in Aqueous Buffer and in Cells Zijuan Hai, † Yajie Bao, † Qingqing Miao, † Xiaoyi Yi, ‡ Gaolin Liang*,†

†

CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and

Technology of China, Hefei, Anhui 230026, China

‡

College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083,

China

*Corresponding author:

E-mail: [email protected] (G.-L. L.).

ACS Paragon Plus Environment 1


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ABSTRACT Herein, we report a new pyridine-biquinoline-derivative fluorophore L for effectively sensing pyrophosphate (PPi) and monohydrogen sulfide (HS-) in aqueous buffer and in living cells. L could selectively coordinate with metal ions (Mn+) in IB and IIB groups to form L-Mn+ complexes with 1:1 stoichiometry, resulting in fluorescence quenching via photoinduced electron transfer (PET) mechanism. L-Zn2+ complex was applied to competitively coordinate with PPi to form a new “ate”-type complex, turning on the fluorescence by a 21-fold-increase. The limit of detection (LOD) of this assay for PPi detection in aqueous buffer is 0.85 µM. L-Cu2+ complex was applied for highly selective detection of HS- with an excellent sensitivity by 25-fold decomplexation-induced fluorescence increase. LOD of L-Cu2+ complex for HS- detection in aqueous buffer is 2.24 µM. With the in vitro data obtained, we successfully applied these two complexes for sequential imaging Zn2+ and PPi, Cu2+ and HS- in living cells, respectively. Since PPi and HS- occur in vascular calcification in positive correlation, our multifunctional probe L might help doctors to more precisely diagnose this disease in vivo in the future. For example, we could use radioactive tracer L-64Cu for qualitative and quantitative positron emission tomography/computed tomography (PET/CT) imaging of HS- in vivo.

INTRODUCTION

Anions are important to industrial and biological processes, playing crucial roles in both environment and health. Thus, sensing of anions is receiving more attention.1 Among the anions, pyrophosphate (PPi) is one type of biologically significant anions.2 It is the product of adenosine triphosphate (ATP) hydrolysis under cellular conditions. PPi concentration provides critical information for some important


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biological processes such as DNA replication.3 Moreover, PPi can act as a potential biomarker for some diseases such as chondrocalcinosis or calcium pyrophosphate dihydrate (CPPD) crystal deposition.4,5 Up to date, several techniques have been developed for the detection of PPi such as electrooptical method,6 colorimetric assays,7 and fluorescence assays.8,9 In terms of sensitivity, spatial resolution, response time, and cost, fluorescent chemosensors are very attractive for PPi detection.10 But a majority of these fluorescence assays are limited to organic or mixed aqueous media because of strong hydration of PPi and low water solubility of the organic fluorescent probes.11,12 Nevertheless, due to the strong binding affinity between metal ions and PPi, utilization of a metal ion complex as a fluorescent chemosensor has been found to be the most successful strategy for the detection of PPi in 100% aqueous solution.13

Besides PPi, monohydrogen sulfide (HS-) is also one type of biologically important anions. Its protonated form, hydrogen sulfide (H2S), has been identified as the third gasotransmitter (the other two are NO and CO) exerting a series of biological effects on various biological targets.14 In mammalian cells, the majority of H2S is synthesized by four enzymes when they are under tightly regulated conditions.15 H2S contributes to various physiological processes,16 including relaxation of vascular smooth muscles,17 mediation of neurotransmission,18 inhibition of insulin signaling,19 regulation of inflammation,20,21 and O2 sensing.22 In aqueous media, hydrogen sulfide is essentially present as both its protonated H2S and deprotonated HS- forms (in water, pKa = 6.76 at 37 °C). Hence, the detection of hydrogen sulfide could be achieved by sensing of HS-.23 There are a large number of methods reported for the detection of

sulfide such as chemiluminescence,24 colorimetric assays,25 iodimetry,26



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electrochemical method,27 ion chromatography,28 polarographic sensors,29 and gas chromatography.30 But these assays often result in sample destruction and/or are limited to extracellular detection. Compared with these methods, fluorescent assay shows its advantages in non-invasiveness and aptness for living cells.31 Recently, a lot of fluorescence probes, utilizing the three specific characters of H2S (i.e., reducibility,32,33 nucleophilicity,31 or strong Cu2+-complexation ability34,35), have been developed for the sensing of H2S. Due to the very low-solubility product of CuS (Ksp = 6.3 × 10−36), using Cu2+-fluorophore complex to selectively react with sulfide and thereafter turn-on the fluorescence of the fluorophore has been an important method for hydrogen sulfide detection.36 Vascular calcification is a common complication in atherosclerosis, whose pathogenesis is the osteoblastic differentiation of vascular smooth muscle cells (VSMCs).37 H2S is a potent inhibitor of vascular calcification by suppressing the induction of the alkaline phosphatase (ALP). ALP hydrolyzes PPi into phosphate (Pi) which is an essential compound for osteoblastic differentiation.38 Therefore, developing a chemosensor for sensitive and simultaneous detections of H2S and PPi should help doctors to diagnose vascular calcification more precisely. But, to the best of our knowledge, there is no report of using single fluorescent chemosensor to detect these two important anions simultaneously.

In 2004, Valliant and co-workers reported a lysine-derived bis(quinoline) amine as a single amino acid chelate quinoline (SAACQ). SAACQ could be used to prepare complementary fluorescent and radioactive probes, allowing direct correlation between aqueous buffer and in vivo imaging studies.39 Based on this work, we recently developed a fluorescent switch SAACQ-deFmoc for fast and selective


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detection of Hg2+ in water and in living cells.40 We also constructed a fluorescent switch of Ac-SAACQ-Gly-Gly-Gly-Lys(FITC)-OH for sequentially sensing Cu2+ and L-His in aqueous buffer and in living cells.41 Rissanen and co-workers developed a simple terpyridine-Zn2+ complex for nanomolar PPi detection in water.9 Inspired by these pioneering studies, as illustrated in Figure 1, we rationally designed a new fluorescent pyridine-biquinoline probe (i.e., L) for Zn2+ or Cu2+ chelation. Upon addition of Zn2+ or Cu2+, the fluorescence of L is quenched by the formation of L-Mn+ complexes via photoinduced electron transfer (PET) mechanism. Interestingly, addition of PPi to the L-Zn2+ complex yields another fluorescent “ate”-type complex by recovering 64% of original fluorescence of L. But addition of HS- to L-Cu2+ complex results in the decomplexation and turns on 77% of original fluorescence of L. Employing this fluorescence “off” and “on”, we successfully applied L for selectively sensing PPi and HS- in aqueous buffer and in living cells.

EXPERIMENTAL SECTION Materials. All the starting materials were obtained from Sigma or Sangon Biotech. Commercially available reagents were used without further purification, unless noted otherwise. All chemicals were reagent grade or better. General methods. 1

H NMR spectra were obtained on a Bruker AV 300. ESI mass spectra were obtained on a Finnigan

LCQ Advantage ion trap mass spectrometer (ThermoFisher Corporation) equipped with a standard ESI source, respectively. Fluorescence spectra were recorded on a F-4600 fluorescence spectrophotometer



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(Hitachi High-Technologies Corporation, Japan) with excitation wavelengths set to 340 nm. Cell images were obtained on the IX71 fluorescence microscope (Olympus, Japan). Transmission electron microscope (TEM) images and Energy-dispersive X-ray spectrometer (EDS) spectra were obtained on a JEOL 2010 electron microscope equipped with an energy dispersive spectrometer (EDS), operating at 200 kV. Syntheses and Characterizations. Synthesis of L. 2-acetylquinoline (85.5 mg, 0.500 mmol) was added to a solution of 4-dimethylaminobenzaldehyde (37.5 mg, 0.252 mmol) in ethanol (1.25 mL). KOH pellets (42.5 mg, 0.759 mmol) and aqueous NH3 (725 µL) were added to above solution and the resulting mixture was stirred at room temperature (RT) for 30 hours. Then the reaction mixture was filtered and the precipitate was washed with ethanol for several times. The precipitate was then recrystallized in chloroform/n-hexane (1:3, v/v), filtered, washed with n-hexane, and dried in air to yield L (33.9 mg, 30.0%) (Scheme S2, Supporting Information). Characterization of L. 1H NMR (300 MHz, d6-DMSO) δ (ppm): 8.94 (s, 2 H), 8.92 (d, J = 9.0 Hz, 2 H), 8.63 (d, J = 9.0 Hz, 2 H), 8.23 (d, J = 8.0 Hz, 2 H), 8.10 (d, J = 8.0 Hz, 2 H), 7.92 (d, J = 9.0 Hz, 2 H), 7.87 (t, J = 8.0 Hz, 2 H), 7.69 (t, J = 8.0 Hz, 2 H), 6.97 (d, J = 9.0 Hz, 2 H), 3.05 (s, 6 H) (Figure S2, Supporting Information). MS of L: calculated for C31H24N4, [(M+H)+]: 453.20; obsvd. ESI-MS: m/z 453.30 (Figure S3, Supporting Information).


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Preparation of L-Mn+ complex. The L-Mn+ complexes system were simply prepared by mixing L with 1 equiv. of metal ion in situ and diluting with 10 mM HEPES buffer (pH 7.4), then the mixture was sonicated for 5 minutes without any additional purification procedures.

Cell Culture. The hepatocellular carcinoma HepG2 cells were cultured in Dulbecco’s modiﬁed eagle medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO) and streptomycin (100 µg/mL). The cells were expanded in tissue culture dishes and kept in a humid atmosphere of 5% CO2 at 37 °C. The medium was changed every other day. Cell Imaging. The hepatocellular carcinoma HepG2 cells were seeded into glass bottom cell culture dish (3.5 cm) and incubated at 37 °C in a CO2 incubator for one day. Then the HepG2 cells were washed for three times with phosphate buffered saline (PBS, pH 7.4) and incubated with 10 µM L in serum-free DMEM at 37 °C for 1 hour in a CO2 incubator. The cells were again washed for three times with PBS to remove the free L prior to imaging. After the HepG2 cells were treated with 10 µM L, the cells were then incubated with 10, 20, or 30 µM Zn2+ (or Cu2+) in serum-free DMEM for 20 minutes prior to imaging. For PPi imaging, after the HepG2 cells were treated with 10 µM L for 1 hour at 37 °C followed by addition of 30 µM Zn2+ for 20 minutes, various concentrations of PPi (30 µM, 60 µM, or 90 µM) in serum-free DMEM were incubated with the cells for 20 minutes at 37 °C prior to imaging. For HSimaging, after the HepG2 cells treated with 10 µM L for 1 hour at 37 °C followed by addition of 30 µM Cu2+ for 20 minutes, various concentrations of HS- (60 µM, 90 µM, or 120 µM) in serum-free DMEM were incubated with the cells for 20 minutes at 37 °C prior to imaging.



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RESULTS AND DISCUSSION

Figure 1. Schematic illustration of fluorescence “off” and “on” of L for selective detections of PPi and HS- in aqueous buffer and in cells.

Syntheses and Rationale of the Design. We began the study with the syntheses of 2-acetylquinoline and L. Preparation of 2-acetylquinoline from 2-methylquinoline was following the literature42 (Scheme S1, Supporting Information). L was synthesized with a facile one-pot reaction according to the literature43 (Scheme S2, Supporting Information): the reaction was started with 4-dimethylaminobenzaldehyde and two equiv. of 2-acetylquinoline. After aldol condensation and Michael addition between these two reactants to yield the diketone intermediate, the intermediate reacts with aqueous ammonia nitrogen source to complete the formation of the central pyridine ring of L. L was designed to have two rationales as following: (1) the conjugated pyridine-biquinoline structure of L itself is a fluorophore with fluorescence emission at 525 nm; (2) the pyridine-biquinoline motif


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could chelate metal ions (Mn+) to form L-Mn+ complexes, quenching the fluorescence of L. As formed L-Mn+ complexes could be used to detect anions by either decomplexation or forming a new complex which results in fluorescence “turn-on” again.

Fluorescence property of L. After synthesis, we firstly investigated the fluorescence property of L. In 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM, pH 7.4, 3% DMSO) at RT, L has a fluorescence emission maximum at 525 nm. Interestingly, when different metal ions were added, fluorescence emission of L only responds to those metal ions in IB and IIB groups (e.g., Zn2+, Cu2+, Ag+, Cd2+, and Hg2+,) by an obvious 33~37-fold decrease (Figure 2) along with a 39 nm blue shift from 525nm to 486nm (Figure 3). Other metal ions in VIB group (e.g., Cr2+), VIII group (e.g., Fe2+, Fe3+, Co2+, and Ni2+), IIA group (e.g., Ca2+, Sr2+, and Ba2+), or IVA group (e.g., Pb2+) would not induce obvious fluorescence change of L (Figure 2). This indicates that L could selectively coordinate with metal ions in IB or IIB groups by forming L-Mn+ complexes and thereafter its fluorescence is quenched via PET mechanism.



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Figure 2. Fluorescence responses of L to different metal ions. Fluorescence emissions of L were measured with 30 µM L before and after additions of 5 equiv. different metal ions in HEPES buffer (10 mM, pH 7.4, 3% DMSO) at RT. F0 and F are the fluorescence intensities of L at 486 nm before and after metal ion addition, respectively. λex = 340 nm.

Characterizations of L-Zn2+ and L-Cu2+ complexes. In the presence of Zn2+ or Cu2+ at RT in HEPES buffer (10 mM, pH 7.4, 3% DMSO), the fluorescence intensity (FI) of 30 µM L gradually decreased with the increase of Zn2+ or Cu2+ concentration, accompanied by a 39 nm-blue shift from 525 nm to 486 nm (Figure 3). When the concentration of Zn2+ or Cu2+ was increased to 30 µM (L:Mn+ = 1:1), FI of L dropped to its bottom and further addition of the metal ions would not induce any more decrease of the FI (Figure S4, Supporting Information). Thus, we assume that the binding stoichiometry between L and Zn2+ (or Cu2+) is 1:1. We then used electrospray ionization mass (ESI-MS) spectra to study the binding stoichiometry between L and Zn2+ (or Cu2+). Upon addition of 1 equiv. Zn2+, the ESI-MS spectrum of L clearly shows that the dominant ionic peak in the spectrum has a m/z value of 630.14, corresponding to [L-Zn2+-TFA]+ (Figure S5, Supporting Information). ESI-MS spectrum of L after addition of 1 equiv. Cu2+ also shows that the dominant ionic peak in the spectrum has a m/z value of 628.14 which corresponds to [L-Cu2+-TFA-]+ (Figure S6, Supporting Information). Moreover, the isotopic mass peaks of the L-Cu2+-TFA- complex are identical to those of Cu2+. Therefore, the ESI-MS spectrum results confirmed the binding stoichiometry between L and Zn2+ (or Cu2+) to be 1:1. Following the methods reported44 and using the data in Figure S4, we calculated the binding constants of L for


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Zn2+ and Cu2+ to be 2.27 × 105 M-1 and 3.33 × 105 M-1, respectively.

Figure 3. Fluorescence spectra of 30 µM L in the presence of (a) Zn2+ or (b) Cu2+ at different concentrations of 0, 5, 10, 20, 30, or 40 µM in HEPES buffer (10 mM, pH 7.4, 3% DMSO) at RT. λex = 340 nm.

We also conducted 1H NMR spectroscopic analyses to confirm the coordination between L and Zn2+ (or Cu2+). Since Cu2+ was a paramagnetic ion which heavily interferes with 1H NMR signals, only Zn2+ was chosen to study its coordination with L. Compared with the 1H NMR spectrum of L, the proton resonances on the pyridine-biquinoline rings of L after 1 equiv. of Zn2+ addition became broader and shifted to downfield (Figure S7, Supporting Information), which persuasively confirmed the coordination of Zn2+ to L. To further confirm the formation of the complexes, we directly synthesized



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ZnCl2L and CuCl2L by heating and stirring the dichloromethane/methanol solution of L with ZnCl2 or CuCl2 (1:1.2) at 50 °C for 3 hours9 (Supporting Information). Transmission electron microscope (TEM) images revealed the nanosheet structure of ZnCl2L with an average width of 28.6 ± 3.6 nm and nanorod structure of CuCl2L with an average diameter of 463.2 ± 6.5 nm (Figure S8a & c, Supporting Information). Energy-dispersive X-ray spectrometer (EDS) spectra of ZnCl2L and CuCl2L proved the existence of Zn and Cu in the complexes, respectively (Figure S8b & d, Supporting Information). These above results echoed that the decrease of FI of L upon addition of Zn2+ or Cu2+ was actually induced by the coordination between the Zn2+ (or Cu2+) and the pyridine-biquinoline moieties of L at 1:1 stoichiometry. Therefore, quenching of the fluorescence emission of L should be induced via PET mechanism. Detection of PPi in aqueous buffer. Due to the strong binding affinity between Zn2+ and PPi, the L-Zn2+ complex could be used to detect PPi in aqueous buffer by fluorescence “turn-on”. After the formation of 30 µM L-Zn2+ complex as mentioned above, progressive addition of PPi (0–3 equiv. of L-Zn2+) into the system gradually turned on the fluorescence emission at 477 nm, reaching 21-fold-increase plateau at the PPi concentration of 3 equiv. of L-Zn2+ (i.e., 90 µM) (Figure 4a). This was attributed to the competitive coordination of PPi with the Zn2+ in the L-Zn2+ complex by forming a new “ate”-type complex, thus suppressing the PET process and consequently turning on the fluorescence. Job’s plot analysis indicated that the binding stoichiometry between PPi and L-Zn2+ is 1:1 (Figure S9a, Supporting Information). The Kd of L-Zn2+-PPi was calculated to be 1.83 × 10-5 M,45 and the Kd (herein reciprocal of binding constant) of L-Zn2+ is 4.4 × 10−6 M . This further confirmed the formation of the


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new “ate”-type complex L-Zn2+-PPi instead of complex PPi-Zn2+.

Figure 4. (a) Fluorescence spectra of 30 µM L-Zn2+ complex upon addition of different concentrations of PPi at 0, 5, 10, 15, 20, 30, 40, 60, or 90 µM respectively in HEPES buffer (10 mM, pH 7.4, 3% DMSO) at RT. Inset: The fitted calibration line in the liner region of 0~30 µM PPi. (b) Fluorescence intensity (FI) of 30 µM L-Zn2+ complex at 477 nm without or with addition of 5 equiv. of various common anions.

By correlating the FI of L-Zn2+ complex at 477 nm with the concentration of PPi, we obtained a calibration curve for the determination of PPi in aqueous buffer. A linear relationship between the FI and PPi concentration (Y = 46.757 + 12.606*X, R2 = 0.995) was obtained over the range of 0~30 µM. LOD of PPi in this assay is 0.85 µM (S/N = 3), which is comparable to those of recently reported fluorescence probes for PPi detection (Table S1, Supporting Information) . We then studied the selectivity of L-Zn2+ complex to PPi among various anions. As shown in Figure 4b, it was PPi but not the non-phosphate common anions (e.g., SO42-, NO3-, I-, F-, Cl-, Br-, or AcO-) that induced obvious FI increase of the



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L-Zn2+ complex. But regretfully, the L-Zn2+ complex did not show advantageous selectivity to PPi among inorganic phosphates or nucleotide phosphates (Figure S9b, Supporting Information).

Imaging Zn2+ and PPi in living cells. Based on the fluorescence “off” and “on” property of L upon sequential additions of Zn2+ and PPi aqueous buffer, we applied L for imaging Zn2+ and PPi in living cells. At first, healthy HepG2 cells were incubated with 10 µM L in serum-free DMEM at 37 °C for 1 hour and washed with PBS for three times to remove the free L prior to imaging. Bright blue fluorescence of L in cells was observed (upper row of Figure 5). And the healthy cell morphology suggested that the L was suitable for living cell imaging at this concentration. After that, the cells were respectively incubated with Zn2+ at 0, 10, 20, or 30 µM for 20 minutes at 37 °C for Zn2+ imaging. Interestingly, fluorescence from the cells induced by 10 µM L could not be quenched by either 10 µM or 20 µM Zn2+ added (Figure S10, Supporting Information), suggesting that intracellular phosphates competitively bind with Zn2+. And we need 30 µM Zn2+ to effectively quench the fluorescence of L. The process of the fluorescence quenching of L by Zn2+ in cells was clearly observed (Figure S10, Supporting Information). The average FI of the HepG2 cells in Figure S9 was measured with Image J and summarized in Figure S15a. We found that 30 µM Zn2+ quenched the FI of L in cells by 14-fold (middle row of Figure 5). This property of fluorescence quenching of L by Zn2+ for cell imaging is a good supplementary to those of excellent Zn2+ probes developed by groups of Lippard and O’Halloran.46-50 After that, we incubated the 30 µM Zn2+-treated cells with PPi at 0, 30, 60, or 90 µM for another 20 minutes at 37 °C respectively for PPi imaging. Clearly we observed the fluorescence inside


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cells was gradually turned on again (Figure S11, Supporting Information). The average FI of the HepG2 cells in Figure S11 was measured with Image J and summarized in Figure S15b. The results indicated that 90 µM PPi could effectively turn on the fluorescence of the 30 µM Zn2+-treated cells by 12-fold (low row of Figure 5).

Figure 5. Differential interference contrast (DIC) images (left), and fluorescence images (right, DAPI channel) of HepG2 cells incubated with 10 µM L in serum-free DMEM for 1 hour at 37 °C, washed with PBS for three times prior to imaging (top row), incubated with 30 µM Zn2+ in serum-free DMEM for 20 minutes at 37 °C prior to imaging (middle row), then incubated with 90 µM PPi in serum-free DMEM for 20 minutes at 37 °C prior to imaging (bottom row), respectively. Scale bar: 20 µm.



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Detection of hydrogen sulfide in aqueous buffer. Sulfide is known to react with Cu2+ to form a very stable CuS solid precipitate (Ksp = 6.3 × 10−36), and the Kd of L-Cu2+ complex is calculated to be 3.0 × 10−6 M. Therefore, the L-Cu2+ complex could be used to sense hydrogen sulfide. As shown in Figure 6a, progressive addition of HS- (0-4 equiv. of L-Cu2+) into 30 µM L-Cu2+ gradually turned on the fluorescence emission at 480 nm, reaching 25-fold-increase plateau at the HS- concentration of 4 equiv. of L-Cu2+ (i.e., 120 µM). The fluorescence turn-on was ascribed to the decomplexation of the L-Cu2+ complex by HS- to yield two products of fluorophore L and CuS.

Figure 6. (a) Fluorescence spectra of 30 µM L-Cu2+ complex upon addition of different concentrations of HS- at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 120 µM respectively in HEPES buffer (10 mM, pH 7.4, 3% DMSO) at RT. Inset: The fitted calibration line in the liner region of 30~90 µM HS-. (b) Fluorescence intensity (FI) of 30 µM L-Cu2+ complex at 480 nm without or with addition of 5 equiv. of various common anions. (c) Fluorescence intensity (FI) of 30 µM L-Cu2+ complex at 480 nm without or with addition of 5 equiv. of various sulfur oxyanions and biothiols.


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By correlating the fluorescence intensity at 480 nm with the concentration of HS-, we obtained a calibration curve for the determination of HS- in aqueous buffer. A linear relationship between the fluorescence intensity and HS- concentration (Y = 7.907*X - 186.857, R2 = 0.988) was obtained over the range of 30~90 µM. LOD of HS- in this assay is 2.24 µM (S/N = 3), which is comparable to those of recently reported H2S probes (Table S2, Supporting Information).

The selectivity of L-Cu2+ complex to various anions was investigated. Negligible changes in the FI of the complex were observed in the presence of 5 equiv. various common anions such as H2PO4-, AcO-, CO32-, F-, NO3-,Cl-, Br-, or I- (Figure 6b). Interestingly, neither various sulfur oxyanions (e.g., SO32-, SO42-, S2O32-, S2O52-) nor biothiols (e.g., glutathione (GSH), cysteine (Cys), Homocysteine (Hcy)) exerted obvious changes of the FI of the complex, compared to the 25-fold increase of FI induced by HS- (Figure 6c). This indicated that L-Cu2+ complex had an excellent in vitro selectivity to HS- among other possibly competitive anions. Since GSH is the most abundant cellular thiol with intracellular concentration ranging from 1 to 10 mM,51 we further studied the FI recovery of L-Cu2+ complex by high concentrations of GSH. The results showed that, compared with 25-fold increase of the 30 µM L-Cu2+ complex by 120 µM HS-, 4 mM GSH only resulted in 4-fold increase of the fluorescence and even 10 mM GSH only resulted in 7-fold increase of the fluorescence of 30 µM L-Cu2+ (Figure S12, Supporting Information). Therefore, the L-Cu2+ complex still shows good selectivity to HS- for intracellular applications.



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Imaging Cu2+ and HS- in living cells. The potential utility of L for sensing Cu2+ and HS- in living cells was further evaluated. Healthy HepG2 cells were incubated with 10 µM L in serum-free DMEM at 37 °C for 1 hour and washed with PBS for three times to remove the free L. After that, the cells were respectively incubated with Cu2+ at 0, 10, 20, or 30 µM for 20 minutes at 37 °C for Cu2+ imaging. The process of the fluorescence quenching of L by Cu2+ in cells was also clearly observed (Figure S13, Supporting Information). The average FI of the HepG2 cells in Figure S13 was measured with Image J and summarized in Figure S15c. We found that 30 µM Cu2+ could effectively quench the FI of L in cells by 18-fold (middle row of Figure 7). After that, we incubated the 30 µM Cu2+-treated cells with HS- at 0, 60, 90, or 120 µM for another 20 minutes at 37 °C prior to imaging. Clearly we observed the fluorescence inside cells was gradually turned on again (Figure S14, Supporting Information). The average FI of the HepG2 cells in Figure S13 was measured with Image J and summarized in Figure S15d. The results indicated that 120 µM HS- could effectively turn on the fluorescence of the 30 µM Cu2+-treated cells by 17-fold (bottom row of Figure 7).


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Figure 7. Differential interference contrast (DIC) images (left), and fluorescence images (right, DAPI channel) of HepG2 cells incubated with 10 µM of L in serum-free DMEM for 1 hour at 37 °C, washed with PBS for three times prior to imaging (top row), incubated with 30 µM Cu2+ in serum-free DMEM for 20 minutes at 37 °C prior to imaging (middle row), then incubated with 120 µM HS- in serum-free DMEM for 20 minutes at 37 °C prior to imaging (bottom row), respectively. Scale bar: 20 µm.

CONCLUSIONS In conclusion, we rationally designed a pyridine-biquinoline fluorophore L for metal ions (Mn+) complexation and used the L-Mn+ complexes for effectively sensing PPi and HS- in aqueous buffer and in living cells. L could selectively coordinate with metal ions in IB and IIB groups to for L-Mn+ complexes with 1:1 stoichiometry, resulting in fluorescence quenching via PET mechanism. In aqueous buffer, PPi could competitively coordinate with the Zn2+ in the L-Zn2+ complex by forming a new



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“ate”-type complex and turn on the fluorescence again by a 21-fold-increase. LOD of this assay for the detection of PPi in aqueous buffer is 0.85 µM. Thus, we successfully applied the L-Zn2+ complex for sequential imaging Zn2+ and PPi in living cells. Due to the high stability of CuS species, L-Cu2+ complex was applied for highly selective detection of HS- with an excellent sensitivity by decomplexation-induced fluorescence turn-on. LOD of this assay for the detection of HS- in aqueous buffer is 2.24 µM, which is comparable to those of recently reported H2S probes. We also successfully applied L-Cu2+ complex for sequential imaging Cu2+ and HS- in living cells. Due to the positive correlation of the occurrence of PPi and HS- in vascular calcification, we envision herein that our multifunctional probe L might help doctors to diagnose this chronic disease more precisely in the future. For example, we could label L with radioactive isotope 64Cu to get L-64Cu complex for qualitative and quantitative positron emission tomography/computed tomography (PET/CT) imaging of in vivo HS-.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (G.-L. L.).

ACKNOWLEDGMENT This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the National Natural Science Foundation of China (Grants 21175122, 91127036, 21375121), and the Fundamental Research Funds for Central Universities (WK2060190018).


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ASSOCIATED CONTENT

Supporting Information Available

Additional experimental details as described in text. Synthesis and characterizations of 2-acetylquinoline, L, ZnCl2L, and CuCl2L. Scheme S1, S2; Figure S1-S15, Table S1, S2. This information is available free of charge via the Internet at http://pubs.acs.org/.

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