Dyes and Pigments - download.xuebalib.comdownload.xuebalib.com/bczcezTPW3t.pdf · ice-bath for 20 min. To prepare the probe 1, methyl oxalyl chloride (55 mL, 0.59 mmol) along with

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Dyes and Pigments 133 (2016) 127e131

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Dyes and Pigments

journal homepage: www.elsevier .com/locate/dyepig

A cyanine-based colorimetric and fluorescent probe for highlyselective sensing and bioimaging of phosphate ions

Bo Liu a, Huan Wang a, Dan Yang b, Rui Tan a, Rui Rui Zhao b, Rui Xu a, Zhang Jian Zhou a,Jun Feng Zhang a, *, Ying Zhou b, **

a College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming, 650500, Chinab College of Chemical Science and Technology, Yunnan University, Kunming, 650091, China

a r t i c l e i n f o

Article history:Received 18 March 2016Received in revised form18 April 2016Accepted 20 April 2016Available online 26 May 2016

Keywords:Cyanine dyeNear-infrared chemosensorPi detectionCell imaging

* Corresponding author.** Corresponding author.

E-mail addresses: [email protected] (J.Fcn (Y. Zhou).

http://dx.doi.org/10.1016/j.dyepig.2016.04.0320143-7208/© 2016 Published by Elsevier Ltd.

a b s t r a c t

This paper reports a near-infrared chemosensor for phosphate ions (Pi) based on Pi-controlled fluores-cence off-on switching mechanism. The Pi sensor displayed colorimetric responses towards Pi with adistinct color change from green to yellow in aqueous media. Apyrase, a hydrolytic enzyme, was used toaccelerate the endogenous Pi production for evaluating in vivo fluorescent sensing ability of the newcyanine based probe (1). In addition, the present study demonstrated potential of the newly developedchemosensor in bioimaging by performing experiments in Chinese hamster ovary (CHO) cells bothin vitro and in vivo.

© 2016 Published by Elsevier Ltd.

1. Introduction

Near-infrared (NIR) fluorescent probes can detect molecularactivity in vivo because of the following advantages associated withsuch probes: deep penetration of NIR photons in tissues, weakphoto-damage to biological samples, and low auto-fluorescencebackground [1]. Recently significant efforts have been successfullymade towards the use of NIR probes for bioanalytical applications[2]. With the relatively large extinction coefficient and high quan-tum yield, cyanine dye is an ideal NIR fluorophore that has beenwidely employed for NIR in vivo imaging [3].

Phosphate ions (Pi, PPi, and nucleotides) play vital roles in livingsystems. For example, pyrophosphate (PPi), being the product ofATP hydrolysis under cellular conditions, is an important target inbioenergetic and metabolic processes [4]; phosphate ion (Pi), animportant downstream metabolic product of nucleotides, playspivotal roles in energy transduction and storage in biological sys-tems [5]. However, compared with the numerous studies on PPi [6],the number of studies focusing on Pi is relatively low. Recently, a

. Zhang), [email protected].

series of reaction-based chemodosimeters, displaying highly se-lective fluorescence changes towards Pi over PPi and ATP, was re-ported [7]. In our previous study, methoxy oxalyl group has beenshown as an effective reaction site for the selective Pi sensing.Inspired by the results demonstrated by our previously developedchemodosimeters, we thought that a cyanine-based and Pi-targeted chemodosimeter with colorimetric or ratiometric NIRproperties would be highly attractive. Herein, we designed a newcyanine based probe 1 for Pi sensing and examined its Pi sensingability in vitro.

Probe 1 showed a strong NIR absorption band from 600 to850 nm and exhibited specific recognition ability of Pi among allthe tested anions and structural analogues including PPi, ATP, ADP,AMP, GTP, GDP, and GMP. Upon addition of Pi to the probe 1 solution(DMSO/HEPES ¼ 4:6, pH ¼ 7.4), a dramatic color change from deepgreen to light yellow, accompanied by an increase intensity of thefluorescence peak at 560 nm, was observed. In addition, the probewas tested for in vivo bioimaging of exogenous and endogenous Pisin Chinese hamster ovary (CHO) cells.

2. Experimental section

2.1. General procedures and materials

Unless otherwise specified, all chemicals were purchased from

Scheme 1. Synthesis route of compound 1.

B. Liu et al. / Dyes and Pigments 133 (2016) 127e131128

commercial sources and used without further purification. The 1Hand 13C NMR spectra were measured on a Bruker 500 MHz mag-netic resonance spectrometer. Chemical shifts were expressed inppm and coupling constants (J) in Hz. Mass spectrometry wasrecorded with a Xevo TQ-S mass spectrometer and a Q-TOF B.05.01mass spectrometer. The UVeVis spectra were obtained using UV-240 IPC spectrophotometer. The fluorescence spectra were ob-tained with F-4500 FL spectrometer with a 1 cm standard quartzcell. Flash chromatography was carried out on silica gel(100e200 mesh).

2.2. Synthesis of the cyanine based probe 1

The compound 2 was synthesized following a previous reportwith modification [8], and the yield was 81%. Under argon atmo-sphere, compound 2 (100 mg, 0.15 mmol) was dissolved in 30 mLanhydrous dichloromethane (DCM), and the solution was kept inice-bath for 20 min. To prepare the probe 1, methyl oxalyl chloride(55 mL, 0.59 mmol) along with triethyl amine (Et3N) (86 mL,0.59mmol) in 10mL anhydrous DCMwas added to the solution of 2dropwise. The reaction mixture was stirred at 0 �C for 1.5 h andlater kept at room temperature overnight. The solvent wasremoved under reduced pressure, and the resulting solid was pu-rified by column chromatography (silica) by eluting with ethyl ac-etate and methanol mixture (EA: MeOH,120:1). 1H NMR (500 MHz,CDCl3) d (ppm) 7.63 (d, J ¼ 14.1 Hz, 2H), 7.38 (ddd, J ¼ 21.9, 10.9,4.0 Hz, 4H), 7.24 (d, J ¼ 7.5 Hz, 2H), 7.16 (d, J ¼ 7.9 Hz, 2H), 6.30 (d,J ¼ 14.1 Hz, 2H), 4.27e4.16 (m, 4H), 3.84 (q, J ¼ 7.2 Hz, 2H), 3.63 (s,3H), 2.88e2.78 (m, 2H), 2.65e2.57 (m, 2H), 1.92 (dd, J¼ 14.8, 7.4 Hz,6H),1.69 (s, 6H),1.66 (s, 6H),1.33 (t, J¼ 7.2 Hz, 3H),1.07 (t, J¼ 7.4 Hz,6H). 13C NMR (126 MHz, CDCl3) d (ppm) 172.34, 162.10, 150.97,142.33, 142.01, 141.27, 129.04, 128.76, 125.40, 122.21, 111.05, 102.35,60.41, 53.43, 52.75, 49.28, 46.64, 45.22, 28.19, 27.83, 25.58, 21.02,19.17, 14.21, 13.70, 11.72. MS m/z 634.38 Mþ (Calcd for C41H52N3O3

þ

634.40).

2.3. UV/vis and fluorescence measurements

Stock solutions (1� 10�2 M) of the sodium salts of Pi, P2O74�, ATP,

ADP, AMP, GTP, GDP, GMP, TTP, TDP, TMP, UTP, UDP and UMP wereprepared in deionized water. Fluorescence spectra were recordedwith the slit width 5/5 nm.

2.4. Culture of CHO cells and fluorescent imaging

CHO was cultured in Dulbecco’s modified Eagle’s medium

Fig. 1. (a) Absorbance spectra of 1 (2.0 � 10�5 M) in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/GMP, TTP, TDP, TMP, UTP, UDP and UMP. (b) Absorbance intensity of 1 (2.0 � 10�5 M) at 430 nselected molecules: 1: blank, a: Pi, b: P2O7

4�, c: ATP, d: ADP, e: AMP, f: GTP, g: GDP, h: GMP

(DMEM, Invitrogen) supplemented with 10% FBS (fetal bovineserum) in an atmosphere of 5% CO2 and 95% air at 37 �C. The cellswere seeded in 24-well flat-bottomed plates and then incubated for24 h at 37 �C under 5% CO2. Then the cells were incubated with500.0 mM Pi and ATP in an atmosphere of 5% CO2 and 95% air for2 h at 37 �C, respectively. Then the cells treated with ATP wereincubation with 1 U and 1.5 U and 2 U of apyrase for 1 h, respec-tively. Then the cells were incubation with 20.0 mM 1 for 1 h. Washcells twice with 1 mL deionized water at room temperature, cellswere imaged using an Olympus BX51 inverted fluorescencemicroscopy.

3. Results and discussion

3.1. Synthesis and characterizations

Scheme 1 depicts the synthesis route for probe 1. Withcommercially available IR-780 as starting materials, a precursor 2first prepared following a previously reported method with somemodification [8]. Next, the target probe 1was easily prepared in thepresence Et3N in dry DCM by acylation of methoxy oxalyl chloridewith 2, followed by column purification. The probe 1 was charac-terized using different spectroscopic techniques such as 1H NMR,13C NMR, and ESI-MS. The detailed synthetic procedure and char-acterization of the new compound are provided in the SupportingInformation (SI).

3.2. UVevis absorption and fluorescent emission spectra

To investigate the selectivity of probe 1 for Pi ions, the spectralchanges (UVeVis and fluorescence) of 1 were monitored in thepresence of various anions (as sodium salts) in DMSO-HEPES buffer

V ¼ 4:6) before and after the addition of 250 equiv of Pi, P2O74�, ATP, ADP, AMP, GTP, GDP,

m in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6) after the addition of 250 equiv of, i: TTP, j: TDP, k: TMP, l: UTP, m: UDP n: UMP.

Fig. 2. (a) Fluorescence spectra of 1 (2.0 � 10�5 M) in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6) before and after the addition of 250 equiv of Pi, P2O74�, ATP, ADP, AMP, GTP,

GDP, GMP, TTP, TDP, UTP, UDP and UMP (lex ¼ 440 nm). (b) Fluorescence intensity of 1 (2.0 � 10�5 M) at 558 nm in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6) after theaddition of 250 equiv of selected molecules: 1: blank, a: Pi, b: P2O7

4�, c: ATP, d: ADP, e: AMP, f: GTP, g: GDP, h: GMP, i: TTP, j: TDP, k: TMP, l: UTP, m: UDP, n: UMP.

Fig. 3. (a) Absorbance titration spectra of 1 (2.0 � 10�5 M) in the presence of varying concentrations of Pi in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6). (inset) Color changesof 1 (20 mM) in the presence of PO4

3� (0, 50, 100, 150, 200 and 250 equiv) in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6). (b) Absorbance intensity of 1 (2.0 � 10�5 M) at 430 nmand 799 nm as a function of varying concentrations of Pi.

B. Liu et al. / Dyes and Pigments 133 (2016) 127e131 129

(0.02 M, pH 7.4) (V/V ¼ 4:6). Fig. 1 shows that the probe 1 initiallyexhibits a major absorption band centered at 799 nm. Among allthe tested anions, only Pi promotes a distinct color change of theprobe 1 from deep green to light yellow. As evident from Fig. 2, thecolor change is easily distinguished by the naked eyes and isassociated with a 369 nm hypochromatic shift of the maximum ofthemajor absorption band at 799 nme430 nm. Fig. 2 illustrates thatthe probe 1 weakly fluoresces at 520 nm in DMSO-HEPES buffered(0.02 M, pH 7.4) (V/V ¼ 4:6) solution. However, a significant fluo-rescence enhancement was observed at 560 nm upon addition ofPi, while in presence of other anions, such as P2O7

4�, ATP, ADP, AMP,

Fig. 4. (a) Fluorescence emission titration spectra of 1 (2.0 � 10�5 M) in the presence o(lex ¼ 440 nm). (b) Fluorescence intensity of 1 (2.0 � 10�5 M) at 560 nm as a function of

GTP, GDP, GMP, TTP, TDP, UTP, UDP, and UMP; however, negligiblespectral changes were noticed even at concentration as high as5 mM concentration (Figs. 1 and 2). This result indicates highselectivity of the probe 1 towards Pi over other anions.

To verify whether Pi can trigger amide bond cleavage and inducefluorophore release, 1 was titrated in DMSO-HEPES buffer by add-ing different equivalents of Pi and monitored the spectral changesusing UVeVis and fluorescence. Fig. 3a shows that the color of thesolution of 1 changes from dark green to yellow with increasing Piconcentration. Such colorimetric changes ensured the visualdetection of Pi in the present study. It was found that upon addition

f varying concentrations of Pi in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6)varying concentrations of Pi.

Fig. 5. Time-dependent change (A430 nm/A795 nm) of 1 (20 mM) with the addition of Pi,P2O7

4�, and ATP (250 equiv) in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V ¼ 4:6).

B. Liu et al. / Dyes and Pigments 133 (2016) 127e131130

of Pi to a buffered solution of 1, the intensity of absorptionmaximum at 799 nm decreased accompanied by appearance of anew absorbance band at 430 nm simultaneously with a ratio ofintensities (A799/A430) ranging from 937 to 1.5 (Fig. 3b). The probe1 itself was found to be very weakly fluorescent. Fig. 4 shows that Piaddition causes a dramatic increase in the intensity of fluorescenceat 560 nm. The solution was found to emit towards orange-yellowwith increasing Pi concentration. In addition, it was observed thatthe increase in the absorbance caused by the addition of Pi to 1waslinearly proportional to Pi concentration in the 0e2.51 mM range(Fig. S1). The detection limit was calculated as 9.37 � 10�7 M from3s/k, where s is the standard deviation of a blank measurement,

Fig. 6. Fluorescence imaging (top and middle) and phase contrast (bottom) for Pi in CHO ce(5 mM); (c, h) probe 1 (20 mM), ATP (5 mM) and apyrase (1 U); (d, i) probe 1 (20 mM), ATP (5emission from the green channel; (fej): emission from the red channel. (For interpretation oof this article.)

and k is the slope of the plot of fluorescence intensity vs. Piconcentration.

3.3. The reaction mechanism of compound 1 with Pi

The sensing mechanism is based on the cleavage ester bond of 1in presence of Pi, as shown in Scheme S1. This mechanism is inaccordance with results obtained in our other works [9], however,we found that compound 2 in present paper is not very stable likeother reported ones, because of the cyanine dyes in it. To establishthis mechanism further, mass spectrometry (TOF-MS) study wasperformed in the mixture of 1 with 250 equiv of Pi and 2 with 250equiv of Pi (both in DMSO-HEPES buffer (0.02 M, pH 7.4), (V/V ¼ 4:6)) after stirring for 1 h (Figs. S6 and S7). It showed peaks atm/z 375.2791 and 261.0848, which are attributed to the hydroly-sates of 2. The tests of optical spectra of 2 and 2with Pi also gave thesupports to the deduction (Figs. S8 and S9). That is compound 1reacted with Pi to give compound 2, which later hydrolyzed in thepresence of Pi. Additionally, the time-dependent absorbancespectra of 1 in the presence of Pi, PPi and ATP in DMSO-HEPESbuffer (0.02 M, pH 7.4) (V/V ¼ 4:6) were recorded. Among thethree tested anions, only Pi was found to lead to an increase of theA430/A799 ranging from 0.01 to 0.56, which reached themaximumand stopped within 6 min (Fig. 5). This indicates that the sensingreaction might complete in a few minutes with high selectivity,allowing a real-time in vivo Pi imaging study.

3.4. Imaging Pi in CHO cells

Prompted by its high sensitivity and selectivity, the probe 1wastested for practical application of the endogenous and exogenous Pifluorescent sensing in CHO cells. Fluorescent imaging was carried

lls treated with 1 and apyrase. (a, f) probe 1 (20 mM) only; (b, g) probe 1 (20 mM) and PimM) and apyrase (1.5 U); (e, j) probe 1 (20 mM), ATP (5 mM) and apyrase (2 U). (aee):f the references to colour in this figure legend, the reader is referred to the web version

B. Liu et al. / Dyes and Pigments 133 (2016) 127e131 131

out using CHO cells incubated with 20 mM 1 (e) or with 5mM Pi (e)or with 5 mM ATP and different amount of apyrase (Fig. 6cee, h-j).As the emission red-shifted from 520 nm to 560 nm after theaddition of Pi, the emissions were recorded in green and redchannels at the same time. Apyrase, a hydrolytic enzyme, convertsboth ATP and ADP into AMP and Pi [10]. In the present study, theenzyme was used to accelerate the endogenous Pi production forevaluating the fluorescent sensing ability of 1 for in vivo imaging.Fig. 6aef shows that the CHO cells are weakly fluorescent whentreated with 1 alone. However, the fluorescent intensity increasessharply with the addition of exogenous Pi, as evident fromFig. 6beg. In the second group, the cells were first treated with 1and ATP, followed by different amounts of apyrase. Fluorescenceintensities in both green channel and red channel were increasedwith increasing apyrase concentration, and the strongest fluores-cence appeared in presence of 2 U apyrase in all the tested samples(Fig. 6eej). Importantly, compared with the fluorescent intensity ingreen channel, the fluorescence in red channel was much brighterin each case. This suggests that the emission at 558 nm is the mainemission, which is consistent with results obtained in the in-vitroexperiments. Thus, the current study demonstrated the change-inducing process of Pi occurring inside CHO cells through directfluorescent evidence using the newly developed chemodosimeter1.

4. Conclusions

This paper introduces a new strategy to design a NIR functionalprobewith Pi-controlled fluorescence off-on switchingmechanism.As predicted, the results showed that Pi promoted ester bondcleavage in 1, thus liberating the fluorophore, accompanied by ablue shift in themain absorptionmaximum from 799 nm to 430 nmand up to a 30-fold enhancement in the fluorescence intensity at560 nm. The fluorescence imaging studies in CHO cells demon-strated that 1 can detect exogenous and endogenous Pi in vitro.Therefore, cyanine based probe 1 has potential to be used as NIRfunctional probe for colorimetric and fluorescent Pi sensing withsignificant responses both in vitro and in vivo.

Acknowledgements

This work was supported by the Natural Science Foundation ofChina (21262045, 21262050, 21302165, 21462050 and 41561108),the Foundation of the Department of Science and Technology ofYunnan Province of China (2013HB062, 2014HB008), and theFoundation of the Department of Education of Yunnan Province ofChina (2015z062).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.dyepig.2016.04.032.

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