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Sensors and Actuators B 203 (2014) 833–847

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo ur nal home page: www.elsev ier .com/ locate /snb

ed turn-on fluorescent phenazine-cyanine chemodosimeters foryanide anion in aqueous solution and its application for cell imaging

in Yanga, Xin Lib, Yi Quc, Weisong Qua, Xiao Zhanga, Yandi Hanga,ans Ågrenb, Jianli Huaa,∗

Laboratory for Advanced Materials and Institute of Fine Chemicals, 130 Meilong Road, East China University of Science and Technology, Shanghai 200237,hinaDivision of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal Institute of Technology, SE-10691 Stockholm, SwedenDepartment of Chemistry and Laboratory of Advanced Materials, Fudan University, 220 Handan Road, Shanghai 200433, China

r t i c l e i n f o

rticle history:eceived 23 May 2014eceived in revised form 4 July 2014ccepted 12 July 2014vailable online 21 July 2014

eywords:luorescent chemodosimeteryanidehenazine-cyanine

a b s t r a c t

Two chemodosimeters PDMI and PMI for cyanide detection were designed and synthesized based onphenazine-cyanine dyes with N-methyl indolium group as receptor unit. According to the specific reac-tivity of indolium C N+ bond against cyanide anion, both of them featured high sensitivity with detectionlimit of 1.4 �M and 200 nM, respectively, and high selectivity against other anions. The quenching effecton phenazine-cyanine fluorophore by strong intramolecular charge transfer (ICT) from phenazine donorto indolium receptor made both PDMI and PMI non-emissive at the original state. After addition ofcyanide, the ICT effect decreased and vanished leading to dramatic “off–on” fluorescence enhancement.PDMI which proceeded bilateral electrophilic reaction toward cyanide anion provided an emission sig-nal at 580 nm in HEPES buffer with naked-eye detectable color change. Probe PMI utilized an unreactive

ell imagingear-infrared

formyl group instead of one reactive N-methyl indolium group as the electron-withdrawing compo-nent. Due to the unilateral recognition process for cyanide the ICT orientation of PMI was redirectedthus exhibited fluorescence enhancement with maximum emission at 630 nm. Meanwhile, PMI wasapplied for monitoring intracellular cyanide in Hela cells and proved to achieve “off–on” fluorescentsignal confirmed by confocal laser scanning microscopic imaging.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Cyanide recognition has been widely concerned according tohe detrimental toxicity of cyanide substances to animals anduman beings as well as the widespread application in industrialroduction [1–3]. Therefore, chemosensors and chemodosimeterspplied for detection of cyanide have been attracting considerablettention and quickly developed in cyanide assay [4,5]. Com-ared to traditional methods, chemosensors and chemodosimetersre characterized by their better selectivity against other anions6–8], sensitivity with much lower detection limit [9–11], control-able structures satisfied for different environmental requirements12–14] and possibilities for bioanalysis [15,16].

For design of a cyanide sensor, the special properties of cyanidenion must be firstly considered [17–19]. Strong hydrogen bond-ng affinity [20], lewis acid–base binding effect [21–26] and

∗ Corresponding author. Tel.: +86 21 64250940; fax: +86 21 64252758.E-mail address: jlhua@ecust.edu.cn (J. Hua).

ttp://dx.doi.org/10.1016/j.snb.2014.07.045925-4005/© 2014 Elsevier B.V. All rights reserved.

nucleophilic reactivity [27–32] are the most important charac-ters of cyanide anion and decided the kind of receptors. Recentyears, compared to the hydrogen bond donor–receptor and lewisacid–base pair recognition, cyanide chemodosimeters based on thenucleophilic reaction have attracted increasing attention [33–37],because specificity of nucleophilic reaction provides higher sensi-tivity and excellent selectivity leading to more reliable assay resultsespecially for luminescent bioimaging and in vivo/vitro analysis[38–41].

The second point for molecular design of chemosensor andchemodosimeter is the structure of fluorophore which directlyrelate to the photochemical mechanism of recognition and thesignal processing. “Off–on” fluorescence sensors always obtainedmore advantages over “on–off” type, because of their higher signal-to-noise ratio and more recognizable optical changes suitable fornaked-eye observation [42–44]. In consideration of cyanide analy-

sis in biosample, red to near infrared (NIR) emission is preferableto ultra-visible range for the low phototoxicity to living cells andthe negligible autofluorescence interference [45,46]. Large stokesshift is also preferred for anti-interference of incident light [47].

8 Actuat

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34 L. Yang et al. / Sensors and

A variety of fluorophores have been applied in constructingyanide sensor, such as coumarin [48–50], BODIPY [51–53] andxazine [54,55], but their green to yellow emission wavelength450–550 nm) limited the application in bioimaging. Cyanine [56]nd hemicyanine [57] dyes were also applied, however, few of themisplayed “off–on” fluorescence emission [58–62]. Water-solubilityf the sensor compound is always quitely concerned for molecu-ar design of a cyanide sensor in aqueous solution or living cells,ecause solubility greatly affected the reaction rate and cell per-eability which definitely influenced the response time [63–66].Recently, we have reported a sensing platform for cyanide

ased on phenazine derivatives realizing near infrared ratio-etric emission profile and nanomolar detection limit [67]. The

henazine fluorophore possessed large stokes shift with excita-ion wavelength at 540 nm and emission at 730 nm due to thetrong intramoleculer charge transfer process. However, their rel-tive sparing solubility in water lowered the response rate inater-containing solution and thus limited their application in

ell imaging. In this paper, vinyl-indolium moiety was introducedo phenazine skeleton replacing the former used dicyano-vinylroup as receptor for cyanide anion. Phenazine-cyanines PMI andDMI were designed as shown in Fig. 1. Short N-alkylchain kepthe solubility of phenazine in organic solvent, at the same time,he vinyl-indolium moiety largely enhanced their water-solubility.ompared to PDMI with two indolium moieties on both sides ofhenazine, the unilateral compound PMI showed largely enhancedmission around 560–800 nm and realized “off–on” signaling in cellmaging.

. Results and discussion

.1. Molecular design and synthesis

Dicyano-vinyl group was an effective receptor for cyanidenion since the strong electrophilic reactivity toward nucleophiles.owever, according to our former report, when dicyano-vinylas introduced to phenazine fluorophore, sensor application was

imited for the reluctant solubility in water solution [67]. Tomprove the solubility as well as maintaining the electrophilic reac-ivity, indolium group came into view and took place the rolef dicyano-vinyl as cyanide receptor. Via Knoevenagel reaction,,2,3,3-tetramethyl-3H-indol-1-ium iodide (4) was connected tohenazine fluorophore (3) through C C double bond. The indoliumroup has stronger electron-withdrawing effect because of theitrogen atom of positive charge and provided an electron accep-or. Meanwhile, as shown in Scheme 1, the presence of n-butylroups linked to nitrogen atoms of phenazine changed the initiallectron-deficient phenazine group into an electron-rich conju-ated donor group. Therefore, intramolecular charge transfer (ICT)appened from alkyl phenazine donor to the indolium acceptor.ifferent from the acceptor-donor-acceptor (A-D-A) structure ofDMI with two indolium groups on both sides of phenazine, theetained carbaldehyde moiety on PMI made an acceptor-donor-nother acceptor (A-D-A′) structure with two different routes ofCT process. Cyanide attack toward C N+ electrophilic double bond

as expected to reduce the electon-withdrawing effect of indoliumroup and disturb the ICT process due to the gradually destruc-ion and neutralization of the C N+ double bond, at the same time,oming out with corresponding optical changes.

Before nucleophilic addition of cyanide anion toward thehemosensors, PMI and PDMI were at the “off” state without emis-

ion around the visible and near-infrared wavelength, because thetrong ICT effect of A-D-A or A-D-A′ from phenazine donor (D) to thendolium acceptor (A) widely quenched their fluorescence emissionFig. 2 and theoretical calculation). Taken in this sight, the indolium

ors B 203 (2014) 833–847

group played the crucial role of fluorescence quencher functionalunit. After addition of cyanide anion, the conjugated C N+ doublebond was neutralized into C N single bond, disrupting the mainstructure of indolium quencher group thus impaired the quench-ing effect. Along with the reaction of cyanide with sensor, thephenazine fluorophore recovered its emission and the optical signalshifted into the “on” state (Fig. 2).

In PDMI, the conjugate bonds on both sides were disturbed bycyanide addition toward the indolium C N+ bond. The signalingunit was alkylated phenazine subject with conjugated double bondon each sides. On the other hand, sensing process of PMI was differ-ent from that of PDMI, because the unsymmetrical A-D-A′ systemwith unilateral receptor (Scheme 1, Figs. 12 and 13). Under neutralconditions with pH at 7.4, carbaldehyde substitute on cycloben-zene were not an ideal receptor for cyanide although the carbonylgroup has relative electrophilic reactivity [68]. The fluorophore inPMI of which the emission was originally quenched off by indoliummoiety was a carbaldehyde phenazine with one conjugated doublebond on the other side. Formyl group produced relatively weakerintramolecular charge transfer (ICT) effect, thus bring in an obviousbathochromic shifted emission into red region than PDMI. This redsignaling profile made PMI into a promising sensing platform forapplication in cell imaging.

The synthesis of PMI and PDMI both started from reductionand alkylation of phenazine, derivatives of which are common innature products and act important role in biosysthesis (Scheme 1).And then formylation of the side phenyl rings were conductedthrough Vilsmeier–Haack reaction. After Knoevenagel reactionwith methyl indolium iodide, the desirable products were obtained,respectively. The structures were confirmed by 1H and 13C NMRspectroscopy and MS-HRMS analysis. The details of synthesis andthe structural identification spectra were formulated in the sup-porting information.

2.2. Optical response of PDMI toward cyanide anion

We first explored optical response of PDMI by monitoring thechanges in the absorption and fluorescence spectra. PDMI exhib-ited maximum absorption band centering at 716 nm and anothermain absorption band at 426 nm (Fig. 4). However, no fluorescenceemission was found in PDMI. In order to profile the reactivity ofPDMI toward cyanide, time dependent absorption spectra of PDMIwith cyanide were firstly examined by adding 3, 5 and 10 equiva-lents of cyanide anion into PDMI solution during 1 h, respectively(Fig. 3, Fig. S10). As the reaction progressed, the absorbance at both716 nm and 426 nm gradually decreased. According to the timedependent absorbance bleaching efficiency at 716 nm, the reac-tion nearly reached equilibrium with 10 eq. CN− after 30 min (seegreen down triangular curve in Fig. 3b). This indicated the suit-able electrophilic reactivity of indolium toward cyanide in buffersolution.

Furthermore, the titration experiment of PDMI was carried outby added 0.0–6.0 equivalents of CN− in sensory system. Two mainabsorption bands at 716 nm and 426 nm were used to monitorthe changes of spectra both of which decreased gradually withobviously naked-eye recognized color change from deep green tolight yellow (inset in Fig. 4b). As shown in Fig. 4, the absorbancebleaching efficiency increased and plateaued after 5 equiv. of CN−

was added.The fluorescence change was a bit different from the absorbance

response. Very weak emission signal can be observed respon-ding to the beginning two equivalents of cyanide which indicated

that when such amount of cyanide was added, only one side ofindolium receptor participated into the addition reaction and thesensory system was still at quenching state. After 3.0 equivalents,the emission intensity at 580 nm quickly enhanced due to the

L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847 835

Fig. 1. Molecular structures of PDMI and PMI.

Scheme 1. The synthesis procedures of PMI and PDMI and expected recognition toward cyanide anion; (i) Na2S2O4, EtOH, H2O, reflux, 2 h; (ii) 1-iodobutane, sodiumhydroxide, TBAB, DMSO, H2O, r.t., 5 h; (iii) phosphorus oxychloride, DMF, ice bath to 80 ◦C, 7 h; (iv) piperidine, acetonitrile, reflux, 8 h; (v) ammonium acetate, acetic acid,reflux, 8 h.

Fig. 2. Normalized fluorescence emission spectra of PMI (a) and PDMI (b) before and after reaction with cyanide. �Ex. = 530 nm (a), 425 nm (b). Inset: correspondingphotographs of PMI and PDMI, both in HEPES buffer/DMSO (7:3, v/v, pH 7.4) solution under a UV lamp at 365 nm.

836 L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847

Fig. 3. (a) Time-dependent absorption spectra of PDMI with 10.0 equivalents of cyanide; (b) bleaching efficiency plots of absorbance at 716 nm against reaction time with3.0, 5.0 and 10.0 equivalents of cyanide anion at 37 ◦C. PDMI (10−5 M) was in HEPES buffer/DMSO (7:3, v/v, pH 7.4). Cyanide anion was dissolved in distilled water astetrabutylammonium salt and added by microsyringe.

F 3, v/v)a tion b3

rlm

af

F�p

ig. 4. (a) Absorption titration spectra of PDMI (10−5 M) in HEPES buffer/DMSO (7:bsorbance at 716 nm against cyanide equivalents. Inset: photographs of PDMI solu0 min after addition.

eaction conducting on both side of PDMI and reached the equi-ibrium point after 7.0 equivalents addition. As a result, the

acroscopic yellow fluorescence was obtained in Fig. 5b.

To further examine the sensitivity of PDMI toward cyanide

nion, smaller range of titration experiment was conductedrom 0.0 to 1.9 equivalents (0.0–19.0 �M) of cyanide. A linear

ig. 5. (a) Titration fluorescence spectra of PDMI (10−5 M) in HEPES buffer/DMSO (7:3,

Em. = 580 nm; (b) corresponding fluorescence emission enhancement at 580 nm againhotograph of PDMI before and after addition of cyanide under a UV lamp with 365 nm i

, pH 7.4 upon addition of 0.0–6.0 equivalents of cyanide; (b) bleaching efficiency ofefore and after addition of cyanide under natural light. Each sample was recorded

correlation plot of absorbance at 716 nm against cyanide concentra-tion ranging from 0.0 to 19.0 �M was obtained from the absorbancetitration spectra which indicated a detection limit of 1.4 �M, less

than the maximum contaminant level (MCL) for cyanide in drinkingwater at 0.2 ppm (8.46 �M) [69] set by the United States Envi-ronmental Protection Agency (EPA). Corresponding fluorescence

v/v, pH 7.4) upon addition of 0.0–8.0 equivalents of cyanide anion, �Ex. = 425 nm,st equivalents of cyanide. The time interval of titration tests was 30 min. Inset:ncident light.

L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847 837

F −5 O (7:3c ide cos

ePcibatdt5eg

twnrcieaCtF

2

Pt

ig. 6. (a, b) Absorption titration spectra of PDMI (10 M) in HEPES buffer/DMSorresponding linear fitted plot of change ratio of absorbance at 716 nm against cyanegmental linear fit of intensity change plot at 580 nm.

mission enhancement profile was also provided. However, sinceDMI expressed very weak emission response to small amount ofyanide below 10 �M which is vulnerable to the instrument noisenterference, the advantage of fluorescence signal which shoulde more sensitive has been partially hidden. On the other hand,s shown in Fig. 6d, the crossing point at about 1.0 equivalent ofwo linear correlation lines verified the two steps reaction proce-ure. Before 1.0 equivalent of cyanide, the reaction mostly occurredoward one side of PDMI, the fluorescence intensity growth at80 nm was very slow. After more cyanide participation, the lin-ar slope became much larger and the intensity at 580 nm quicklyrew up.

The selectivity of PDMI was investigated to measure its applica-ion ability. As a reaction based chemosensor, major interferencesere generally coming from other anions which also possessucleophilic reactivity. Fortunately, due to the specific electrophiliceactivity of PDMI toward cyanide, identical response of PMI foryanide was obtained against other interference anions. Changesn absorption and fluorescence spectra caused by CN− (3.0 and 10.0quivalents) and miscellaneous interference anions (20.0 equiv-lents) including F−, Cl−, Br−, I−, OH−, PO4

3−, HPO42−, H2PO4

−,O3

2−, SO42−, NO3

−, SCN−, AcO−, HSO3−, NO2

− and HCO3− with

heir Na+ or K+ salts in aqueous solutions were recorded shown inig. 7a as well as the actual images in Fig. 7c.

.3. Optical response of PMI toward cyanide anion

After investigation and verification of the optical response ofDMI toward cyanide, PMI with one indolium receptor was syn-hesized. This molecular design was carried out to avoid the

, v/v, pH 7.4) upon addition of 0.0–1.9 equivalents (0.0–19.0 �M) of cyanide andncentration (0.0–19.0 �M); (c, d) correlated fluorescence spectra and corresponding

non-emissive response vulnerability of PDMI against trace amountof cyanide.

Firstly, we also tested time dependent absorption spectra of PMItoward cyanide by adding 1.0, 2.0 and 5.0 equivalents of cyanideinto PMI (10−5 M) in HEPES buffer/DMSO (7:3, v/v, pH 7.4), respec-tively (Fig. 8 and Fig. S11). Different from PDMI, PMI exhibited twomajor absorption bands centering at 670 nm and 416 nm. Solutioncolor was still shown in dark green. A shoulder absorption bandaround 470 nm was also observed. As shown in Fig. 8b, by adding5.0 equivalents of cyanide anion, the absorbance change rate at670 nm obviously decreased after 30 min (1800 s), and plateauedafter 50 min (3000 s) which indicated the similar reactivity of PMIwith one receptor compared to PDMI with two receptors. The solu-tion color changes from dark green into light yellow similar as PDMIand also can be detected by naked-eye. The remained aldehydegroup did not affect the reaction rate which therefore can be usedfor realizing a longer wavelength emission response.

We next studied the titration spectra of PMI against graduallyaddition of cyanide. The absorption titration spectra were recordedand the absorbance change ratio against equivalence of cyanide wasplotted in Fig. 9. The absorbance at 670 nm gradually decreased asthe cyanide concentration increased. At the same time, the absorp-tion band at 416 nm (Fig. 9a) slightly declined as well. The bleachingcurve leveled off after 2.6 equivalents of cyanide and graduallyreached the saturation point of 3.5 equivalents.

As expected, one-side indolium acceptor almost quenched

the emission of phenazine based fluorophore. Upon titration ofcyanide anion, the initially non-emissive PMI solution exhibited an“off–on” fluorescence signal around the wavelength at 560–800 nmwith nearly 8-fold intensity enhancement at the maximum

838 L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847

F HEPESe p: bl

eocp

F2

ig. 7. (a, b) Absorption and fluorescence emission spectra of PDMI (10−5 M) in

quivalents of other various anions; (c) corresponding images under 365 nm UV lam

mission wavelength of 630 nm due to recovering of the emission

f formyl-dialkylphenazine fluorophore. Intensity–concentrationhange was plotted as shown in Fig. 10b. The unilateral reactionroperty greatly optimized the intensity plot into a linear fitted

ig. 8. (a) Time-dependent absorption spectra of PDMI with 5.0 equivalents of cyanide; (.0 and 5.0 equivalents of cyanide anion at 37 ◦C. PDMI (10−5 M) was in HEPES buffer/DM

buffer/DMSO (7:3, v/v, pH 7.4) toward 3.0, 10.0 equivalents of cyanide or 20.0ank, with 10.0 equivalents, with 20.0 equivalents of other anions.

correlation against cyanide concentration with a first placid then

acute slope variation. After addition of 3.0 equivalents of cyanide,the plot plateaued which indicated the saturation point in accor-dance with the absorbance change plot at 670 nm. Benefiting from

b) bleaching efficiency plot of absorbance at 670 nm against reaction time with 1.0,SO (7:3, v/v, pH 7.4).

L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847 839

F , v/v, pa tion b3

tbfiot

aaHan

1

cw

2

twcsaWqtt

Fca

ig. 9. (a) Absorption titration spectra of PMI (10−5 M) in HEPES buffer/DMSO (7:3bsorbance at 670 nm against cyanide equivalents. Inset: photographs of PMI solu0 min after addition.

he unilateral receptor structure, PMI expressed better sensitivityased on the emission spectra than PDMI. According to the lineartting calculation, a much lower detection limit reaching 200 nMf PMI than that of PDMI was obtained, which is much lower thanhe MCL set by EPA.

PMI also exhibited high selectivity against other interferencenions. As shown in Fig. 11 and Fig. S12, miscellaneous interferencenions (20.0 equivalents) including F−, Cl−, Br−, I−, OH−, PO4

3−,PO4

2−, H2PO4−, CO3

2−, SO42−, NO3

−, SCN−, AcO−, HSO3−, NO2

−

nd HCO3− with their Na+ or K+ salts in aqueous solutions could

ot cause significant optical response from PMI.For investigation into recognition mechanism of PDMI and PMI,

H NMR and HRMS titration were conducted and further proved theyanide nucleophilic addition mechanism. Theoretical calculationas used to support the spectra changes.

.4. 1H NMR and HRMS titration

As a result of two reactive C N+ double bond receptors respec-ively located on each side, the 1H NMR spectral change of PDMIas more complicated than that of PMI. As shown in Fig. 12,

yanide addition made all the peaks around lower aromatic fieldhift to higher field. This phenomenon indicated gradually dis-ppearance of the original strong electron-withdrawing effect.

ith cyanide added bit by bit, the electron-withdrawing effect

uickly decreased and finally vanished, which induced the shif-ing of aromatic protons around the chemical shift of 8.2–6.7 ppmoward high field of NMR spectrum. After 2.0 equivalents, the

ig. 10. (a) Fluorescence titration spectra of PMI (10−5 M) in HEPES buffer/DMSO (7:3, v/orresponding fluorescence emission intensity at 620 nm against equivalents of cyanide. Tddition of cyanide under a UV lamp with 365 nm incident light.

H 7.4) upon addition of 0.0–3.5 equivalents of cyanide; (b) bleaching efficiency ofefore and after addition of cyanide under natural light. Each sample was recorded

peaks of aromatic protons carried no longer obvious changes andfinally located at 7.2–6.1 ppm. Therefore, all peaks of the aro-matic protons upfield shifted. Particularly, the resonance peaks ofHa (ı = 8.17 ppm, J = 15.8 Hz) and Hb (ı = 7.24 ppm, J = 15.9 Hz) ofthe cis-ethylene double bond dramatically shifted upfield to H′

a(ı = 6.78 ppm, J = 16.4 Hz) and H′

b (ı = 6.13 ppm, J = 16.0 Hz). Mean-while, because of neutralization of the adjacent positive charge,the impact on chemical shift of indolium methyl protons was thegreatest of all. The single peak of the indolium methyl protons Hc

at 4.03 gradually disappeared and a new single peak correspondingto reduced indole methyl proton H′

c at 2.71 emerged at the sametime which is a common chemical shift of N–C–H proton (Fig. S14).Appearance of the tetrabutyl protons of TBA+ (tetrabutyl ammo-nium) illustrated the formation of PDMI-2CN which was furtherconfirmed by the 13C NMR and HRMS analysis.

Further structure identification of PDMI-2CN adduct was con-ducted by 13C NMR as shown in Fig. S15. The newly-presented peakat 80.6 ppm represented the quaternary carbon atom of the five-membered ring of reacted indole moiety and indicated the additiontoward C N+ double bond by cyanide anion. In the ESI-TOF high res-olution mass spectroscopy analysis (HRMS), molecular ion peaksof PDMI performed at m/z = 331.2136 (calcd. = 331.2174) as halfof the original exact molecular mass for the existence of doublecharge (Fig. S9). From the MALDI-TOF analysis spectra, the molec-

ular ion peak of [M−H]2+ at m/z = 661.4359 (calcd. = 661.4348) wasobviously confirmed (Fig. S17). After cyanide addition, the productpeak of PDMI-2CN appeared at m/z = 714.4452 (calcd. = 714.4410).In addition, the peak at m/z = 688.4327 corresponding to unilateral

v, pH 7.4) upon addition of 0.0–3.5 equivalents of cyanide anion, �Ex. = 530 nm; (b)he time interval of titration was 30 min. Inset: photograph of PMI before and after

840 L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847

Fig. 11. (a) Fluorescence spectra of PMI with 3.0 equivalents of cyanide and 20.0 equivalents of miscellaneous interference anions; (b) fluorescence anti-interferenceexperiment for PMI: blue bars represent PMI with various anions (blank or 20.0 equiv. of various anions); red bars represent following addition and reaction with 3.0 equiv.C − −5 spondt

rmbnw

ticteJ(ttiaaeo

N for 1.0 h. PMI (10 M) was in HEPES buffer/DMSO (7:3, v/v, pH 7.4); (c) correhis figure legend, the reader is referred to the web version of the article.)

eactive product PDMI-CN (calcd. = 688.4379) was also found in theass spectra. This product further proved and explained the side

y side addition reaction of cyanide toward PDMI and the relativeon-emissive optical response when only one equivalent of cyanideas added.

PMI possessed a unilateral receptor which also induced elec-rical deshielding effect on the aromatic donor group. As shownn Fig. 13, 1H NMR spectrum of PMI before addition of cyanidelearly showed the chemical shifts of single peak of aldehyde pro-on (ı = 9.63 ppm), phenazine ring (ı from 7.83 to 6.60 ppm) and thethylene protons Hd (ı = 8.15 ppm, J = 15.6 Hz) and He (ı = 7.19 ppm,

= 15.6 Hz). The characteristic peak of indolium methyl protonsHf) located at 4.02 ppm as a single peak without coupling split-ing. That was lower than ordinary N–C–H chemical shift dueo the strong electron inducing effect from the positive chargedndolium group. After addition by 1.6 equivalents of cyanide

nion, all peaks of the aromatic protons showed upfield shiftnd located around 6.12–7.17 ppm. The initial peaks of ethyl-ne protons Hd and He disappeared, replacing by new groupsf peaks of Hd′ (ı = 6.78 ppm, J = 16.1 Hz) and He′ (ı = 6.14 ppm,

ing images under 365 nm UV lamp.(For interpretation of the references to color in

J = 16.1 Hz). Similar as PDMI, the N+-methyl proton of PMI was at4.02 ppm. After reaction, the N-methyl protons’ resonance signalof PMI-CN emerged at 2.70 ppm and was still a single peak. Thenegligible change of aldehyde proton (Hg) chemical shift provedthat cyanide addition reaction took little effect on the aldehydegroup.

The structure of PMI-CN was also confirmed by 13C NMR spec-trum. As shown in Fig. S16, PMI possessed quaternary carbonatom of indolium five-membered ring was at 179.6 ppm (Fig. S5)due to the deshielding effect by positive charged C N+ doublebond. After addition by cyanide, the unsaturated quaternary carbonwas changed into a saturated one with an upfield shifted peak at80.6 ppm. Meanwhile, similar as the 1H NMR spectra, the aldehydecarbon at 190.1 was nearly not affected. ESI-TOF and MALDI-TOFHRMS analysis were also used to further confirm the reaction pro-cess. As shown in Fig. S18, the molecular ion peak at m/z = 506.3166

(calcd. = 506.3171) corresponded to PMI without cyanide. And thepeak at m/z = 532.3298 (calcd. = 532.3202) represented the adductPMI-CN. Jobs’ Plot of PMI toward cyanide anion was also conductedand proved the 1:1 reaction adduct (Fig. S13).

L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847 841

F um wH

2

wmaP

4Tas

TClP

a

C

ig. 12. 1H NMR spectra of PDMI upon addition of cyanide in DMSO-d6. Each spectra/a′ (from 8.17 ppm to 6.78 ppm) and Hb/b′ (from 7.24 ppm to 6.14 ppm).

.5. Theoretical calculations

To gain further insight into the cyanide-sensing mechanism,e employed density functional theory (DFT) calculations to opti-ize the ground state geometries of compounds PDMI and PMI,

nd corresponding adducts with cyanide anion, PDMI-CN andMI-CN.

The absorption spectrum of compound PDMI has two bands at

28 nm and 716 nm, respectively, which are well reproduced byD DFT calculations with an error of around 0.1 eV (Table 1). Thebsorption band at 716 nm corresponds to HOMO → LUMO tran-ition or charge-transfer from dihydrophenazine to cyanine dye,

able 1omputed absorbance and emission wavelength, oscillator strengths and molecu-

ar orbital (MO) compositions for the low-lying excited singlet states of compoundDMI and PDMI-CN.

State Eabs/em (eV)c �abs/em (nm)d fe MOf

PDMI-S1a 1.84 674 0.000 H − 0 → L + 1 (97%)

PDMI-S2a 1.85 671 1.811 H − 0 → L + 0 (99%)

PDMI-S3a 2.98 415 1.551 H − 1 → L + 1 (90%)

PDMI-CNb 2.12 584 0.000 H − 0 → L + 0 (98%)

a The low-lying excited singlet state of PDMI.b The first excited singlet state of PDMI-CN.c Computed absorbance energy of PDMI or computed emission energy of PDMI

nd PDMI-CN.d Computed absorbance wavelength of PDMI or emission wavelength of PDMI-N.e Oscillator strengths.f Molecular orbital compositions.

as recorded 10 min after addition. The arrows indicated change of chemical shift of

while the absorption band at 428 nm arises from local excitationswithin the central dihydrophenazine or peripheral cyanine dye(Fig. S19). As shown in Table 1 and Fig. 14, the HOMO → LUMOtransition of PDMI-CN at the first excited single state geometryis largely localized at the dihydrophenazine core. Although theS1 → S0 transition show zero oscillator strength, it can possibly bor-row intensity from the S2 → S0 transition through Herzberg–Tellercoupling [67], resulting in the experimentally observed fluores-

cence at around 567 nm. The observed fluorescence of PDMI-CNis in higher energy than that of PMI-CN, since no charge-transfer from dihydrophenazine to aldehyde is present (Table 2).

Table 2Computed absorbance and emission wavelength, oscillator strengths and molecularorbital (MO) compositions for the low-lying excited singlet states of compound PMIand PMI-CN.

State Eabs/em (eV)c �abs/em(nm)d fe MOf

PMI-S1a 1.81 684 0.709 H-0 → L + 0 (97%)

PMI-S2a 2.71 457 0.522 H-0 → L + 1 (94%)

PMI-S3a 3.02 410 0.907 H-1 → L + 0 (97%)

PMI-S1b 1.45 852 0.777 H-0 → L + 0 (98%)

PMI-CNb 1.97 628 0.062 H-0 → L + 0 (97%)

a The low-lying excited singlet state of PMI.b The first excited singlet state of PMI and PMI-CN.c Computed absorbance energy of PMI or computed emission energy of PMI and

PMI-CN.d Computed absorbance wavelength of PMI or emission wavelength of PMI and

PMI-CN.e Oscillator strengths.f Molecular orbital compositions.

842 L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847

F m waH tons Ht

Mlra

ig. 13. 1H NMR spectra of PMI upon addition of cyanide in DMSO-d6. Each spectrud/d′ (from 8.15 ppm to 6.78 ppm), He/e′ (from 7.19 ppm to 6.14 ppm), N-methyl pro

o 9.54 ppm).

oreover, the emission from the PDMI-CN compound exhibits

ower energy compared with the 1:2 adduct of the previouslyeported dihydrophenazine-based cyanide sensor and cyanidenions [67], since the �-conjugation of the dihydrophenazine core

Fig. 14. Contour plots of frontier molecular orbitals of compound PDMI at the gro

s recorded 10 min after addition. The arrows indicated change of chemical shift off/f′ (from 4.02 ppm to 2.70 ppm) and aldehyde proton peak of Hg/g′ (from 9.63 ppm

in PDMI-CN is augmented by the two C C double bond substitut-

ions.

The computed absorption wavelengths, oscillator strengths andmolecular orbital (MO) compositions for the low-lying excited

und state geometry and PDMI-CN at the first excited singlet state geometry.

L. Yang et al. / Sensors and Actuators B 203 (2014) 833–847 843

e gro

ssab

FH3t

Fig. 15. Contour plots of frontier molecular orbitals of compound PMI at th

inglet states of compound PMI are listed in Table 2. We canee that the experimentally observed absorption peaks at 670 nmnd 416 nm and shoulder absorption band at around 470 nm haveeen nicely reproduced by TD DFT calculations. In particular, the

ig. 16. Confocal laser scanning imaging of Hela cells with PMI incubated in RPMI-1640ela cells incubated with PMI (10 �M) for 30 min. Second row: bright field (d), dark field0 min. �Ex. = 543 nm, Emission was collected by red channel from 650 to 750 nm.(For inthe web version of the article.)

und state geometry and PMI-CN at the first excited singlet state geometry.

absorption band at 670 nm corresponds to the HOMO → LUMOtransition with oscillator strength of 0.709, which shows charge-transfer character from the dihydrophenazine core to the cyaninedye (see Fig. 15). The shoulder band at around 470 nm results from

, pH 7.4, 37 ◦C. First row: bright field (a), dark field (b) and merged (c) images of (e) and merged (f) images with following incubated with CN− (2.0 eq.) for anothererpretation of the references to color in this figure legend, the reader is referred to

8 Actuat

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44 L. Yang et al. / Sensors and

OMO → LUMO + 1 transition which consists of charge-transferrom dihydrophenazine to the aldehyde substitution. The absorp-ion band at 416 nm arises from HOMO − 1 → LUMO transitionhich largely takes place within the cyanine dye (Fig. S20). Geom-

try optimization of the first excited singlet state gives an emissionavelength of 852 nm, or emission energy lower than 1.5 eV

Table 2), in accordance with the non-emissive nature of the PMIompound. The HOMO and LUMO at the optimized first excitedinglet state show strong resemblance to those of the groundtate (Fig. S21), indicating that the emission of the PMI compoundt the excited state is quenched by charge-transfer from dihy-rophenazine to cyanine dye. Addition of cyanide anions destroyshe conjugation between cyanine dye and the dihydrophenazineore, hence the ICT emission from dihydrophenazine to alde-yde at around 620 nm is recovered (see Table 2, Fig. 15 andef. [67]).

.6. Intracellular cyanide detection by PMI

Many phenazine derivatives were found in nature and were pro-uced by bacteria such as Pseudomonas spp., Streptomyces spp.nd Pantoea agglomerans, which have been implicated in the vir-lence and competitive fitness of producing organisms [70,71]. Inrder to evaluate the bioapplicated actions of man-made phenazineerivatives, PDMI and PMI was designed, synthesized and exploreds a series of red/NIR fluorescent probe for cyanine with large stokeshift. Compared to PDMI, PMI has larger stokes shift of more than0 nm and enhanced fluorescence response in red/NIR channel,hus was more suitable for cell analysis and imaging.

Confocal laser scanning microscopy (CSLM) images of PMI inela cells were demonstrated in Fig. 16 and Fig. S20. The Hela cellsere primarily incubated with 10−5 M PMI at 37 ◦C for 30 min. As

an be seen, extremely weak fluorescence was observed from the

ig. 17. Hela cells co-staining confocal fluorescence imaging of PMI-CN and xzross-sectional image. (a) Blue channel: image of nucleus stained by Hochest 33258,Ex. = 405 nm, emission was collected by blue channel from 370 to 470 nm; (b) redhannel: co-stained with PMI-CN, �Ex. = 543 nm, emission was collected by red chan-el from 650 to 750 nm; (c) overlay of (a) and (b); (d) xz cross-sectional image.(For

nterpretation of the references to color in this figure legend, the reader is referredo the web version of the article.)

ors B 203 (2014) 833–847

CSLM image corresponding to the fluorescence quenched statusof PMI. Then 2.0 equivalents cyanide was added and incubated foranother 30 min. As shown in Fig. 16, distinct fluorescence enhance-ment can be observed showing sharp contrast to the previous dimlight, which indicated a fast recognition procedure toward cyanideby intracellular PMI. To investigate distribution of PMI in Helacells, co-staining with nucleus specific dye Hochest 33258 and xzcross-sectional image were conducted. As shown in Fig. 17, PMI-CN well-distributed throughout the cytoplasm of Hela cells as wellas Hochest 33258 which was well-located in nucleus. These resultsconfirmed the promising application prospect of PMI in the appli-cation of intracellular detection of cyanide.

3. Conclusion

In summary, cyanide anion is a common toxic source in cir-cumstance and some organisms such as Madagascar bamboo etc.Quick and inexpensive analysis of cyanide urgent requires thedevelopment of colorimetric and fluorescent sensors with highsensitivity and selectivity. In this work, two phenazine-cyaninechemodosimeters PDMI and PMI were synthesized through sim-ply introduction of methyl indolium group to phenazine core.PDMI and PMI implemented an obvious progress in applica-tion of “off–on” fluorophore and provided new cell imaging andcyanide detection tools. Each chemodosimeter showed high sensi-tivity and selectivity enabled by the specific reactivity of indoliumelectrophilic bond. PDMI exhibited a distinct enhancement of emis-sion at 580 nm and achieved a detection limit of 1.4 �M fromthe absorbance change plot. On the other hand, PMI revealed“off–on” red emission response toward cyanide anion accordingto the maintained aldehyde group on the other side of phenazine.Because of the unilateral reaction pattern of PMI, lower detectionlimit was obtained as 200 nM, which is much lower than 0.2 ppm,the MCL standard set by EPA. Sensing mechanisms of these twochemodosimeters were investigated by 1H NMR, HRMS titrationexperiment and TD DFT theoretical calculations. Benefit from thelarge stokes shift and red to NIR emission, application of PMI wassuccessfully expanded to intracellular cyanide detection which wasconfirmed by confocal laser scanning microscopy imaging. Fromthe above, we anticipate that superior properties and relativelysimple synthesis method of PDMI and PMI will make them of greatresearch tools in cyanide detection and of potential use in moni-toring cyanide in biological system.

4. Experimental

4.1. Synthesis of 5,10-dihydrophenazine (1)

Phenazine (2.84 g, 13.8 mmol) was dissolved in 70 mL ethanol.Under argon atmosphere, the mixture was heated to refluxing,then, added Na2S2O4 (28.4 g, 0.16 mol) dissolved in water. The colorof the mixture changed immediately into blue, then, came out largeamount of white precipitation. About 2 h later, the reaction mixturewas stopped heating and cooled into room temperature, then fil-tered, washed with water and dried in vacuo. The light green solidwas collected and weighed 3.82 g (0.02 mol). Since the unstabilityin air, it was not characterized by NMR spectra and quickly put intothe next step reaction.

4.2. Synthesis of 5,10-dibutyl-5,10-dihydrophenazine (2)

A mixture of sodium hydroxide (2.4 g, 0.06 mol), tetrabutyl-ammonium bromide (0.4 g, 1.2 mmol) and 1 (3.82 g, 0.02 mol) crudeproduct was dissolved in 70 mL DMSO and 1.5 mL H2O. After stir-ring for 5 min, the 1-iodobutane (4.7 mL, 0.04 mol) was added.

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L. Yang et al. / Sensors and

he mixture was then heated to 40 ◦C and continued stirring for h. After cooling to room temperature, the reaction mixture wasoured into water, extracted by dichloromethane (150 mL), therganic layer was then combined and evaporated. The crude prod-ct was purified by neutral aluminum oxide chromatography, usingetroleum ether as an eluent to isolate pure compound 2 (3.5 g,6%) as a dim green crystal. 1H NMR (400 MHz, C6D6), ı (ppm):.64 (m, 4H), 6.21 (m, 4H), 3.11 (m, 4H), 1.42 (m, 4H), 1.07 (m, 4H),.74 (t, J = 7.3 Hz, 6H). 13C NMR (101 MHz, C6D6) ı (ppm): 137.50,21.12, 110.96, 45.00, 26.63, 20.15, 13.77. MS (ESI, m/z): calcd. for20H26N2: 294.21; found: 294.22.

.3. Synthesis of 5,10-dibutyl-5,10-dihydrophenazine-,7-dicarbaldehyde (3)

2 (1.4 g, 4.75 mmol) was dissolved in 12 mL DMF and stirrednder ice bath. Then phosphorus oxychloride (1 mL, 10.92 mmol)as added dropwise. The resulting mixture stirred at 80 ◦C for 7 h,

nd then cooled into room temperature. After pouring into iceater and stirred until the ice melted, sodium hydroxide solutionas added dropwise until the pH value of the mixture was adjusted

o 7.0. The solvent was evaporated and the crude product was thenurified by silica gel chromatography using petroleum/CH2Cl2 (1:1)s an eluent to isolate pure compound 3 (550 mg, 33%). 1H NMR400 MHz, CDCl3) ı (ppm): 9.59 (s, 2H), 7.10 (dd, J = 8.1, 1.6 Hz, 2H),.72 (d, J = 1.6 Hz, 2H), 6.27 (d, J = 8.2 Hz, 2H), 3.44 (m, 4H), 1.63 (m,H), 1.48 (m, 4H), 1.04 (t, J = 7.3 Hz, 6H). 13C NMR (101 MHz, C6D6) ıppm): 189.89, 142.61, 135.27, 130.24, 109.85, 108.29, 45.59, 26.32,0.13, 13.90. MS (ESI, m/z): [M+H]+ calcd. for C22H27N2O2

+: 351.2;ound: 351.2.

.4. Synthesis of 1,2,3,3-tetramethyl-3H-indol-1-ium iodide (4)

2,3,3-trimethyl-3H-indole (2.50 mL, 19.15 mmol) was dissolvedn 5 mL CH3CN and stirred under ice bath. Under argon atmosphere,odomethane (0.8 mL, 16.23 mmol) was added. The mixture washen stirred up to 80 ◦C and reflux overnight. After cooled intooom temperature, the mixture was precipitated and washed byH2Cl2 until the precipitation was light pink in color. The productas then dried in vacuo and obtained as light pink solid (5.20 g)

n 90% yield. 1H NMR (400 MHz, DMSO-d6) ı (ppm): 7.92 (m, 1H),.83 (m, 1H), 7.63 (m, 2H), 3.97 (s, 3H), 2.77 (s, 3H), 1.53 (s, 6H). 13CMR (101 MHz, DMSO-d6) ı (ppm): 195.97, 142.08, 141.57, 129.28,28.79, 128.28, 115.11, 53.90, 34.76, 21.68, 14.22. MS (ESI, m/z):ald. for C12H16N+: 174.13; found: 174.13.

.5. Synthesis of 2-(2-(5,10-dibutyl-7-formyl-5,10-ihydrophenazin-2-yl)vinyl)-1,3,3-trimethyl-3H-indol--ium iodide (PMI)

A mixture of 4 (130 mg, 0.43 mmol) and 3 (150 mg, 0.43 mmol)as dissolved in 10 mL acetonitrile with two drops of piperidine.

hen the mixture was stirred and refluxed under argon atmo-phere for 8 h. After cooling to room temperature, the solvent wasvaporated and the crude product was purified by silica gel chro-atography using CH2Cl2/ethanol as eluent. The pure product was

lack green in color (82 mg, 30%). 1H NMR (400 MHz, DMSO-d6), ıppm): 9.63 (s, 1H), 8.15 (d, J = 15.6, 1H), 7.82 (d, J = 7.3 Hz, 1H), 7.78d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.2 Hz, 2H), 7.54 (t, J = 7.3 Hz, 1H), 7.30d, J = 8.4 Hz, 1H), 7.19 (d, J = 15.8 Hz, 1H), 6.96 (s, 1H), 6.83 (s, 1H),.63 (dd, J = 11.7 Hz, 8.7, 2H), 4.01 (s, 3H), 3.68 (m, 2H), 3.61 (m, 2H),.76 (s, 6H), 1.57 (m, 4H), 1.50 (m, 4H), 1.01 (dd, J = 13.9 Hz, 6H). 13C

MR (101 MHz, DMSO-d6) ı (ppm): 190.07, 179.61, 152.03, 142.83,42.40, 141.96, 141.63, 134.37, 133.55, 131.47, 129.75, 129.59,29.50, 128.76, 128.08, 127.94, 122.70, 114.06, 111.38, 111.05,09.66, 108.43, 107.54, 54.90, 51.18, 44.38, 43.83, 33.37, 29.01,

[

ors B 203 (2014) 833–847 845

25.93, 22.07, 19.25, 13.77, 13.66. HRMS (ESI, m/z): [M + H]+calcd.for C34H40N3O+: 506.3171; found: 506.3166.

4.6. Synthesis of 2,2′-((5,10-dibutyl-5,10-dihydrophenazine-2,7-diyl)bis(ethene-2,1-diyl)) bis(1,3,3-trimethyl-3H-indol-1-ium)iodide (PDMI)

A mixture of 4 (257 mg, 0.85 mmol), ammonium acetate (5 mg)and 3 (150 mg, 0.43 mmol) was dissolved in 10 mL acetic acidthen refluxed under argon atmosphere for 8 h. After coolinginto room temperature, the solvent was evaporated. The crudeproduct was purified by silica gel chromatography usingCH2Cl2/ethanol as eluent. The pure product was black green incolor (124 mg, 32%). 1H NMR (400 MHz, DMSO-d6), ı (ppm): 8.17(d, J = 15.8 Hz, 2H), 7.83 (d, J = 7.1 Hz, 2H), 7.79 (d, J = 7.8 Hz, 2H), 7.68(d, J = 8.4 Hz, 2H), 7.60 (m, 2H), 7.54 (m, 2H), 7.24 (d, J = 15.9 Hz, 2H),7.08 (s, 2H), 6.72 (d, J = 8.7 Hz, 2H), 4.03 (s, 6H), 3.79 (m, 4H), 1.77(s, 12H), 1.62 (m, 4H), 1.54 (m, 4H), 1.02 (t, J = 7.2 Hz, 6H). 13C NMR(100 MHz, CDCl3) ı (ppm): 143.16, 141.89, 129.01, 128.70, 128.07,127.98, 122.73, 112.67, 58.54, 51.77, 29.76, 27.66, 20.15, 18.50,14.32. HRMS (ESI, m/z): [M]2+ calcd. for C46H54N4

2+: 331.2174;found: 331.2136.

Acknowledgments

This work was supported by NSFC/China (21372082,2116110444, 91233207 and 21172073) and the National BasicResearch 973 Program (2013CB733700). J.-L. Hua appreciatesProf. H. Tian very much for his helpful discussion and valuablecomments.

Appendix A. Supplementary data

Supplementary material related to this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2014.07.045.

References

[1] D.W. Boening, C.M. Chew, A critical review: general toxicity and environmentalfate of three aqueous cyanide ions and associated ligands, Water Air Soil Pollut.109 (1999) 67–79.

[2] J.L. Gerberding, Toxicological Profile for Cyanide, U.S. Department of Health andHuman Services, Atlanta, 2006.

[3] J.D. Pritchard, Hydrogen Cyanide Toxicological Overview, U.K. Health Protec-tion Agency, 2007.

[4] F. Wang, L. Wang, X.Q. Chen, J.Y. Yoon, Recent progress in the developmentof fluorometric and colorimetric chemosensors for detection of cyanide ions,Chem. Soc. Rev. 43 (2014) 4312–4324.

[5] Y. Yang, Q. Zhao, W. Feng, F.Y. Li, Luminescent chemodosimeters for bioimaging,Chem. Rev. 113 (2012) 192–270.

[6] E.M. Nolan, S.J. Lippard, Tools and tactics for the optical detection of mercuricion, Chem. Rev. 108 (2008) 3443–3480.

[7] J. Chan, S.C. Dodani, C.J. Chang, Reaction-based small molecule fluorescentprobes for chemoselective bioimaging, Nat. Chem. 4 (2012) 973–984.

[8] T. Ueno, T. Nagano, Fluorescent probes for sensing and imaging, Nat. Methods8 (2011) 642–645.

[9] Z.C. Xu, X.Q. Chen, H.N. Kim, J.Y. Yoon, Sensors for the optical detection ofcyanide ion, Chem. Soc. Rev. 39 (2010) 127–137.

10] L. Ding, Z.Y. Zhang, X. Li, J.H. Su, Highly sensitive determination of low-levelwater content in organic solvents using novel solvatochromic dyes based onthioxanthone, Chem. Commun. 49 (2013) 7319–7321.

11] Y.B. Ding, Y.S. Xie, X. Li, J.P. Hill, W.B. Zhang, W.H. Zhu, Selective and sensitive“turn-on” fluorescent Zn2+ sensors based on di- and tripyrrins with readilymodulated emission wavelengths, Chem. Commun. 47 (2011) 5431–5433.

12] L.M. Wysocki, L.D. Lavis, Advances in the chemistry of small molecule fluores-cent probes, Curr. Opin. Chem. Biol. 15 (2011) 752–759.

13] X.J. Peng, Z.G. Yang, J.Y. Wang, J.L. Fan, Y.X. He, F.L. Song, B.S. Wang, S.G. Sun, J.L.Qu, J. Qi, M. Yan, Fluorescence ratiometry and fluorescence lifetime imaging:

using a single molecule sensor for dual mode imaging of cellular viscosity, J.Am. Chem. Soc. 133 (2011) 6626–6635.

14] H. Zhang, J.L. Fan, J.Y. Wang, S.Z. Zhang, B.R. Dou, X.J. Peng, An off-on COX-2-specific fluorescent probe: targeting the Golgi apparatus of cancer cells, J. Am.Chem. Soc. 135 (2013) 11663–11669.

8 Actuat

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

46 L. Yang et al. / Sensors and

15] H.B. Yu, X.F. Zhang, Y. Xiao, W. Zou, L.P. Wang, L.J. Jin, Targetable fluorescentprobe for monitoring exogenous and endogenous NO in mitochondria of livingcells, Anal. Chem. 85 (2013) 7076–7084.

16] B. Liu, B.Z. Tang, Fluorescent sensors, Macromol. Rapid Commun. 34 (2013)704.

17] P.C.A. Swamy, S. Mukherjee, P. Thilagar, Dual binding site assisted chromogenicand fluorogenic recognition and discrimination of fluoride and cyanide by aperipherally borylated metalloporphyrin: overcoming anion interference inorganoboron based sensors, Anal. Chem. 86 (2014) 3616–3624.

18] Q. Zou, X. Li, J.J. Zhang, J. Zhou, B.B. Sun, H. Tian, Unsymmetrical diarylethenesas molecular keypad locks with tunable photochromism and fluorescence viaCu2+ and CN− coordinations, Chem. Commun. 48 (2012) 2095–2097.

19] Y. Qu, B. Jin, Y. Liu, Y.Q. Wu, L. Yang, J.C. Wu, J.L. Hua, A new triphenylaminefluorescent dye for sensing of cyanide anion in living cell, Tetrahedron Lett. 54(2013) 4942–4944.

20] N. Kumari, S. Jha, S. Bhattacharya, Colorimetric probes based on anthraimida-zolediones for selective sensing of fluoride and cyanide ion via intramolecularcharge transfer, J. Org. Chem. 76 (2011) 8215–8222.

21] T.W. Hudnall, F.P. Gabbaï, Ammonium boranes for the selective complexationof cyanide or fluorede ions in water, J. Am. Chem. Soc. 129 (2007) 11978–11986.

22] Y.M. Kim, H.Y. Zhao, F.P. Gabbaï, Sulfonium boranes for the selective capture ofcyanide ions in water, Angew. Chem. Int. Ed. 48 (2009) 4957–4960.

23] X.D. Lou, L.Y. Zhang, J.G. Qin, Z. Li, The chaotic sequences in the Bray-Liebhafskyreaction in an open reactor, Chem. Commun. 44 (2008) 5848–5858.

24] R. Guliyev, O. Buyukcakir, F. Sozmen, O.A. Bozdemir, Cyanide sensing via metalion removal from a fluorogenic BODIPY complex, Tetrahedron Lett. 50 (2009)5139–5141.

25] Y.H. Tang, Y. Qu, Z. Song, X.P. He, J. Xie, J.L. Hua, G.R. Chen, Discovery of a sensi-tive Cu(II)-cyanide “off-on” sensor based on new C-glycosyl triazolyl bis-aminoacid scaffold, Org. Biomol. Chem. 10 (2012) 555–560.

26] X.D. Lou, Q. Zeng, Y. Zhang, Z.M. Wan, J.G. Qin, Z. Li, Functionalized poly-acetylenes with strong luminescence: “turn-on” fluorescent detection ofcyanide based on the dissolution of gold nanoparticles and its application inreal samples, J. Mater. Chem. 22 (2012) 5581–5586.

27] P. Singh, M. Kaur, CN− scavenger: a leap towards development of a CN− anti-dote, Chem. Commun. 47 (2011) 9122–9124.

28] H.J. Kim, H. Lee, J.H. Lee, D.H. Choi, J.H. Jung, J.S. Kim, Bisindole anchoredmesoporous silica nanoparticles for cyanide sensing in aqueous media, Chem.Commun. 47 (2011) 10918–10920.

29] S.J. Hong, J. Yoo, S.H. Kim, J.S. Kim, J.Y. Yoon, C.H. Lee, Beta-vinyl substitutedcalix[4]pyrrole as a selective ratiomatric sensor for cyanide anion, Chem. Com-mun. 45 (2009) 189–191.

30] A.S.F. Farinha, A.C. Tome, J.A.S. Cavaleiro, (E)-3-(meso-Octamethyl-calix[4]pyrrol-2-yl)propenal: a versatile precursor for calix[4]pyrrole-basedchromogenic anion sensors, Tetrahedron Lett. 51 (2010) 2184–2187.

31] S.J. Hong, C.H. Lee, Nitrovinyl substituted calix[4]pyrrole as a unique, reaction-based chemosensor for cyanide anion, Tetrahedron Lett. 53 (2012) 3119–3122.

32] S.H. Kim, S.J. Hong, J. Yoo, S.K. Kim, J.L. Sessler, C.H. Lee, Strappedcalix[4]pyrroles bearing a 1,3-indanedione at a beta-pyrrolic position: chemo-dosimeters for the cyanide anion, Org. Lett. 11 (2009) 3626–3629.

33] Y.H. Jeong, C.H. Lee, W.D. Jang, A diketopyrrolopyrrole-based colorimet-ric and fluorescent probe for cyanide detection, Chem. Asian J. 7 (2012)1562–1566.

34] C.H. Zong, L.R. Zheng, W.H. He, X.Y. Ren, C.H. Jiang, L.H. Lu, In situformation of phosphorescent molecular gold(I) cluster in a macroporous poly-mer film to achieve colorimetric cyanide sensing, Anal. Chem. 86 (2014)1687–1692.

35] Y.L. Liu, X. Lv, Y. Zhao, J. Liu, Y.Q. Sun, P. Wang, W. Guo, A Cu(II)-basedchemosensing ensemble bearing rhodamine B fluorophore for fluorescenceturn-on detection of cyanide, J. Mater. Chem. 22 (2012) 1747–1750.

36] S. Park, H.J. Kim, Highly selective chemodosimeter for cyanide based on a dou-bly activated Michael acceptor type of coumarin thiazole fluorophore, Sens.Actuators B, Chem. 161 (2012) 317–321.

37] S. Goswami, A. Manna, S. Paul, A.K. Das, K. Aich, P.K. Nandi, Resonance-assisted hydrogen bonding induced nucleophilic addition to hamper ESIPT:ratiometric detection of cyanide in aqueous media, Chem. Commun. 49 (2013)2912–2914.

38] J.L. Liu, Y. Liu, Q. Liu, C.Y. Li, L.N. Sun, F.Y. Li, Iridium(III) complex-coatednanosystem for ratiometric upconversion luminescence bioimaging of cyanideanions, J. Am. Chem. Soc. 133 (2011) 15276–15279.

39] L.M. Yao, J. Zhou, J.L. Liu, W. Feng, F.Y. Li, Iridium-complex-modified upcon-version nanophosphors for effective LRET detection of cyanide anions in purewater, Adv. Funct. Mater. 22 (2012) 2667–2672.

40] X.S. Zhou, H. Yang, J. Hao, C.X. Yin, D.S. Liu, W. Guo, A fluorescence turn-onsensor for cyanide anion based on exciplex signaling mechanism, Chem. Lett.41 (2012) 518–520.

41] Y.M. Kim, H.S. Huh, M.H. Lee, I.L. Lenov, H. Zhao, F.P. Gabbaï, Turn-on flu-orescence sensing of cyanide ions in aqueous solution at parts-per-billionconcentrations, Chem. Eur. J. 17 (2011) 2057–2062.

42] L.H. Feng, Y. Wang, F. Liang, W. Liu, X.J. Wang, H.P. Diao, A specific sensingensemble for cyanide ion in aqueous solution, Sens. Actuators, B Chem. 168

(2012) 365–369.

43] H.T. Niu, X.L. Jiang, J.Q. He, J.P. Cheng, A highly selective and synthetically facileaqueous-phase cyanide probe, Tetrahedron Lett. 49 (2008) 6521–6524.

44] G. Absalan, M. Asadi, S. Kamran, S. Torabi, S. Leila, Design of a cyanide ionoptode based on immobilization of a new Co(III) Schiff base complex on

[

ors B 203 (2014) 833–847

triacetylcellulose membrane using room temperature ionic liquids as modi-fiers, Sens. Actuators, B Chem. 147 (2010) 31–36.

45] X.M. Wu, X.R. Sun, Z.Q. Guo, J.B. Tang, Y.Q. Shen, T.D. James, H. Tian, W.H.Zhu, In vivo and in situ tracking cancer chemotherapy by highly photo-stable NIR fluorescent theranostic prodrug, J. Am. Chem. Soc. 136 (2014)3579–3588.

46] G. Qian, X.Z. Li, Z.Y. Wang, Visible and near-infrared chemosensor for col-orimetric and ratiometric detection of cyanide, J. Mater. Chem. 19 (2009)522–530.

47] J.Y. Shao, H.Y. Sun, H.M. Guo, S.M. Ji, J.Z. Zhao, W.T. Wu, X.L. Yuan, C.L. Zhang,T.D. James, A highly selective red-emitting FRET fluorescent molecular probederived from BODIPY for the detection of cysteine and homocysteine: an exper-imental and theoretical study, Chem. Sci. 3 (2012) 1049–1061.

48] G.J. Kim, H.J. Kim, Doubly activated coumarin as a colorimetric and fluorescentchemodosimeter for cyanide, Tetrahedron Lett. 51 (2010) 185–187.

49] H.S. Jung, J.H. Han, Z.H. Kim, C. Kang, J.S. Kim, Coumarin-Cu(II) ensemble-basedcyanide sensing chemodosimeter, Org. Lett. 13 (2011) 5056–5059.

50] H.J. Kim, K.C. Ko, J.H. Lee, J.Y. Lee, J.S. Kim, KCN sensor: unique chromogenicand ‘turn-on’ fluorescent chemodosimeter: rapid response and high selectivity,Chem. Commun. 47 (2011) 2886–2888.

51] C.H. Lee, H.J. Yoon, J.S. Shim, W.D. Jang, A boradiazaindacene-based turn-onfluorescent probe for cyanide detection in aqueous media, Chem. Eur. J. 18(2012) 4513–4516.

52] L.J. Jiao, M.M. Liu, M. Zhang, C.J. Yu, Z.Y. Wang, E.H. Hao, Visual and colorimetricdetection of cyanide anion based on a “turn-off” daylight fluorescent molecule,Chem. Lett. 40 (2011) 623–625.

53] R. Guliyev, S. Ozturk, E. Sahin, E.U. Akkaya, Expanded bodipy dyes: anionsensing using a bodipy analog with an additional difluoroboron bridge, Org.Lett. 14 (2012) 1528–1531.

54] Z.P. Liu, X.Q. Wang, Z.H. Yang, W.J. He, Rational design of a dual chemosensorfor cyanide anion sensing based on dicyanovinyl-substituted benzofurazan, J.Org. Chem. 76 (2011) 10286–10290.

55] Y.K. Tsui, S. Devaraj, Y.P. Yen, Azo dyes featuring with nitrobenzoxadiazole(NBD) unit: a new selective chromogenic and fluorogenic sensor for cyanideion, Sens. Actuators B Chem. 161 (2012) 510–519.

56] H.T. Niu, X. Jiang, J. He, J.P. Cheng, Cyanine dye-based chromofluorescent probefor highly sensitive and selective detection of cyanide in water, TetrahedronLett. 50 (2009) 6668–6671.

57] X. Lv, J. Liu, Y. Liu, Y. Zhao, Y.Q. Sun, P. Wang, W. Guo, Ratiometric fluores-cence detection of cyanide based on a hybrid coumarin-hemicyanine dye:the large emission shift and the high selectivity, Chem. Commun. 47 (2011)12843–12845.

58] B. Chen, Y.B. Ding, X. Li, W.H. Zhu, J.P. Hill, K. Ariga, Y.S. Xie, Steric hindrance-enforced distortion as a general strategy for the design of fluorescence “turn-on” cyanide probes, Chem. Commun. 49 (2013) 10136–10138.

59] S.K. Kwon, S. Kou, H.N. Kim, X.Q. Chen, H. Hwang, S.W. Nam, S.H. Kim, K.M.K.Swamy, S. Park, J. Yoon, Sensing cyanide ion via fluorescent change and itsapplication to the microfluidic system, Tetrahedron Lett. 49 (2008) 4102–4105.

60] M. Dong, Y. Peng, Y.M. Dong, N. Tang, Y.W. Wang, A selective, colori-metric, and fluorescent chemodosimeter for relay recognition of fluorideand cyanide anions based on 1,1′-binaphthyl scaffold, Org. Lett. 14 (2012)130–133.

61] S. Goswami, S. Paul, A. Manna, Carbazole based hemicyanine dye for both“naked eye” and ‘NIR’ fluorescence detection of CN- in aqueous solution:from molecules to low cost devices (TLC plate sticks), Dalton Trans. 42 (2013)10682–10686.

62] Y. Zhang, D.H. Yu, G.Q. Feng, Colorimetric and near infrared fluorescent detec-tion of cyanide by a new phenanthroimidazole–indolium conjugated probe,RSC Adv. 4 (2014) 14752–14757.

63] X.L. Feng, L.B. Liu, S. Wang, D.B. Zhu, Water-soluble fluorescent conjugated poly-mers and their interactions with biomacromolecules for sensitive biosensors,Chem. Soc. Rev. 39 (2010) 2411–2419.

64] E. Galbraith, T.D. James, Boron based anion receptors as sensors, Chem. Soc.Rev. 39 (2010) 3831–3842.

65] S. Rochat, T.M. Swager, Water-soluble cationic conjugated polymers: responseto electron-rich bioanalytes, J. Am. Chem. Soc. 135 (2013) 17703–17706.

66] S.J. Chen, Y.N. Hong, Y. Liu, J.Z. Liu, C.W.T. Leung, M. Li, R.T.K. Kwok, E.G.Zhao, J.W.Y. Lam, Y. Yu, B.Z. Tang, Full-range intracellular pH sensing byan aggregation-induced emission-active two-channel ratiometric fluorogen, J.Am. Chem. Soc. 135 (2013) 4926–4929.

67] L. Yang, X. Li, J.B. Yang, Y. Qu, J.L. Hua, Colorimetric and ratiometric near-infrared fluorescent cyanide chemodosimeter based on phenazine derivatives,ACS Appl. Mater. Interfaces 5 (2013) 1317–1326.

68] X. Zhang, L. Guo, F.Y. Wu, Y.B. Jiang, Development of fluorescent sensing ofanions under excited-state intermolecular proton transfer signaling mecha-nism, Org. Lett. 5 (2003) 2667–2670.

69] T.F. Robbins, H. Qian, X. Su, R.P. Hughes, I. Aprahamian, Cyanide detection usinga triazolopyridinium salt, Org. Lett. 15 (2013) 2386–2389.

70] M. McDonald, D.V. Mavrodi, L.S. Thomashow, H.G. Floss, Phenazine biosyn-thesis in pseudomonas fluorescens: branchpoint from the primary shikimate

biosynthetic pathway and role of phenazine-1,6-dicarboxylic acid, J. Am. Chem.Soc. 123 (2001) 9459–9460.

71] L.E.P. Dietrich, C. Okegbe, A.P. Whelan, H. Sakhtah, R.C. Hunter, D.K. Newman,Bacterial community morphogenesis is intimately linked to the intracellularredox state, J. Bacteriol. 195 (2013) 1371–1380.

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L. Yang et al. / Sensors and

iographies

in Yang received his BS degree in fine chemical from the East China Universityf Science and Technology (ECUST, Shanghai, China). He is now a doctor candidatet the Key Laboratory for Advanced Materials in ECUST, under the supervision ofro. Jianli Hua. His research focused on the development of novel chemodosimetersased on phenazine derivatives.

in Li is a postdoctoral researcher in Division of Theoretical Chemistry andiology at KTH Royal Institute of Technology, Sweden. His current research

nterest is theoretical simulations of optical properties of supramolecularystems.

i Qu received his Ph.D. degree in 2012 in applied chemistry from East China Uni-ersity of Science and Technology (Shanghai, China). He is currently a postdoctoralellow in the group of Professor Fuyou Li at the Fudan University. (Shanghai, China).is work focuses on the design and synthesis of fluorescent dye and conjugatedolymers. His main research interests include the design, synthesis, and spectro-copic evaluation of molecular probes that are responsive to toxic ions and biological

olecules.

eisong Qu obtained his bachelor degree in 2012 in Applied Chemistry from Easthina University of Science and Technology (Shanghai, China). He is at the momentursuing a MS degree under the supervision of Prof. Jianli Hua at ECUST. His research

ors B 203 (2014) 833–847 847

is focused on the synthesis of organic materials derived from diketopyrrolopyrrole-based systems and their applications.

Xiao Zhang received his BS degree in fine chemistal from the East China Universityof Science and Technology in 2013. He is now pursuing his master degree at theKey Laboratory for Advanced Materials in ECUST, under the supervision of Pro. JianliHua.

Yandi Hang received her BS degree in applied chemistry from the East China Uni-versity of Science and Technology (ECUST, Shanghai, China) in 2012. She is now aPh.D. candidate at the Key Laboratory for Advanced Materials in ECUST, under thesupervision of Pro. Jianli Hua. Her research focused on the development of novelfluorescent dye and evaluation of its optical and electronic properties.

Hans Ågren is a professor and head of Division of Theoretical Chemistry and Biol-ogy at KTH Royal Institute of Technology, Sweden. His current research interest ispredictive multiscale modeling that combines the accuracy of quantum mechanicsand the applicability of classical physics.

Jianli Hua received her Ph.D. degree in Organic Chemistry from Wuhan University in2002 from 2002 to 2004, Dr. Hua was a postdoctoral fellow at East China University

of Science and Technology (ECUST) and she was a visiting scholar at the Hong KongUniversity of Science and Technology in 2005. Since 2002, she has been full professorand works at ECUST now. Her current research interests cover the developmentof new molecules and polymers with novel structures and electronic and opticalproperties.