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A ruthenium(II) complex-based lysosome-targetable multisignal chemosensor for in vivo detection of hypochlorous acid

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Although considerable efforts have been made for the development of ruthenium(II) complex-based chemosensors and bioimaging reagents, the multisignal chemosensor using ruthenium(II) complexes as the reporter is scarce. In addition, the mechanisms of
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  A ruthenium(II) complex-based lysosome-targetable multisignalchemosensor for in vivo detection of hypochlorous acid Liyan Cao  1 , Run Zhang  1 , Wenzhu Zhang ** , Zhongbo Du, Chunjun Liu, Zhiqiang Ye,Bo Song, Jingli Yuan * State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, PR China a r t i c l e i n f o  Article history: Received 8 April 2015Received in revised form26 July 2015Accepted 31 July 2015Available online 3 August 2015 Keywords: Multisignal chemosensorRuthenium(II) complexHypochlorous acidBioimagingIn vivo analysis a b s t r a c t Although considerable efforts have been made for the development of ruthenium(II) complex-basedchemosensors and bioimaging reagents, the multisignal chemosensor using ruthenium(II) complexesas the reporter is scarce. In addition, the mechanisms of cellular uptake of ruthenium(II)-based che-mosensors and their intracellular distribution are ill-de fi ned. Herein, a new ruthenium(II) complex-based multisignal chemosensor,  Ru-Fc , is reported for the highly sensitive and selective detection of lysosomal hypochlorous acid (HOCl).  Ru-Fc  is weakly luminescent because the MLCT (metal-to-ligandcharge transfer) state is corrupted by the ef  fi cient PET (photoinduced electron transfer) process from  Fc (ferrocene) moiety to Ru(II) center. The cleavage of   Fc  moiety bya HOCl-induced speci fi c reaction leads toelimination of PET, which re-establishes the MLCT state of the Ru(II) complex, accompanied byremarkable photoluminescence (PL) and electrochemiluminescence (ECL) enhancements. The result of MTT assay showed that the proposed chemosensor,  Ru-Fc,  was low cytotoxicity. The applicability of   Ru-Fc  for the quantitative detection of HOCl in live cells was demonstrated by the confocal microscopyimaging and  fl ow cytometry analysis. Dye colocalization studies con fi rmed very precise distribution of the Ru(II) complex in lysosomes, and inhibition studies revealed that the caveolae-mediated endocytosisplayed an important role during the cellular internalization of   Ru-Fc . By using  Ru-Fc  as a chemosensor,the imaging of the endogenous HOCl generated in live macrophage cells during the stimulation wasachieved. Furthermore, the practical applicability of   Ru-Fc  was demonstrated by the visualizing of HOClin laboratory model animals,  Daphnia magna  and zebra fi sh. ©  2015 Elsevier Ltd. All rights reserved. 1. Introduction Recently, luminescent transition metal complexes werespringing up as attractive candidates for the development of responsive chemosensors and cell imaging probes due to theirabundant photophysical, photochemical, and electrochemicalproperties [1 e 6]. Among these complexes, ruthenium(II) com-plexes with polypyridyl ligands, such as 2,2 0 -bipyridine (bpy),1,10-phenanthroline (phen), and/or bathophenanthroline derivativeshave been constantly reported as luminescent chemosensors/probes forbioactive molecules,metalcations,andanions inthelastfew years [7 e 11]. As one of useful luminescent reporters for bio-sensing and bioimaging, ruthenium(II) complexes offer severalunique advantages including metal-to-ligand charge transfer(MLCT) based visible-light excitation and emission [11 e 13], largeStokes shifts (typically greater than 150 nm) [12,14,15], highchemical and photochemical stabilities [16 e 18], low cytotoxicity[19 e 22], good water solubility [22 e 25], and high response ef  fi -ciency[16,22,26].Inaddition,astheresultofMLCT-basedemission,polypyridyl-ruthenium(II) complexes have also been widelyinvestigated as an important material for use in electrogeneratedchemiluminescence analysis with advantages of high stability,sensitivity, and lower environmental sensitivity [27 e 30].Since the MLCT emission strongly depends on the orbital com-bination of polypyridyl ligands, the emission properties could bereadily modulated by the variation of ligand properties[9,12,17,31,32], which provides a useful approach for the develop-ment of polypyridyl-ruthenium(II) complex-based luminescent *  Corresponding author. **  Corresponding author. E-mail addresses:  wzhzhang@dlut.edu.cn (W. Zhang), jlyuan@dlut.edu.cn(J. Yuan). 1 Equal contribution to this work. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials http://dx.doi.org/10.1016/j.biomaterials.2015.07.0520142-9612/ ©  2015 Elsevier Ltd. All rights reserved. Biomaterials 68 (2015) 21 e 31  chemosensors [33 e 39]. In previous works, we identi fi ed that theMLCT excited state of ruthenium(II) complexes could be corruptedbytheintramolecular photoinduced electrontransfer (PET)processwhen an electron donor or acceptor was attached to polypyridylligands, and the elimination of PET by the analyte-triggered reac-tion could restore the MLCTstate to switch on the luminescence of the complexes [40 e 42]. Using this strategy, our group has devel-oped a series of polypyridyl-ruthenium(II) complex-based lumi-nescent chemosensors/probes for reactive oxygen/nitrogen species(ROS/RNS)[22,28,40,41,43],highlyactiveaminoacids[14,17,30]and ions [42], and successfully demonstrated their applicability forbiosensing and bioimaging [17,22,40,41]. Nevertheless, mecha-nisms for cellular uptake and distribution of ruthenium(II) com-plexes in live cells remained ill-de fi ned [16,44 e 48]. Furthermore,for live cell imaging, it is important to identify the target in theorganelle of interest even though it is still a challenge at themoment, especially for the metal complex-based chemosensors[49 e 54].In living biosystems, it is well known that ROS/RNS play pivotalroles in many biological processes, such as signal transduction forcellular proliferation, migration, apoptosis and bactericidal activityof phagocytes [55,56]. As one of the important ROS, hypochlorousacid (HOCl) can be biologically produced by an oxidation reactionbetweenchlorideion(Cl  )andhydrogenperoxide(H 2 O 2 )catalyzedby myeloperoxidase (MPO) within phagosomes [57 e 61]. Theendogenous HOCl is an essential molecule in immune defenseagainst microorganisms. However, excessive or misplaced HOClcould induce apoptosis of cells through the rupture of lysosomes[22,41]. In this regard, the development of accurate and reliabledetection methods for HOCl in lysosome is fundamentally impor-tant to understand its physiological functions in living organisms.To our knowledge, the lysosome-targetable luminescent chemo-sensors for HOCl are scarce.In this work, we report a novel ruthenium(II) complex-basedluminescent chemosensor,  Ru-Fc , for the photoluminescent (PL)and electrochemiluminescent (ECL) multisignal detection of HOClin living cell and laboratory animal samples. The proposed che-mosensor is weakly luminescent in both PL and ECL analyses. Thespeci fi c reaction between  Ru-Fc  and HOCl leads to the cleavage of the luminescence quencher moiety,  Fc , so that the luminescence of ruthenium(II) complex is turned on. The chemosensor showedhighlysensitiveandselectivePLandECLresponsestowardsHOClina wide pH range, which allowed it to be favorably used for the PL and ECL multisignal detection of HOCl. Using  Ru-Fc  as a chemo-sensor,thequanti fi cationofHOClinlysosomesofasingleintactcellwas realized by confocal microscopy imaging and  fl ow cytometryanalysis, and the process of cellular uptake and sub-cellular local-ization of   Ru-Fc were elucidated. Moreover,  Ru-Fc  was successfullyused for the luminescent imaging of HOCl in live laboratory ani-mals,  Daphnia magna  and zebra fi sh, which demonstrated thepractical applicability and highlighted advantages of   Ru-Fc  for thein vivo bioimaging application. 2. Experimental section  2.1. Materials and instruments DAPI (4 0 ,6-diamidino-2-phenylindole, dihydrochloride), tri-n-propylamine (TPrA), 3-morpholinosydnonimine (ONOO  donor),PI (propidium iodide), NH 4 Cl, chloroquine, chlorpromazine,  fi lipin,nocodazole, colchicines, 4 b -phorbol-12-myristate-13-acetate(PMA), lipopolysaccharide (LPS), interferon- g  (IFN- g ) and 4-aminobenzoic acid hydrazide (4-ABAH) were purchased fromSigma e Aldrich (Hydrazinocarbonyl)ferrocene was purchased fromTCI Shanghai. MitoTracker ® Green FM, ER-Tracker ™  Green,LysoSensor ™ Green, LysoTracker ® Blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Roswell Park Memo-rial Institute's Medium (RPMI-1640), Dulbecco's Modi fi ed EagleMedium (DMEM), fetal bovine serum (FBS),  L  -glutamine, penicillin,and streptomycin sulfate were purchased from Life Technologies(Australia). 4-Carboxylic acid-4 0 -methyl-2,2 0 -bipyridine [22,41],cis-Ru(bpy) 2 Cl 2 $ 2H 2 O [17,42], [Ru(bpy) 2 (COOH-bpy)] 2 þ [22] and 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene (NOC-13,an NO donor) [40] were synthesized by using the literaturemethods. Cultured  D. magna  and zebra fi sh were obtained fromProfessor JingwenChen'sgroupat SchoolofEnvironmentalScienceand Technology, Dalian University of Technology. Unless otherwisestated, all chemical materials were purchased from commercialsourcesandusedwithoutfurtherpuri fi cation.Deionizedwaterwasused throughout.NMR spectra were recorded on Bruker Avance spectrometers(400 MHz for  1 H and 100 MHz for  13 C). Mass spectra weremeasured onan AgilentTechnologies6130QuadrupoleLC/MSDMSspectrometer. Elemental analysis was carried out on a Vario-EL CHN analyzer. Absorption spectra were recorded on an AgilentCary 300 UV  e vis spectrometer. Photoluminescence spectra weremeasured on a Perkin e Elmer LS-55 luminescence spectrometerwith excitation and emission slits of 10 nm. All the ECL measure-ments were carried out on an ECL instrument system (MPI-A,RemexElectronicsInstrumentLtd.Co.)usingasmallquartzECLcellat room temperature. All the spectra measurements were con-ducted in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4. Lumi-nescent live cell imaging measurements were carried out on anOlympus Fluoview FV 1000 IX81 inverted confocal laser-scanningmicroscope equipped with 405, 473, 559 and 635 nm laser di-odes. The relative luminescence intensities, and colocalization of images were analyzed by using Image J software version 1.44p.Flow cytometry analysis was carried out on a BD FACSAria II  fl owcytometer with a 488 nm laser. The data were analyzed with aFlowing software.  2.2. ECL measurements The glassy carbon (3.0 mm in diameter) electrode and KClsaturated Ag/AgCl electrode were used as working electrode andreference electrode, respectively, and a platinum wire (0.3 mm indiameter) was used as the auxiliary electrode. Before measure-ments, the glassy carbon working electrode was soaked in 10%HNO 3  in an ultrasonic water bath for 1 min, polished by an Al 2 O 3 slurry, and thoroughly rinsed with deionized water for 1 min. Thevoltage of the photomultiplier tube was set at 900 V in the detec-tion process while collecting the ECL signals.  2.3. Confocal luminescence imaging of HOCl in live cells 2.3.1. For luminescent imaging of live cells MDA-MB-231 or U-343 MGa cells were typically seeded at adensity of 5  10 4 cells/mL in a 22 mm cover glass bottom culturedishes (ProSciTech, Australia) for the confocal microscopy imaging.After 24 h, the culture media were replaced with fresh RPMI-1640(MDA-MB-231, 2 mL/dish) or DMEM (U-343 MGa, 2 mL/dish)containing100 m M  Ru-Fc ,andthe cellswere incubated at 37  C inahumidi fi ed 5% CO 2 /95% air incubator for another 6 h. Prior to theimaging experiments, the  Ru-Fc -loaded cells were washed withPBS (3    2 mL/dish) and further treated with 10  m M HOCl for15 min. After washing with PBS (3    2 mL/dish), the cells weresubjected to the luminescence imaging measurements on theconfocal microscope. L. Cao et al. / Biomaterials 68 (2015) 21 e  31 22   2.3.2. For luminescent imaging of   fi  xed cells MDA-MB-231 or U-343 MGa cells were washed with PBS(3  2 mL/dish), and then  fi xed with ethanol at room temperaturefor 15 min. After washing with PBS (3  2 mL/dish), the  fi xed cellswere incubated with 50  m M  Ru-Fc  for 1 h at 37   C. The  Ru-Fc- loaded cells were washed with PBS (3    2 mL/dish), and thenincubated with 10  m M HOCl for 15 min. After washing with PBS(3    2 mL/dish), the cells were further stained with 300 nM DAPIfor another 5 min. Then the cells were washed with PBS (3  2 mL/dish)andsubjectedtotheluminescenceimagingmeasurementsonthe confocal microscope.  2.3.3. For colocalization imaging of live cells The  Ru-Fc- loaded MDA-MB-231 or U-343 MGa cells wereincubated with 10  m M HOCl for 15 min. After washing with PBS(3    2 mL/dish), the cells were further stained with MitoTracker ® GreenFM,ER-Tracker ™ Green,LysoSensor ™ GreenorLysoTracker ® Blue according to each protocol from Life Technologies, respec-tively. Then the cells were washed with PBS (3    2 mL/dish) andsubjected to the luminescence imaging measurements on theconfocal microscope.  2.3.4. For live cell imaging after treatment with endocytic inhibitors For investigating temperature-dependent cellular uptake of   Ru-Fc , MDA-MB-231 and U-343 MGa cells were cooled at 4   C for30 min, then 100  m M  Ru-Fc  was added intodishes for an additionalincubation of 6 h under the same conditions. After washing withPBS (3    2 mL/dish), the cells were subjected to the confocal mi-croscopy imaging. For studying the effect of endocytotic inhibitorson cellular uptake of   Ru-Fc , the cells were treated with NH 4 Cl(10 mM), chloroquine (100  m M),  fi lipin (10  m g/mL), nocodazole(10  m M), chlorpromazine (10  m g/mL), or colchicine (10  m M) for30minat37  C,respectively,andthen 100 m M Ru-Fc wasaddedforan additional incubation of 6 h under the same conditions. There-after, the cells were washed with PBS (3    2 mL/dish) and furthertreated with 10  m M HOCl for 15 min. After washing with PBS(3   2 mL/dish), the cells were subjected to the luminescence im-aging measurements on the confocal microscope. For control ex-periments, the cells were prepared by incubating cells with  Ru-Fc under the same conditions. To evaluate the viability of cells, thecells were stained with 1.5  m M PI for 2 min after they were treatedwith inhibitors for 6 h.  2.3.5. For live cell imaging with [Ru(bpy)  2 (COOH-bpy)]   2 þ MDA-MB-231 or U-343 MGa cells were seeded in culture dishesand incubated at 37  C in a humidi fi ed 5% CO 2 /95% air incubator for24 h. After washed with PBS (3  2 mL/dish), the cells were furtherincubated with fresh medium containing 100  m M [Ru(bpy) 2 (COOH-bpy)] 2 þ for another 6 h. The cells werewashed with PBS (3  2 mL/dish) and then subjected to the luminescence imaging measure-ments on the confocal microscope.  2.4. Flow cytometry analysis Cellular uptake of   Ru-Fc  (100  m M) under different conditionsand the intracellular turn-on luminescence response of   Ru-Fc -loaded cells towards HOCl were assessed by  fl ow cytometry anal-ysis. MDA-MB-231 and U-343 MGa cells (1    10 5 cells/mL) wereseeded into a six-chamber culture well and incubated for 24 h. Fortheanalysisof turn-onluminescenceresponsein livecells, thecellswere washed with PBS for three times, incubated with 100  m M  Ru-Fc  for 6 h at 37   C, and then treated with 10  m M HOCl for another15 min. For the analysis of turn-on luminescence response in  fi xedcells, the suspension cells were  fi xed with ethanol and incubatedwith 50  m M  Ru-Fc  for 1 h, then the  Ru-Fc- loaded cells weretreatedwith 10  m M HOCl for 15 min. For studying cellular uptake of   Ru-Fc ,the cells were co-incubated with  Ru-Fc  and different endocyticinhibitors for 6 h, and further treated with 10  m M HOCl for another15 min. For studying cellular uptake of [Ru(bpy) 2 (COOH-bpy)] 2 þ ,the cells were incubated with 100  m M [Ru(bpy) 2 (COOH-bpy)] 2 þ for6 h. After washing with PBS (3    2 mL/well), the cells were sub- jected to the  fl ow cytometry analysis. The cells incubated withculture medium for 24 h were used as the control for all experi-ments. All the cells were detached from the well using 0.05%trypsin-EDTA solution and washed with PBS for three times beforethe  fl ow cytometry analysis.  2.5. Imaging of HOCl in live D. magnaD. magna  were cultured at 20   C in nonchlorinated tap waterthat was aerated for 3 days and saturated oxygen under cool-white fl uorescent light with a 14:10 h light:dark photoperiod. The culturemedium was renewed three times a week.  Scenedesmus obliquus were fed to  D. magna  daily. The newborn  D. magna  (age  <  48 h)were incubated with 100  m M  Ru-Fc  for 2 h and 4.5 h, respectively.After washing three times with the culture medium,  Ru-Fc -loaded D. magna  were incubated with 15  m M HOCl for another 20 min.After washing with PBS for three times,  D. magna weresubjectedtothe imaging measurements on a laboratory-use luminescence mi-croscope (excitation  fi lter, 450 e 490 nm; dichroic mirror, 505 nm;emission  fi lter,  > 520 nm). 3. Results and discussion  3.1. Design, synthesis and characterization of chemosensor for HOCl In previous work, although we have demonstrated the appli-cability of ruthenium(II) complexes as luminescent chemosensorsfor the detection of HOCl in live cells [22,41], the more reliablemethod that can be used for multisignal detection of HOCl is un-explored. In addition, the distribution of ruthenium(II) complexesinlivecellsandpathwaysforcellularuptakeofthesecomplexesareunclear [19,44,48]. Towards these ends, in this work, we synthe-sized a novel ruthenium(II) complex,  Ru-Fc , that can act as a che-mosensor for the highly sensitive and selective PL and ECL dual-signal detection of HOCl in living biosystems.As shown in Scheme 1, the new chemosensor,  Ru-Fc , wasdesigned according to the  “ PL and ECL reporter-reactive linker-quencher ”  sandwich approach [41]: a bipyridine-ruthenium(II)complex core was used as a PL and ECL reporter, a ferrocenylmoiety as signal quencher [62,63], and a hydrazine as the reactivelinker. The choice of bipyridine-ruthenium(II) complex as a signalreporter is due to its rich PL and ECL properties reported in litera-ture [12,27], and the choice of ferrocenyl moiety as a signalquencher is based on the consideration that ferrocenyl derivativescaneffectivelyquenchtheluminescencethroughthePETprocesstocorruptthe excitedstate of aluminophore [63]. We envisioned thatthe designed chemosensor,  Ru-Fc,  should be weakly luminescentowing tothe quenching of ferrocenyl moiety, and a HOCl promotedspeci fi c oxidation reaction of hydrazine will cleave ferrocenylmoiety from  Ru-Fc  to turn on the luminescence of the bipyridine-ruthenium(II) complex. Different from our previously reportedprobe [22], in this work,  Ru-Fc  was designed to be as the  fi rst PL and ECL multisignal chemosensor for the detection of HOCl withhigh sensitivity and selectivity, which was anticipated to be appli-cable for the quantitative detection of HOCl in both live cells andorganisms.AsoutlinedinSupplementary, Ru-Fc wasfacilelysynthesizedbyatwo-stepreactionprocedure.Brie fl y,4-carboxylicacid-4 0 -methyl-2,2 0 -bipyridine was reacted with (hydrazinocarbonyl)ferrocene in L. Cao et al. / Biomaterials 68 (2015) 21 e  31  23  CH 2 Cl 2  to form the new ligand, 4-ferrocenecarbonylhydrazinocarbonyl-4 0 -methyl-2,2 0 -bipyridine(bpy-Fc). Then the chemosensor,  Ru-Fc,  was synthesized by thecoordination of Ru(bpy) 2 Cl 2  with bpy-Fc in ethanol.  Ru-Fc  waspuri fi ed by silica gel column chromatography, and isolated as itshexa fl uorophosphatesalt.The structureofligand Fc-bpyand Ru-Fc was well-characterized by ESI-HRMS, NMR and elemental analyses(Figs.S1 e S6).Theproposedcleavageof  Ru-Fc inducedbyHOClwassupported by the results of ESI-MS studies. As shown in Fig. S7,when HOCl was added into the solution of   Ru-Fc , the signals of cleaved products [Ru(bpy) 2 (COOH-bpy)] 2 þ and [Ru(bpy) 2 (COONa-bpy)] 2 þ , were clearly observed (peaks of   m /  z   at 314.1 and 325.1).The luminescence properties of two ruthenium(II) complexes, Ru-Fc  and [Ru(bpy) 2 (COOH-bpy)](PF 6 ) 2 , were examined at roomtemperature in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4(Table S1, Figs. S8 e S9). As shown in Fig. S8, the most intense peaks below 300 nm of   Ru-Fc  and its cracked product are dominated bythe ligand-centered  p / p * transition, while the absorption bandsin the visible region are the MLCT bands of ruthenium(II) com-plexes.Sincethed e dabsorptionbandofferrocene(around460nm[62]) is close to the MLCT transition band of Ru(II) complexes, theabsorption centered at 456 nm of   Ru-Fc  is stronger( ε  ¼  2.06    10 4 cm  1 M  1 ) than that of [Ru(bpy) 2 (COOH-bpy)] 2 þ ( ε  ¼  1.60    10 4 cm  1 M  1 ). Due to the presence of effective PETprocess in  Ru-Fc , the luminescence of   Ru-Fc  is almost negligiblewith excitation at 456 nm. However, upon reaction with HOCl toform [Ru(bpy) 2 (COOH-bpy)] 2 þ , the complex becomes stronglyluminescent, and emits bright red luminescence centered at626 nm with excitation at 456 nm (Fig. S9). The luminescencequantum yield ( f ) of [Ru(bpy) 2 (COOH-bpy)] 2 þ is ~39.5-fold higherthan that of   Ru-Fc .  3.2. PL response of Ru-Fc towards HOCl At  fi rst, the PL response dynamics of   Ru-Fc  towards HOCl wasevaluated in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4.Fig. 1A represents the changes of PL intensity of   Ru-Fc  at 626 nmupon multistep additions of HOCl. As shown in Fig. 1,  Ru-Fc exhibited a weak and stable luminescence under the excitation at456 nm. Upon addition of HOCl, remarkable luminescenceenhancement was observed within a few seconds, and thenreached a platform and maintained relatively stable. Anotheraddition of HOCl led to the increase of luminescence again andreached another maximum value. This result indicates that  Ru-Fc can quickly respond to HOCl, which will be bene fi cial for the real-time detection of short-lived HOCl in complicated biosystems.Toevaluate the effectof pH onthe PLresponseof   Ru-Fc towardsHOCl, changes of PL intensities of   Ru-Fc  in the absence andpresence of HOCl at different pH values were recorded in 25 mMPBS-ethanol (3:1, v/v) buffer. As shown in Fig. S10, the PL intensityof   Ru-Fc  was pH-independent in the range of pH 4 e 11. Upon re-actionwith HOCl,theluminescenceenhancementwassigni fi cantly Scheme 1.  Design of luminescent chemosensor,  Ru-Fc , for selective and sensitive detection of HOCl. Fig.1.  (A) Time course of the luminescence response of   Ru-Fc  (10  m M) towards HOCl in25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4 (2.0  m M HOCl solution was added  fi vetimes). (B) PL intensities of   Ru-Fc  (10  m M) upon reactionwith various ROS/RNS (60  m M)and metal ions (60  m M) in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4. (a) blank,(b) HOCl, (c)   OH, (d) H 2 O 2 , (e) O 2  , (f)  1 O 2 , (g) ONOO  , (h) NO 2  , (i) NO 3  , (j) NO, (k)Fe 2 þ , (l) metal ions (including Mg 2 þ , Zn 2 þ , Fe 3 þ , Mn 2 þ , Na þ , K þ , Ni 2 þ , Cd 2 þ , Co 2 þ , Pb 2 þ and Cu 2 þ ). L. Cao et al. / Biomaterials 68 (2015) 21 e  31 24  higher at pH 7 e 8 than at other pH values. Nevertheless, 10.4-foldand 50.0-fold enhancements of PL intensities were still observedat pH 4 and pH 11, respectively, indicating that  Ru-Fc  can be usedforthedetectionofHOClinweaklyacidic,neutralandbasicbuffers.The PL response speci fi city of   Ru-Fc  towards HOCl was evalu-ated in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4. As shownin Fig. 1B, the PL intensity of   Ru-Fc  was greatly increased uponreactionwith HOCl,while noobvious changes ofPL intensityof   Ru-Fc  were observed in the presence of other reactive species(including H 2 O 2 , O 2  ,   OH, 1 O 2 , ONOO  , NO, NO 3  , NO 2  , Fe 2 þ ) andbiologically relevant metal ions (including Mg 2 þ , Zn 2 þ , Fe 3 þ , Mn 2 þ ,Na þ , K þ , Ni 2 þ , Cd 2 þ , Co 2 þ , Pb 2 þ and Cu 2 þ ). These results reveal thatthe PL response of   Ru-Fc  towards HOCl is highly speci fi c withoutinterferences of other species, which enables it to be used for thehighly selective detection of HOCl in complicated bioisystems.To evaluate the applicability of   Ru-Fc  for the quantitativedetection of HOCl, the emission spectra of   Ru-Fc  in the presence of different concentrations of HOCl were recorded in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4. As shown in Fig. 2A, uponreactionwithdifferentconcentrationsofHOCl,theluminescenceof  Ru-Fc  was gradually increased with up to 60-fold enhancement.The dose-dependent luminescence enhancement showed a goodlinearity in a HOCl concentration range of 2 e 15  m M (Fig. 2B). The detection limit, calculated according to the method de fi ned byIUPAC, is 38.6 nM, indicating that  Ru-Fc  can be used as a chemo-sensor for the highly sensitive detection of HOCl.  3.3. ECL response of Ru-Fc towards HOCl Following the discovery in 1972 by Bard that [Ru(bpy) 3 ] 2 þ iselectrochemiluminescent, considerable efforts have been made indevelopments of the ruthenium(II) complexes so as to employthem on ultrasensitive ECL detections of a wide variety of analytes[64]. In this work, to con fi rm whether the ECL intensity of   Ru-Fc also responds to the HOCl concentration changes, the changes of ECL intensity of   Ru-Fc  upon reaction with different concentrationsof HOCl in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4 con-taining 10 mM of TPrA were recorded. As shown in Fig. 3A, the ECL intensity of   Ru-Fc  (3.33  m M) was also obviously increased with upto 21-fold enhancement when the HOCl concentration wasincreased from 0.33 to 3.33  m M. The detection limit, calculated asthe concentration corresponding to triple standard deviations of  Fig. 2.  (A) Excitation and emission spectra of   Ru-Fc  (10  m M) in the presence of different concentrations of HOCl (0.0, 2.0, 5.0, 8.0, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70,100,150 and 200  m M) in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4. (B) Plot of PL intensity of   Ru-Fc  (10  m M) at 626 nm against HOCl concentration (the inset shows thecalibration curve for the PL detection of HOCl). Fig. 3.  (A) ECL intensity changes of   Ru-Fc  (3.33  m M) upon reaction with differentconcentrations of HOCl (0, 0.33, 0.67, 1.33, 2.00, 2.67 and 3.33  m M) in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4 containing 10 mM TPrA (the inset shows thecalibration curve for the ECL detection of HOCl). The voltage of cyclic voltammetry wasset up between 0.2 and 1.8 V, and scan rate of ECL was 0.15 v/s. (B) ECL intensities of  Ru-Fc  (3.33  m M) upon reaction with HOCl (3.33  m M), various ROS/RNS (16.7  m M) andmetal ions (16.7  m M) in 25 mM PBS-ethanol (3:1, v/v) buffer with pH 7.4 containing10 mM TPrA. (a) blank, (b) HOCl, (c)   OH, (d) H 2 O 2 , (e) O 2  , (f)  1 O 2 , (g) ONOO  , (h)NO 2  , (i) NO 3  , (j) NO, (k) Fe 2 þ , (l) metal ions (including Mg 2 þ , Zn 2 þ , Fe 3 þ , Mn 2 þ , Na þ ,K þ , Ni 2 þ , Cd 2 þ , Co 2 þ , Pb 2 þ and Cu 2 þ ). L. Cao et al. / Biomaterials 68 (2015) 21 e  31  25
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