Bioluminescent Probe for Hydrogen Peroxide Imaging in Vitro and inVivoWenxiao Wu,† Jing Li,† Laizhong Chen,† Zhao Ma,† Wei Zhang,† Zhenzhen Liu,† Yanna Cheng,‡

Lupei Du,† and Minyong Li*,†

†Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan,Shandong 250012, China‡Department of Pharmacology, School of Pharmacy, Shandong University, Jinan, Shandong 250012, China

*S Supporting Information

ABSTRACT: Reactive oxygen species (ROS) often havesignificant roles in mediating redox modifications and otheressential physiological processes, such as biological processregulation and signal transduction. Considering that H2O2 is asubstantial member of ROS, detection and quantitation of H2O2undertakes important but urgent responsibility. In this report, abioluminescent probe for detecting H2O2 was well designed,synthesized, and evaluated. This probe was designed into threeparts: a H2O2-sensitive aryl boronic acid, a bioluminescentaminoluciferin moiety, and a self-immolative linker. Afterextensive evaluation, this probe can selectively and sensitivelyreact with H2O2 to release aminoluciferin. It should be pointedout that this probe is a potential bioluminescent sensor for H2O2since it can provide a promising toolkit for real-time detection of the H2O2 level in vitro, in cellulo, and in vivo.

Reactive oxygen species (ROS) refer to the oxygencontaining substances whose chemical character is active

that exist in the living organisms or the natural environment. Sofar, they are classified into four groups: (a) excited state oxygenmolecule (singlet state oxygen molecule, 1O2); (b) oxygen-related free radicals (superoxide anion free radical O2−•;hydroxyl radical, HO•; hydroperoxyl radical, HO2

•); (c)hyperoxides (hydrogen peroxide, H2O2; lipid peroxide,ROO•); (d) nitrogen oxide (nitric oxide, NO). H2O2 is avital member for cell signaling, energy synthesis, cell growth,living material production, cell metabolism, and so on.1−3

However, the imbalance induced by H2O2 production canimpair tissues and organ systems and then lead to aging,injuries, and diseases, such as malignant tumor, cardiovasculardisease, Alzheimer’s disease, Parkinson’s syndrome, etc.4−6

Therefore, H2O2 is a strategic attention of study into themolecular mechanisms underlying the development andprogression of a disease. Given the short lifetime, lowconcentration of the steady state, and high reactivity of H2O2,it is challenging to capture and detect H2O2 in vivo. In thiscontext, developing a sensitive, selective, and biocompatibleprobe for detecting H2O2 in cells and tissues is one of thethought-provoking topics.7

So far, there are various methods for detecting H2O2,including electron spin resonance, chemiluminescence, spec-trophotometry, fluorescence, bioluminescence, and so on. Inview of the steadfast sensitive, convenient, and noninvasiveadvantages for understanding in vivo biology, fluorescence and

bioluminescence are the most used methods. Moreover, laserconfocal imaging and living imaging technology contribute toaccomplishing active substances visualized in cells but under-standing distinctive characteristics in in vivo biology to be real-time online.8−10

Bioluminescent imaging (BLI) is a reliable, sensitive,appropriate, and noninvasive imaging technique and has beenenormously practical on imaging various processes envisionedin life sciences. Generally, BLI employs firefly luciferase and itshigh-specific substrate luciferin or aminoluciferin to producelight in the presence of ATP, O2, and Mg2+.11 It has well beenrecognized that the 6′-amino (or 6′-hydroxyl) group of D-aminoluciferin (or luciferin) is essential for enzyme combina-tions. Consequently, the caging at 6′-position of aminoluciferin(or luciferin) can hinder its recognition with firefly luciferase aswell as quench the release of bioluminescence.12,13 Forexample, we have successfully developed several APN bio-luminogenic probes by conjugating D-aminoluciferin with theAPN recognition amino acids.14 On the basis of the samestrategy, Chang and co-workers recently have presented ahighly efficient reaction-based approach by utilizing the uniquereaction between H2O2 and the boronate moiety: a H2O2 D-luciferin probe has been devised and used for biologicalapplication.15

Received: June 30, 2014Accepted: September 5, 2014Published: September 5, 2014

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The bioluminescence emission peak for D-luciferin andaminoluciferin were 565 and 584 nm, respectively. It should beunderlined that the above-mentioned red-shifted biolumines-cence profile for aminoluciferin is of significance for penetratingtissues in live animal imaging.13 In the current study, wedesigned a H2O2 bioluminescent probe (probe 3) by couplingboronic acid group with D-aminoluciferin at the 6′-position bya self-immolative linker. This probe is a poor substrate forfirefly luciferase, which leads to the weak bioluminescence;however, in the presence of H2O2, the caging group is proposedto be cleaved to release the free D-aminoluciferin, simulta-neously. The free D-aminoluciferin will experience a subse-quent recognition with the firefly luciferase to produce thebioluminescence (Scheme 1). The quantitative relationshipbetween the bioluminescence intensity and the concentrationof H2O2 has been confirmed herein, as well.

■ EXPERIMENTAL SECTIONSynthesis. The preparation of probe 3 (Figure 1) was

conveniently reached by three steps. Briefly, the synthesis starts

with the preparation of intermediate 1 using an one-potreaction: 5-aminobenzo[d]thiazole-2-carbonitrile as the startingmaterials by reacting with bis(trichloromethyl)carbonate(BTC) and 4-bromobenzyl alcohol. Compound 1 reactedwith bis(pinacolato)diboron via the catalysis of Pd(dppf)Cl2under a dry and anaerobic atmosphere to obtain the pureboronate intermediate 2 after column separation. Probe 3 wasprovided with the cross-coupling reaction of 2 and D-cysteinehydrochloride under N2 atmosphere without light. The detailedpreparation of these probes is described in the SupportingInformation.Bioluminescent Assay Instrumentation. Millipore water

was used to prepare all aqueous solutions. Measurements forbioluminescent assays were performed in 50 mM Tris buffer,

pH 7.4, with 10 mM MgCl2 and 0.1 mM ZnCl2 at 37 °C. Thebioluminescence images of the cells were captured by using aZeiss Observer A1 microscopy implemented with a cooledCCD camera (Hamamatsu ORCA-R2). The bioluminescencespectra were determined with an IVIS Kinetic (Caliper LifeSciences) equipped with a cooled CCD camera that was usedfor bioluminescent imaging. Luciferase was purchased fromPromega. ATP and catalase were purchased from Aladdin. Thepseudocolored bioluminescent images (in photons/s/cm2/scr)were superimposed over the gray scale photographs of theanimals. Circular ROIs were drawn over the areas andquantified using Living Image software. The results werereported as total photon flux within an ROI in photons persecond.

Kinetic Analysis. A volume of 50 μL of probe 3 (10 μM)solution was added to 50 μL of ROS (1 mM) solution in theTris buffer (50 mM, pH 7.4) and incubated at 37 °C for 30 minin 96-well plate. The fluorescent properties of probe 3 andaminoluciferin were recorded. The secondary rate constant (k)was determined by following the methods described in theliterature. A volume of 50 μL of H2O2 probe 3 (10 μM) and 50μL of H2O2 between 1 and 4 mM were used. The assays wereperformed in triplicate.

Selectivity Measurement. A volume of 50 μL of probe 3 (20μM) and the same volume of ROS (200 μM) (H2O2, O2

−,TBHP, t-BuO•, ClO−, •OH) added into a white 96-well plate,buffer solution as a control. After various time (10, 20, 30, 40,50, 60 min) incubation, added commercially available luciferase(2 μg/100 μL) and ATP (2 mM), the mixture solutions cameout to initiate bioluminescence and then measured thebioluminescence intensity. As a blank control, Tris buffer wasadded instead of ROS solution under the same condition.Relative total photon flux for each condition was calculated bydividing the total photon flux for the experimental condition bythe total photon flux for the blank control.

Measurement for Different H2O2 Concentration. A volumeof 50 μL of H2O2 probe 3 (20 μM) was added to 50 μL ofvarious concentrations of H2O2 (0, 10, 20, 50, 100, 200, 500μM) in the Tris buffer (pH 7.4) and incubated at 37 °C for 5,20, 40, and 60 min in a white 96-well plate, then added 100 μLof luciferase (100 μg/mL) with ATP (2 mM), and measuredthe bioluminescence intensity.

Influence of ROS to the Properties of Aminoluciferin andLuciferase Bioluminescence. A volume of 50 μL of amino-luciferin (20 μM) was added to 50 μL of various ROS (200μM) in the Tris buffer (pH 7.4) and incubated at 37 °C for 0, 5,20, 30, 40, 50, and 60 min in a white 96-well plate, then added100 μL luciferase (100 μg/mL) with ATP (2 mM), andmeasured the bioluminescence intensity.

Cell Culture. ES-2 cells (human ovarian cancers cell line)were purchased from the Committee on Type CultureCollection of Chinese Academy of Sciences. ES-2 cellsexpressing firefly luciferase (Fluc) were supplied by Cellcyto.The ES-2-luc cells were cultured in RPMI 1640 supplementedwith 10% fetal bovine serum (FBS) at 37 °C in a humidifiedatmosphere in a 5% CO2 incubator.

Cell Bioluminescence Imaging of Exogenous H2O2. Cellswere grown in black 96-well plates (4 × 105 cells per well).After a 24-h incubation period, the medium was removed andcells were treated with 100 μL of various concentrations ofH2O2 (range 2.5 to 500 μM) and 100 μL of probe 3 (100 μM),luciferase activity was measured 15 min later using a XenogenIVIS Spectrum imaging system. Luminescent signal (photons

Scheme 1. Design Strategy for H2O2-Mediated Release ofAminoluciferin from Probe 3

Figure 1. Structure of the probe 3.

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per second) for each well was measured and plotted as averagevalues (experiments conducted in triplicate).Cell Bioluminescence Imaging of Endogenous H2O2. Cells

were grown in black 96-well plates (4 × 105 cells per well).After a 24-h incubation period, the medium was removed, andcells were treated with 100 μL of various concentrations ofcisplatin (range 0 to 400 μM). After incubating for 12 h in theincubator, 100 μL of probe 3 (100 μM) was added to each welland the luciferase activity was measured 5 min later using aXenogen IVIS Spectrum imaging system. Luminescent signal(photons per second) for each well was measured and plottedas average values (experiments conducted in triplicate).Subcellular Localization Study. Cells were grown in glass-

bottom dishes (4 × 105 cells). The bioluminescence images ofthe cells were captured by using a Zeiss Observer A1microscopy implemented with a cooled CCD camera(Hamamatsu ORCA-R2).(A) After a 36-h incubation period, the medium was

removed, cells were washed with RPMI 1640 (without FBS)twice, 1 mL of probe 3 (20 μM) was added to the dish, andluminescence was recorded for 6 s.(B) After a 36-h incubation period, the medium was removed

and cells were treated with 3 mL of H2O2 (30 μM) for 30 min,the medium was removed, cells were washed with RPMI 1640(without FBS) twice, 1 mL of probe 3 (20 μM) was added tothe dish, and luminescence was recorded for 6 s.(C) After a 24-h incubation period, the medium was

removed and cells were treated with 3 mL of cisplatin (30 μM).After incubating for 16 h in the incubator, the medium wasremoved, cells were washed with RPMI 1640 (without FBS)twice, 1 mL of probe 3 (20 μM) was added to the dish, andluminescence was recorded for 6 s.Mice Model. Balb/c-nu male mice, 8 weeks of age, were

purchased from the Animal Center of China Academy ofMedical Sciences (Beijing, China). To generate tumorxenografts in mice, ES-2-luc cells (1 × 107) were implantedsubcutaneously under the right armpit region of each 4-week-old female nude mouse. Mice were single or group-housed on a12:12 light−dark cycle at 22 °C with free access to food andwater. Tumors were allowed to grow for 2 weeks beforeimaging. All animal studies were approved by the EthicsCommittee of Qilu Health Science Center, ShandongUniversity, and conducted in compliance with Europeanguidelines for the care and use of laboratory animals.In Vivo Bioluminescence Imaging of Exogenous H2O2. Day

1: Mice bearing ES-2-luc subcutaneous tumors wereanesthetized with isoflurane and intraperitoneally injectedwith 0.2 mL of probe 3 (5.3 mM), followed immediately bybioluminescent imaging every 5 min for 30 min. Thebioluminescent signal changed with the time.Day 2: The mice were intraperitoneally injected with 0.2 mL

of H2O2 (98 mM, diluted by normal saline) followed by anintraperitoneal injection of 0.2 mL of probe 3 (5.3 mM, dilutedby normal saline−DMSO 1:1). Controls for the exogenousH2O2 experiment were completed by intraperitoneally injectingvehicle (0.2 mL) followed by the probe. Subsequently, lightproduction was measured 15 min after the injection of probe 3.The relative total photon flux for each condition was calculatedby dividing the total photon flux after the injection ofexogenous H2O2 or vehicle by the total photon flux beforethe injection of exogenous H2O2 or vehicleIn Vivo Bioluminescence Imaging of Endogenous H2O2.

Day 1: Mice bearing ES-2-luc subcutaneous tumors were

anesthetized with isoflurane and intratumorally injected with 50μL of probe 3 (5.3 mM), followed immediately by bio-luminescent imaging every 5 min for 30 min. The bio-luminescent signal changed with the time.Day 2: The mice were intratumorally injected with 50 μL of

cisplatin (333 μM, diluted by normal saline) followed byintratumorally injected with 50 μL of probe 3 (5.3 mM, dilutedby normal saline−DMSO 1:1) 12 h later. Controls for thecisplatin experiment were completed by intratumorally injectingvehicle (50 μL) followed by the probe 3, 12 h later.Subsequently, light production was measured 10 min afterthe injection of probe 3. The relative total photon flux for eachcondition was calculated by dividing the total photon flux afterthe injection of cisplatin or vehicle by the total photon fluxbefore the injection of cisplatin or vehicle.

■ RESULTS AND DISCUSSIONKinetic Analysis. The reactions were monitored by the

change in fluorescence wavelength of the probe before and afterreacting with various ROS (Figure 2). Compared with the

fluorescent property of the aminoluciferin, this evidenceindicated that the caged group of probe 3 was removed uponreaction with ROS and free aminoluciferin was released, in themeanwhile the maximum emission peak experienced a red shiftfrom 450 to 525 nm. As a result, fluorescence intensity waschanged significantly before and after the addition of ROS.

Figure 2. (a) Fluorescent property of aminoluciferin and probe 3, 5μM, λex = 345 nm. (b) The fluorescent intensity of probe 3 added invarious ROS, probe 3, 5 μM; ROS, 500 μM; λex = 345 nm.

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The secondary rate constant (k) was measured based on thechange of the fluorescent property of probe 3 before and afterdegradation. A volume of 50 μL of H2O2 whose concentrationwas from 1 to 4 mM was added to 50 μL of probe 3 (10 μM),then the fluorescence intensity was measured every 5 s for 20min. As time passed, the fluorescent intensity of the mixture at450 nm was decreased and that at 525 nm was increased clearly.Pseudo first order reaction rate eq 1 of the fluorescence is

= − ′F F k tln( / )t max (1)

Ft, the fluorescence intensity (450 nm) at t seconds; Fmax, thefluorescence intensity (450 nm) at the beginning; k′, pseudofirst-order reaction rate constant.The second order reaction rate constant can be obtained

through eq 2 (Figure 3):

′ =k KM (2)

M, the concentration of H2O2; K = 0.52 ± 0.08 M−1 s−1. As weknow, the smaller the K, the slower the reaction. The small K ofthe probe makes the contribution to real-time H2O2 detection.Selectivity Measurement. Next, the activity and selectivity of

the probe was examined via a comparison of the bio-luminescence intensities of the products achieved by theprobe reacted with different ROS. As a result, the solutionmixture came out to initiate bioluminescence. Figure 4exhibited that addition of H2O2 presented a 11-fold increase

than the control in bioluminescent signal over an hour; therewas little to no increase in signal when the boronic acid probereacted with the other ROS. These attractive results indicatethat probe 3 has the reasonable activity and selectivity toidentify H2O2.

Measurement for Different H2O2 Concentration. Tofurther confirm the sensitivity of the probe, the bio-luminescence of the probe was measured by adding diverseconcentrations of H2O2. Figure 5a,b suggested that the

Figure 3. Linear relationship between pseudo first-order rate constant(k′) and the concentration of H2O2.

Figure 4. Relative total photon flux of probe 3 with ROS for varioustime incubation after adding luciferase and ATP.

Figure 5. (a) Change of the probe 3 (10 μM) relative bioluminescenceintensity after added diverse concentrations H2O2; (b) linearrelationship between the relative bioluminescence intensity of theprobe 3 and the concentration of H2O2 (R

2 = 0.993); (c) change ofaminoluciferin (10 μM) relative bioluminescence intensity after addeddiverse ROS at a different point.

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bioluminescence intensity of probe 3 increased following theincreased concentration of H2O2 within a certain range.Moreover, varying from 5 to 100 μM of hydroperoxide, therelative total photon flux could reflect a good “linear” growth(R2 = 0.993), which can be used to quantify the concentrationof H2O2.Influence of ROS to the Properties of Aminoluciferin of

Luciferase Bioluminescence. To determine if the ROS canaffect aminoluciferin and luciferase, their bioluminescenceactivities were collected using the same method as above.Most of the ROS have no obvious influence on aminoluciferinand luciferase; however, after adding 1O2, the bioluminescenceintensity decreased in a time-dependent manner (Figure 5c).We speculated that 1O2 might disturb the excited electronintermediate that was produced during the bioluminescenceprocess; thus the bioluminescence character changed.Cell Bioluminescence Imaging. On the basis of our in vitro

result, the activity of probe 3 was further verified for detectingexogenous H2O2 in living cells. As depicted in Figure 6, the

activity of probe 3 did not change in the living cells; what’smore, the bioluminescence intensity is dose-dependent on theconcentration of H2O2. Given the above results, we do believethat our probe 3 can be employed to trace H2O2 (as low as 2.5μM) in the living cell.We next examined whether the probe could have detection

efficacy for the endogenous H2O2 in living cells. It is well-known that cisplatin is a potent and broad-spectrum antitumoragent, especially for ovarian cancer. Considering that cisplatincan induce the oxidative stress to produce endogenous H2O2for killing tumor cells,16 we choose cisplatin as the cell stimulusfor the endogenous H2O2. The cells were treated with various

concentrations of cisplatin for different incubation times. As aresult, 12 h is the optimum (Figure S1 in the SupportingInformation). Bioluminescence intensity of the cells incubatedwith various concentrations of cisplatin for 12 h was measuredat different time points, and the peak of luminescent signal isfrom 4 to 7 min (Figure S2 in the Supporting Information). Asdepicted in Figure 7, the bioluminescence intensity was

increased with the induction of cisplatin, especially in theconcentration range between 17 and 50 μM; the intensity withinduction is two times than that without induction. However, atthe high concentration of cisplatin (50−400 μM), thebioluminescence signal was gradually decreased, which maybe caused by the strong cytotoxicity of cisplatin that diminishesthe cell number. Therefore, the change of the concentration ofendogenous H2O2 in living cells can be detected by probe 3,even if the H2O2 was produced by cells in normal condition. IfH2O2 was removed by free radical scavenging agents, thebioluminescence intensity is weaker than the control (Figure S3in the Supporting Information). All results indicate that theprobe has potential to quantify the amount of H2O2 in a realtime manner.The bioluminescence imaging ability of probe 3 was well

examined for detecting cell-based exogenous or endogenousH2O2. Figure 8 exhibits the imaging potential of probe 3without and with exogenous H2O2 and endogenous H2O2 inthe ES-2-luc cells, respectively. These interesting resultsindicate that the lipid solubility of probe 3 could lead to thereasonable membrane permeability. This property can ensurethat this probe can easily move across into cells and release thebioluminescence signal upon reacting with H2O2, which impliesthe various concentrations of H2O2 in the cell.

Figure 6. Imaging the exogenous H2O2 activity in ES-2-luc cells (a)bioluminescence imaging of ES-2-luc cells incubated with probe 3 andvarious concentrations of H2O2; (b) quantification of bioluminescentsignal of part a.

Figure 7. Imaging the endogenous H2O2 activity in ES-2-luc cells. (a)Bioluminescence imaging of ES-2-luc cells incubated with probe 3 andvarious concentrations of cisplatin and (b) quantification of bio-luminescent signal from part a.

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In Vivo Bioluminescence Imaging. To investigate whetherthis probe has detection efficacy for H2O2 in vivo, a livinganimal imaging test was well conducted. In such a case, thetumor xenografts in nude mice were used as the animal model.A volume of 0.2 mL of H2O2 (98 mM) solution wasintraperitoneally injected as the exogenous H2O2, and then0.2 mL of probe 3 (5.3 mM) was intraperitoneally injected.Controls for the exogenous H2O2 experiment were completedby intraperitoneally injecting vehicle (0.2 mL). After 15 min,light production was measured. The relative total photon fluxfor each condition was calculated by dividing the total photonflux after the injection of H2O2 or vehicle by the total photonflux before the injection of H2O2 or vehicle. The relative totalphoton flux increased significantly after the injection of H2O2.The results in Figure 9 further confirmed that our probe couldimage exogenous H2O2 activity in xenografted breast cancertumors in mice.Interestingly, mice treated with only probe in the absence of

exogenous H2O2 also displayed modest but measurablebioluminescence in xenografted breast cancer tumors, whichsuggests that H2O2 may detect basal levels of H2O2 produced inthese living animals. Therefore, to determine whether thisemission signal was due in part to the detection of endogenousH2O2, we intratumorally injected 50 μL of cisplatin as theexternal stimulation for endogenous H2O2 before the probe wasintratumorally injected. It is observed that the cisplatin-treatedanimals exhibited a significantly increasing bioluminescentsignal compared to vehicle control animals (Figure 10). Theabove results suggest that probe 3 can release a time-dependence bioluminescence in vivo.

■ CONCLUSIONSIn conclusion, a novel bioluminescent probe was well designed,prepared, and evaluated for H2O2 imaging herein. After carefulevaluation, our probe paraded high selectivity and sensitivity forhydroperoxide in vitro, in cellulo, and in vivo in the presence offirefly luciferase. The boronic acid recognition group in thisprobe can respond to the H2O2 quickly and then the structureand optical property of the probe change timely, and as a result,probe 3 can identify the H2O2 in a real time manner. Inaddition, the reasonable histocompatibility and long bio-

luminescence wavelength of this probe may contribute to thefeasible application in the animals. Overall, all experimentalresults suggest that this bioluminescent probe stipulates aselective and sensitive platform for real-time imaging of bothexogenous and endogenous hydrogen peroxide in living mice.Furthermore, the influence of the levels of hydrogen peroxideon health, aging, and disease may be illuminated in theupcoming future by utilizing such a bioluminescent approach.We do believe that this bioluminescence probe could provide aconvenient toolkit for the longitudinal noninvasive monitoring

Figure 8. Subcellular localization study with probe 3 of ES-2-luc cells:(A) probe 3 (20 μM); (B) probe 3 with exogenous H2O2; (C) probe 3with endogenous H2O2. (a) Bright-field images, expose time,10 ms;(b) cells stained by probe 3, expose time, 6 s; (c) merged images.Objective lens: 40×.

Figure 9. Bioluminescence imaging of exogenous H2O2 activity withES-2-luc tumors in nude mice. (a) Integrated bioluminescenceemission for mice with probe 3, in the presence or absence of theexogenous H2O2. (b,c) Representative time-course images from onemouse treated with H2O2 or the control.

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of oxidative stress caused by the excess amount of H2O2 indiagnostic and therapeutic fields.

■ ASSOCIATED CONTENT*S Supporting InformationThe details for preparation of probe and their NMR and HR-MS spectra. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: mli@sdu.edu.cn. Phone/fax: +86-531-8838-2076.Author ContributionsW.W. and J.L. contributed equally to this work.The manuscript was written through contributions of all

authors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by grants from the National Programon Key Basic Research Project (Grant No. 2013CB734000),the Program of New Century Excellent Talents in University(Grant No. NCET-11-0306), the Program for ChangjiangScholars and Innovative Research Team in University (GrantNo. IRT13028), the Shandong Natural Science Foundation(Grant No. JQ201019), and the Independent InnovationFoundation of Shandong University, IIFSDU (Grant Nos.2014JC008 and 2012JC002).

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Figure 10. Bioluminescence imaging of endogenous H2O2 activitywith ES-2-luc tumors in nude mice. (a) Integrated bioluminescenceemission for mice with probe 3, in the presence or absence of thecisplatin. (b,c) Representative time-course images from one mousetreated with cisplatin or the control.

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