A Doubly-Quenched Fluorescent Probe for Low-Background Detection

Subscriber access provided by UNIV OF LOUISIANA

Article

A Doubly-Quenched Fluorescent Probe for LowBackground Detection of Mitochondrial H2O2 Jun Liu, Jingjing Liang, Chuanliu Wu, and Yibing Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01294 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A Doubly-Quenched Fluorescent Probe for Low-Background Detection of Mitochondrial H2O2

Jun Liu+, Jingjing Liang+, Chuanliu Wu*, and Yibing Zhao*

Department of Chemistry, College of Chemistry and Chemical Engineering, The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Xiamen University, Xiamen, 361005, P.R. China. +These

authors contributed equally to this work

*Corresponding Author; Email: [email protected]; [email protected]

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 32

ABSTRACT: Hydrogen peroxide (H2O2) is an important product of oxygen metabolism which plays a crucial role in regulating a variety of cellular functions. Fluorescent probes have made a great contribution to our understanding of the biological role of endogenous H2O2. However, fluorescent probes for H2O2 featuring aryl boronates might suffer from the moderate turn-on fluorescence responses. Strategies that can reduce the background fluorescence of these boronate-masked probes would significantly improve the sensitivity of the endogenous H2O2 detection. In this work, we proposed a general and reliable double-quenching concept for the design of fluorescent probes with low-background fluorescence. A new fluorescent probe was developed for the detection of endogenous H2O2 in mitochondria of live cancer cells. This probe exploits a boronate-driven lactam formation and an eliminable quenching moiety simultaneously (i.e., the doubly-quenching effect) to reduce the background fluorescence, which ultimately results in the achievement of a >50-fold fluorescence turn-on. A linear concentration range of response between 1 and 60 μM and a detection limit of 0.025 μM can be obtained. This study not only presents a highly sensitive fluorescent probe for the detection of H2O2, but provides a new concept for the design of fluorescent probes with a previously unachievable fluorescence off-on response ratio for other types of ROS and many other biologically relevant analytes.

2

ACS Paragon Plus Environment

Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction As a typical reactive oxygen species (ROS), hydrogen peroxide (H2O2) is an essential oxygen metabolite mainly generated from the active triphosphopyridine nucleotide (NADPH) oxidase enzymes residing in virtually all cells.1-4 Mounting evidence supports its role as an important marker for oxidative stress and a messenger in cellular signal transduction for various physiological processes.5-9 At the level of cell organelles, mitochondria are the major sites for H2O2 production, and mitochondrial H2O2 plays a crucial role in cell survival, growth, differentiation, and maintenance during physiological and pathological conditions.10-11 However, aberrant production or accumulation of H2O2 within cellular mitochondria over time due to oxidative stress and/or genetic mutations is closely related to many human diseases such as cancer, diabetes, obesity, neurodegenerative disorders, and stroke.12-15 Moreover, emerging evidence suggests that mitochondrial H2O2 can also be beneficial to cell survival, growth, differentiation, and maintenance.16-18 Therefore, the crucial effects of H2O2 homeostasis on human health and diseases provide a motivation to develop novel, special, localizable, and readily deployable tools for monitoring and mapping endogenous H2O2 production in mitochondria.19-22

3 ACS Paragon Plus Environment


Page 4 of 32

Many strategies have been explored for the detection of H2O2, among which the approach based on fluorescent probes shows the highest specificity and sensitivity for measuring endogenous H2O2 in live cells.23-48 This approach was originally developed in the Chang laboratory, involving a highly specific reaction of an aryl boronate with H2O2 to produce a phenol.33,

35

Generally, the boronate moiety masks the phenol of a fluorescent probe, which

drives the equilibrium of the molecule from an opened carboxyl form into a closed lactam form (Figure 1a), and thus efficiently eliminating the absorptive and emissive property of the molecule. The first-generation probe (PF1) can produce a large fluorescence turn-on response (>1000-fold), because this probe was masked with dual aryl boronates, which can more efficiently promote the carboxyl closing.34 However, the presence of two aryl boronates significantly slows down the turn-on response kinetics. The next-generation probes exploited a single boronate handle for the fluorescence elimination and H2O2 de-masking, which allows for more rapid accumulation of fluorescence signals, and enables the detection of the endogenous H2O2 for the first time.42-43, 46 Unfortunately, the fluorescence-eliminating effect of the monoboronate has been found to be dependent on the type of the fluorophores. And usually, only moderate fluorescence turn-on response was observed for most probes due to either the inefficient fluorescence elimination of the masked molecules or the incomplete shift 4 ACS Paragon Plus Environment

Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the equilibrium towards the closed lactone form (Figure 1a).33 A general strategy that can reduce the background fluorescence of these boronate-masked probes would significantly improve the sensitivity of the endogenous H2O2 detection. In this work, by proposing a general and reliable double-quenching concept which takes advantage of a boronate-driven lactam formation and an eliminable quenching moiety simultaneously, we developed a new fluorescent probe for the detection of mitochondria H2O2 (Mito-FBN; Figure 1b). We found that the introduction of an eliminable dark quencher onto the monoboronate-masked rhodamine fluorophore leads to >5-fold decrease of the background fluorescence due to the energy transfer from the fluorophore to the quencher. As the quencher, 4-dimethylaminoazobenzene-4'-carboxylic acid (DABCYL),49-51 was appended onto the fluorophore through a built-in self-immolative spacer,45,

52

which features a rapid 1,4-

elimination process (Figure 1b), the large turn-on response to H2O2 (>50-fold) does not involve a compromise on the response kinetics, a feature that is substantially different from the firstgeneration bisboronate probes. By modifying the doubly-quenched rhodamine with a mitochondrion-targetable moiety, Murphy’s phosphonium cation,22, 53 we were able to visualize the endogenous H2O2 production in mitochondria of live cancer cells. We believe that our double-quenching concept would be also applicable to the design of fluorescent probes for 5 ACS Paragon Plus Environment


Page 6 of 32

other types of ROS and some other analytes for the achievement of a large turn-on fluorescence response.


Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Experimental Section

Chemicals and apparatus. All chemicals were of analytical grade and used as received without further purification. Deionized water was used throughout this work and the pH was adjusted using diluted hydrochloric acid or sodium hydroxide solutions. All chemicals were purchased from major suppliers such as Sigma-Aldrich (Beijing), Alfa Aesar (Tianjin), Sangon (Shanghai), and J&K (Guangzhou). Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and phosphate buffered saline (PBS) was obtained from Thermo Fisher Scientific (Beijing, China). Eppendorf tubes and cell culture dishes were purchased from JET BIOFIL (Guangzhou, China). Mito Tracker Red CM-H2Ros was purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK-8) was purchased from Sigma-Aldrich (Shanghai, China). All UV-Vis absorption and fluorescence spectra were recorded using a U-3900H spectrophotometer (Hitachi) and an F-7000 fluorescence spectrophotometer (Hitachi), respectively. The 1H NMR spectra were recorded at 500 MHz on a Bruker Advance-500 spectrometer using tetramethylsilane (TMS) as the internal standard. Electrospray ionization mass spectra (ESIMS) were recorded on a Bruker Esquire 3000 plus mass spectrometer. All measurements were performed at room temperature. Fluorescence images of KB cells were obtained using



Page 8 of 32

an Olympus FV1000-MPE multi-photon laser scanning confocal microscope (Japan). MitoFBN was efficiently synthesized following the synthetic methodology shown in the Supporting Information.

Synthesis of the compounds. Rhodamine derivative was exploited as the scaffold fluorophore due to its excellent absorptive and emission properties and the ease of synthesis and modification. The detailed synthetic procedures and product characterizations (Mito-FBN, Mito-FB, and Mito-FN) were provided in the Supporting Information.

Cell culture and imaging. KB cells were maintained in DMEM medium (high glucose) supplemented with 10% FBS and 1% penicillin/streptomycin (penicillin, 10 000 U/mL; streptomycin, 10 000 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were passaged at about 80% cell confluence using a 0.25% trypsin solution. Fluorescent images of the cells incubated with probes were obtained using a Leica TCS SP5X confocal microscope. KB cells were plated at a density of 1 × 105 cells/well into a glass bottom cell culture dish. After incubation for 24 h to make the cells adhere, the medium was removed and the cells were washed with PBS. Then, the cells were incubated with the probes in DMEM at 37 °C and 5% CO2 for 1 h. Next, the cells were thoroughly washed three times with PBS. In


Page 9 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


order to track the cell organelles, the commercial tracer reagent Mito Tracker Red CM-H2Ros was used to stain the mitochondria and multichannel detection was used (Green channel: λex = 488 nm, λem = 500−570 nm; Red channel: λex = 543 nm, λem = 580−620 nm).



Page 10 of 32

a) O O B

O B O

O

* Large turn-on fluorescence response * Slow response kinetics

O O

A first-generation probe

O O B

N

O

* Moderate turn-on fluorescence response O

* Rapid response kinetics O

A second-generation probe

b) O B

Built-in self-immolative spacer

H2O2-responsive handle O N

N

O

O

N

Rhodamine fluorophore

N

O

Eliminable quencher

O

O

N

P

Lactam

Mitochondrion-targeting reagent

A new probe developed in this work (Mito-FBN) O B

O

N N

O

N

O

N

N

O

N O

O

O OH

N O

P

O P

Mito-FN

Mito-FB

Figure 1. (a) Structure of a typical first- and second-generation fluorescent probe for H2O2. (b) Structure of a new fluorescent probe (Mito-FBN; where F represents the fluorophore, B means


Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


boronate, and N stands for the quenching group DABCYL) developed in this work and its controls (Mito-FN and Mito-FB).

Results and Discussion

Details on the synthesis and characterization of fluorescent probes were given in the Supporting Information (Figures S1‒S20). Rhodamine derivative with excellent fluorescence properties were selected as the fluorophore for the design of H2O2 probe.54-56 The fluorescence spectrum of Mito-FBN in the absence and presence of H2O2 were first examined in 10 mM PBS at pH = 7.4. Mito-FBN shows negligible fluorescent emission before reacting with H2O2. However, the addition of hydrogen peroxide leads to a large fluorescence off-on response at 528 nm in 10 mM PBS at pH 7.4 (Figure 2a), and the intensity increases linearly with the concentration of H2O2 from 1 μM to 100 μM (Figure 2b). This sharp fluorescence off– on response is rather desirable for a sensitive detection. For comparison, the fluorescence of Mito-FB was also characterized in PBS (pH = 7.4) (Figure 2c). The background fluorescence signal of Mito-FB is significantly higher than that of Mito-FBN (>5-fold). As shown in Figure 2d, a 51-fold fluorescence enhancement was observed for Mito-FBN after 50 min reaction with 100 μM H2O2; in contrast, only 9.4-fold fluorescence enhancement was observed for Mito-FB. 11 ACS Paragon Plus Environment


Page 12 of 32

In addition, a control probe without the H2O2-reactive boronate handle (Mito-FN) shows no change in fluorescence after the incubation with 100 μM H2O2 (Figure 2d). These results, taken together, demonstrate that the extremely low background fluorescence of Mito-FBN is resulted from the interplay of the boronate-masking and the built-in appending of the quencher. A regression equation: F = 16.8C + 19.3 (R2 = 0.997), can then be obtained with a linear concentration range between 1 and 60 μM. The detection limit was determined to be 0.025 μM (3δ/κ, where δ is the standard deviation of blank measurements, and κ is the slope of the linear regression equation). These promising results show the potential applicability of MitoFBN for the highly sensitive detection of H2O2 under physiological conditions.


Page 13 of 32

a)

b)

1200

1200

100 M 800 0

400

Intensity

Intensity

800

y=16.8c+19.3 R2=0.997

400

0

0 500

550

600

0

650

Wavelength / nm

c)

20

40

60

80 100

Concentration / M

d) 1200

1200 100 M

400

9-fold

Intensity

0

400

51-fold

800

800

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

0 500

550

600

Wavelength / nm

650

Mito-FBN Mito-FB Mito-FN

Figure 2. (a) Fluorescence emission spectra of 5 μM Mito-FBN reacted with different concentrations of H2O2 (0‒100 μM) in phosphate buffers at pH 7.4 (λex = 470 nm). (b) Plots of fluorescence intensity of Mito-FBN (5 μM; λex = 470 nm, λem = 528 nm) as a function of H2O2 concentrations (0−100 μM). (c) Fluorescence emission spectra of 5 μM Mito-FB reacted with different concentrations of H2O2 (0‒100 μM) in phosphate buffers at pH 7.4 (λex = 470 nm). (d)



Page 14 of 32

Fluorescence recovery ratio of 5 μM Mito-FBN, Mito-FB, and Mito-FN upon the treatment with 100 μM H2O2.

The effect of pH on the response behavior of Mito-FBN to H2O2 was also investigated. As shown in Figure 3a, the probe works over a wide pH range. The background fluorescence of Mito-FBN is not pH sensitive, indicating that neither lactam opening nor the elimination of the quencher makes a response to the pH variation (pH 1‒12). After the reaction with H2O2, though the unmasked fluorophore shows pH-sensitive fluorescence emission, its fluorescence intensity under physiological pH (~7.0) and mildly acidic conditions (pH 4‒7) is still very high, which guarantees the high sensitivity of Mito-FBN the detection of endogenous H2O2 under physiological conditions. However, the fluorescence intensity of the unmasked fluorophore decreases significantly under basic conditions, very likely due to the formation of the lactam.55 The study on the response kinetics under different temperatures (4, 25 and 37 °C) shows that the fluorescence of Mito-FBN increases rapidly (about 8-fold and 16- fold turn-on response after 5 and 10 min respectively at 37 °C) by the reaction with H2O2 (Figure 3b). The reaction rate was not very sensitive to the incubation temperature, as only a slight decrease in the rate was observed when the temperature was decreased to 4 °C. The response kinetics of Mito-FB 14 ACS Paragon Plus Environment

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to H2O2 was also examined for comparison (Figure S21), and we found that the presence of the DABCYL can slightly decrease the reaction rate of the probe with H2O2, probably due to the hydrophobic interaction between the H2O2-reactive boronate handle and DABCYL. However, we can still obtain ~90% of fluorescence dequenching for Mito-FBN after ~30 min incubation with 100 μM of H2O2 (Figure 3b), suggesting that the slightly decreased response kinetics should not affect the fluorescence detection and visualization of H2O2.

The high specificity of H2O2 probes is another very important parameter for their potential applications in complex biological systems. Therefore, the fluorescence response of Mito-FBN towards various typical biologically relevant species was assessed. As shown in Figure 3c, except for H2O2, other biologically relevant analytes, including Mg2+, Ca2+, K+, Na+, Zn2+, glutathione (GSH), and cysteine (Cys), caused negligible recovery in the fluorescence of MitoFBN even at a much higher concentration. The fluorescence response to other ROS, including tert-butyl hydroperoxide (TBHP), nitrogen oxide (NO), potassium superoxide (KO2), and hypochlorous acid (HClO), was also negligible (Figure 3c). Thus, the probe exhibited at least 50-fold selectivity toward H2O2 compared to other analytes.



a)

b)

1200

1200

800

Intensity

Intensity

800 +H2O2

400

400

25 oC 37 oC 4 oC

0

0 2

4

6

8

10 12

0

20

pH

40

60

d)

DABCYL

1200

Mito-F

800

DABCYL

60 min Mito-FBN lO C H

N

O

ys C

2

KO

P

TB H

SH G

N

a

+

Zn

M g K+ 2+ 2

a C

an H k 2 O

Bl

2+

2+

400

0

80

Time / min

c)

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 32

0

2

4

6

8

10

Time / min

Figure 3. (a) Fluorescence response of 5 μM Mito-FBN to 100 μM H2O2 under different pHs (1‒12). (b) Change of fluorescence intensity at 528 nm over time under different incubation temperature (4, 25, and 37 °C); λex=470 nm. (c) Fluorescence of 5 μM Mito-FBN in the presence of different foreign species (100 μΜ; from left to right: blank, H2O2, Mg2+, Ca2+, K+, Na+, Zn2+, GSH, Cys, TBHP, NO, KO2, HClO); λex=470 nm, λem=528 nm. (d) HPLC analysis of Mito-FBN treated with H2O2 for 60 min (red line). The peaks at 4.7, 6.9, and 7.7 min correspond to Mito-FBN, Mito-F, and DABCYL, respectively. The blue line is chromatogram of pure DABCYL. 16 ACS Paragon Plus Environment

Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O B

OH N

O N

N

O

N

O

N

O

H2O2

N

O

O

O

N

O

N

O

N

N

O

O P

P

Mito-FBN N

O

O OH O

H2O

HO

HN

OH

P

O

Mito-F

N

O

N

O

1,4-elimination

N

O

N O

OH N N

N

O

N

O

P

N

O

O

O P

N

DABCYL

Figure 4. Mechanism of the reaction between Mito-FBN and H2O2.

The mechanism by which the reaction between H2O2 and Mito-FBN takes place for the rapid and intense turn-on response was proposed and illustrated in Figure 4. This mechanism was 17 ACS Paragon Plus Environment


Page 18 of 32

also supported by the high performance liquid chromatography (HPLC) and mass spectral analysis of the reaction products. As shown in Figure 3d, HPLC chromatograms clearly indicate the complete conversion of Mito-FBN to the free DABCYL and Mito-F after 60 min of reaction with H2O2 (see ESI-MS characterizations shown in Figure S22). Thus, it is very clear that H2O2 reacts with the aryl boronate handle to trigger the 1,4-elimination of the selfimmolative spacer to release the DABCYL quencher and the opening of the lactam (Figure 4), which leads to rapid complete recovery of fluorescence of the rhodamine fluorophore. Considering the rapid response kinetics, the large turn-on fluorescence response, the stable emission property under physiological conditions, and the high specificity to H2O2 response, Mito-FBN should be a low-background and sensitive fluorescent probe for visualizing the endogenous H2O2 in live cells.

KB cells (an epidermoid stomatocarcinoma heteroploid cell line) were selected as model cancer cells for the study. The cytotoxicity of Mito-FBN was first evaluated using CCK-8 assays.57 The viability of cells treated with 5.0 μM Mito-FBN is still high (>80%), suggesting that the probe has a good biocompatibility (Figure S23). The cellular uptake of Mito-FBN and its intracellular response to endogenous H2O2 was then investigated using confocal laser


Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


scanning microscopy. After the incubation with 0.2 μM Mito-FBN for 1 h, green fluorescence was observed inside KB cells (Figure 5a). We observed no obvious change in fluorescence intensity in the cells after washing the probe away and adding 100 µM of exogenous H2O2 for 20 min incubation (Figure 5b). This result suggests very likely that all of the probe molecules taken up by the KB cells had already reacted with the endogenous H2O2. When the probe concentration was increased to 2.0 µM, we found that not only the fluorescence intensity of cells incubated with only the Mito-FBN probe obviously increased, but the addition of exogenous hydrogen peroxide H2O2 can further enhance the cellular fluorescence (Figures 5c and 5d). By contrast, we observed no fluorescence emission from cells incubated with the control probe (Mito-FN) without the boronate handle (Figure S24). These results suggest that our Mito-FBN probe can make a sensitive and rapid response the endogenous H2O2 in live cancer cells. To examine the mitochondrion-targeting capability of Mito-FBN, we carried out a co-localization experiment in KB cells. Co-staining with commercially available mitochondrionspecific probe Mito Tracker Red CM-H2Ros was performed to confirm the subcellular location of the Mito-FBN probe. There are obvious overlaps between the red and green fluorescent regions (Figure 5e), implying that Mito-FBN can efficiently diffuse into the mitochondria, and the intense green fluorescence was an indication of the mitochondrial H2O2 production. These 19 ACS Paragon Plus Environment


Page 20 of 32

results suggest that cancer cells can produce a large amount of endogenous H2O2 in mitochondria without external stimulations.

Temperature is one of the most important parameters affecting the behaviors of live cells; for instance, temperature can influence the bioactivity of biomacromolecules contributing in many cellular processes.58-60 To date, the effect of temperature change on the production of mitochondrial H2O2 still remains unexplored. By taking advantage of our highly sensitive and low-background Mito-FBN, in this work we examined the H2O2 productivity in mitochondria of live cancer cells. First, we incubated cells with Mito-FBN (5 µM) in Dulbecco’s Modified Eagle Medium (DMEM) at 4 °C for 60 min, and we found that the cells are weakly fluorescent (Figures 6a‒6c; three parallel experiments). Then, we washed the probes away and increased the temperature of the cell culture to 25, 37 and 45 °C, respectively. We found that the cell fluorescence increased significantly after ~20 min under the three different temperatures (Figures 6d‒6f). Considering that the kinetics of reaction between Mito-FBN and H2O2 were not sensitive to temperature (Figure 3b), these results suggest that the production of mitochondrial H2O2 in live cancer cells is strongly dependent on the cell culture temperature. As the H2O2 production is an energy-dependent, it is not unexpected that the temperature has


Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a significant effect on the productivity of endogenous H2O2. However, the capability of the Mito-FBN probe to monitor the change of H2O2 production under different temperatures underscores its high sensitivity and specificity for the detection of endogenous H2O2 in complex living systems.



Page 22 of 32

Figure 5. Confocal fluorescence images of KB cells; (a) cells treated with 0.2 μM Mito-FBN for 1 h; (b) cells treated with 0.2 μM Mito-FBN for 1 h first, and then washed away the probes and added exogenous H2O2 (100 µM) for 20 min of incubation; (c) cells treated with 2.0 μM MitoFBN for 1 h; (d) cells treated with 2.0 μM Mito-FBN for 1 h first, and then washed away the probes and added exogenous H2O2 (100 µM) for 20 min of incubation; (e) co-localization experiments of Mito-FBN and MitoTracker Red CM-H2Ros in KB cells. The cells were incubated with Mito-FBN (2.0 µM) for 1 h at 37 ℃ (from left to right: bright-field microscopy image; red fluorescence of MitoTracker Red CM-H2Ros; green fluorescence of Mito-FBN; 22 ACS Paragon Plus Environment

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


merged green and red fluorescence image; co-localization Pearson correlation coefficients: 0.86).



Page 24 of 32

Figure 6. Confocal fluorescence images of KB cells; (a‒c) cells treated with 5 µM Mito-FBN at 4°C for 1 h (three parallel experiments); (b‒f) cells treated with 5 µM Mito-FBN at 4°C for 1 h first, and then washed away the probes and incubated the cells at 20 (b), 37 (e) and 45 (f) °C respectively for 20 min.


Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusions

In summary, we have demonstrated a general and reliable double-quenching strategy for developing a new fluorescent probe for the highly sensitive detection of endogenous H2O2 in mitochondria of live cancer cells. This study not only presents a highly sensitive fluorescent probe for the detection of H2O2, but provides a new concept for the design of fluorescent probes with a previously unachievable fluorescence off-on response ratio for other types of ROS and some other analytes. As new fluorescent tools with improved sensitivities are important to uncovering the specific functions of the analytes (e.g., H2O2) in living systems and to deciphering the possible links between the analytes and specific cellular behaviors, this work would greatly benefit the development of new-generation fluorescent probes with large turn-on responses and the reliable application of these probes in complex living systems.



Page 26 of 32

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website, including the synthesis of fluorescent probes (Mito-FBN, Mito-FB, and Mito-FN) and their MS and NMR characterizations (PDF). ACKNOWLEDGMENT We would like to acknowledge the financial support from the National Natural Science Foundation of China (21675132), the Program for Changjiang Scholars and Innovative Research Team in University (13036) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21521004).


Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References 1.

Stone, J. R.; Yang, S. P., Hydrogen peroxide: A signaling messenger. Antioxid. Redox Signal.

2006, 8 (3-4), 243-270. 2.

Veal, E. A.; Day, A. M.; Morgan, B. A., Hydrogen peroxide sensing and signaling. Mol Cell

2007, 26 (1), 1-14. 3.

Fruehauf, J. P.; Meyskens, F. L., Reactive oxygen species: A breath of life or death? Clin.

Cancer Res. 2007, 13 (3), 789-794. 4.

Bedard, K.; Krause, K. H., The NOX family of ROS-generating NADPH oxidases: Physiology

and pathophysiology. Physiol. Rev. 2007, 87 (1), 245-313. 5.

Sablina, A. A.; Budanov, A. V.; Ilyinskaya, G. V.; Agapova, L. S.; Kravchenko, J. E.;

Chumakov, P. M., The antioxidant function of the p53 tumor suppressor. Nat. Med. 2005, 11 (12), 1306-1313. 6.

Poole, L. B.; Nelson, K. J., Discovering mechanisms of signaling-mediated cysteine oxidation.

Curr. Opin. Chem. Biol. 2008, 12 (1), 18-24. 7.

Schoenfeld, J. D.; Sibenaller, Z. A.; Mapuskar, K. A.; Wagner, B. A.; Cramer-Morales, K. L.;

Furqan, M.; Sandhu, S.; Carlisle, T. L.; Smith, M. C.; Abu Hejleh, T.; Berg, D. J.; Zhang, J.; Keech, J.; Parekh, K. R.; Bhatia, S.; Monga, V.; Bodeker, K. L.; Ahmann, L.; Vollstedt, S.; Brown, H.; Kauffman, E. P. S.; Schall, M. E.; Hohl, R. J.; Clamon, G. H.; Greenlee, J. D.; Howard, M. A.; Schultz, M. K.; Smith, B. J.; Riley, D. P.; Domann, F. E.; Cullen, J. J.; Buettner, G. R.; Buatti, J. M.; Spitz, D. R.; Allen, B. G., O-2(center dot-) and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate (vol 31, pg 487, 2017). Cancer Cell 2017, 32 (2), 268-268. 8.

Schumacker, P. T., Reactive Oxygen Species in Cancer: A Dance with the Devil. Cancer Cell

2015, 27 (2), 156-157. 9.

Groeger, G.; Quiney, C.; Cotter, T. G., Hydrogen Peroxide as a Cell-Survival Signaling

Molecule. Antioxid. Redox Signal. 2009, 11 (11), 2655-2671. 10.

Banh, S.; Treberg, J. R., The pH sensitivity of H2O2 metabolism in skeletal muscle

mitochondria. Febs Lett. 2013, 587 (12), 1799-1804. 11.

Murphy, M. P., How mitochondria produce reactive oxygen species. Biochem. J. 2009, 417, 1-

13. 12.

Balaban, R. S.; Nemoto, S.; Finkel, T., Mitochondria, oxidants, and aging. Cell 2005, 120 (4),

483-495. 13.

Mattson, M. P., Pathways towards and away from Alzheimer's disease. Nature 2004, 430 (7000), 27 ACS Paragon Plus Environment


Page 28 of 32

631-639. 14.

Pop-Busui, R.; Sima, A.; Stevens, M., Diabetic neuropathy and oxidative stress. Diabetes-Metab.

Res. Rev. 2006, 22 (4), 257-273. 15.

Ishikawa, K.; Takenaga, K.; Akimoto, M.; Koshikawa, N.; Yamaguchi, A.; Imanishi, H.; Nakada,

K.; Honma, Y.; Hayashi, J., ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 2008, 320 (5876), 661-664. 16.

Sauer, H.; Rahimi, C.; Hescheler, J.; Wartenberg, M., Role of reactive oxygen species and

phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells. Febs Lett. 2000, 476 (3), 218-223. 17.

Li, J.; Stouffs, M.; Serrander, L.; Banfi, B.; Bettiol, E.; Charnay, Y.; Steger, K.; Krause, K. H.;

Jaconi, M. E., The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation. Mol Biol Cell 2006, 17 (9), 3978-3988. 18.

Niethammer, P.; Grabher, C.; Look, A. T.; Mitchison, T. J., A tissue-scale gradient of hydrogen

peroxide mediates rapid wound detection in zebrafish. Nature 2009, 459 (7249), 996-U123. 19.

Xu, J.; Zhang, Y.; Yu, H.; Gao, X. D.; Shao, S. J., Mitochondria-Targeted Fluorescent Probe for

Imaging Hydrogen Peroxide in Living Cells. Anal. Chem. 2016, 88 (2), 1455-1461. 20.

Ren, M. G.; Deng, B. B.; Zhou, K.; Kong, X. Q.; Wang, J. Y.; Lin, W. Y., Single Fluorescent

Probe for Dual-Imaging Viscosity and H2O2 in Mitochondria with Different Fluorescence Signals in Living Cells. Anal. Chem. 2017, 89 (1), 552-555. 21.

Wen, Y.; Liu, K. Y.; Yang, H. R.; Liu, Y.; Chen, L. M.; Liu, Z. K.; Huang, C. H.; Yi, T.,

Mitochondria-Directed Fluorescent Probe for the Detection of Hydrogen Peroxide near Mitochondria! DNA. Anal. Chem. 2015, 87 (20), 10579-10584. 22.

Dickinson, B. C.; Chang, C. J., A targetable fluorescent probe for imaging hydrogen peroxide in

the mitochondria of living cells. J. Am. Chem. Soc. 2008, 130 (30), 9638-9639. 23.

Chen, X. Q.; Wang, F.; Hyun, J. Y.; Wei, T. W.; Qiang, J.; Ren, X. T.; Shin, I.; Yoon, J., Recent

progress in the development of fluorescent, luminescent and colorimetric probes for detection of reactive oxygen and nitrogen species. Chem. Soc. Rev. 2016, 45 (10), 2976-3016. 24.

Dou, B. T.; Yang, J. M.; Yuan, R.; Xiang, Y., Trimetallic Hybrid Nanoflower-Decorated MoS2

Nanosheet Sensor for Direct in Situ Monitoring of H2O2 Secreted from Live Cancer Cells. Anal. Chem. 2018, 90 (9), 5945-5950. 25.

Shen, R.; Liu, P. P.; Zhang, Y. Q.; Yu, Z.; Chen, X. Y.; Zhou, L.; Nie, B. Q.; Zaczek, A.; Chen,

J.; Liu, J., Sensitive Detection of Single-Cell Secreted H2O2 by Integrating a. Microfluidic Droplet Sensor and Au Nanoclusters. Anal. Chem. 2018, 90 (7), 4478-4484. 28 ACS Paragon Plus Environment

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

26.


Kitte, S. A.; Gao, W. Y.; Zholudov, Y. T.; Qi, L. M.; Nsabimana, A.; Liu, Z. Y.; Xu, G. B.,

Stainless Steel Electrode for Sensitive Luminol Electrochemiluminescent Detection of H2O2, Glucose, and Glucose Oxidase Activity. Anal. Chem. 2017, 89 (18), 9864-9869. 27.

Li, R. X.; Liu, X. M.; Qui, W. L.; Zhang, M. N., In Vivo Monitoring of H2O2 with

Polydopamine and Prussian Blue-coated Microelectrode. Anal. Chem. 2016, 88 (15), 7769-7776. 28. and

Xu, S. X.; Li, X. M.; Li, C. B.; Li, J. L.; Zhang, X. F.; Wu, P.; Hou, X. D., In Situ Generation Consumption

of

H2O2

by

Bienzyme-Quantum

Dots

Bioconjugates

for

Improved

Chemiluminescence Resonance Energy Transfer. Anal. Chem. 2016, 88 (12), 6418-6424. 29.

Wang, L. L.; Zheng, J.; Li, Y. H.; Yang, S.; Liu, C. H.; Xiao, Y.; Li, J. S.; Cao, Z.; Yang, R. H.,

AgNP-DNA@GQDs Hybrid: New Approach for Sensitive Detection of H2O2 and Glucose via Simultaneous AgNP Etching and DNA Cleavage. Anal. Chem. 2014, 86 (24), 12348-12354. 30.

Chen, S.; Hai, X.; Chen, X. W.; Wang, J. H., In Situ Growth of Silver Nanoparticles on

Graphene Quantum Dots for Ultrasensitive Colorimetric Detection of H2O2 and Glucose. Anal. Chem. 2014, 86 (13), 6689-6694. 31.

Zhou, J.; Liao, C. N.; Zhang, L. M.; Wang, Q. G.; Tian, Y., Molecular Hydrogel-Stabilized

Enzyme with Facilitated Electron Transfer for Determination of H2O2 Released from Live Cells. Anal. Chem. 2014, 86 (9), 4395-4401. 32.

Zhou, Y. J.; Li, L.; Wan, Y. H.; Chen, T. T.; Chu, X., 2D g-C3N4-MnO2 nanocomposite for

sensitive and rapid turn-on fluorescence detection of H2O2 and glucose. Anal. Methods 2018, 10 (42), 5084-5090. 33.

Lippert, A. R.; De Bittner, G. C. V.; Chang, C. J., Boronate Oxidation as a Bioorthogonal

Reaction Approach for Studying the Chemistry of Hydrogen Peroxide in Living Systems. Acc. Chem. Res. 2011, 44 (9), 793-804. 34.

Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J., A selective, cell-permeable optical

probe for hydrogen peroxide in living cells. J. Am. Chem. Soc. 2004, 126 (47), 15392-15393. 35.

Brewer, T. F.; Garcia, F. J.; Onak, C. S.; Carroll, K. S.; Chang, C. J., Chemical Approaches to

Discovery and Study of Sources and Targets of Hydrogen Peroxide Redox Signaling Through NADVII Oxidase Proteins. Annu Rev Biochem 2015, 84, 765-790. 36.

Chung, C. Y. S.; Timblin, G. A.; Saijo, K.; Chang, C. J., Versatile Histochemical Approach to

Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining. J. Am. Chem. Soc. 2018, 140 (19), 6109-6121. 37.

Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T., Development of a Highly

Sensitive Fluorescence Probe for Hydrogen Peroxide. J. Am. Chem. Soc. 2011, 133 (27), 10629-10637. 29 ACS Paragon Plus Environment


38.

Page 30 of 32

Liu, J.; Ren, J.; Bao, X. J.; Gao, W.; Wu, C. L.; Zhao, Y. B., pH-Switchable Fluorescent Probe

for Spatially-Confined Visualization of Intracellular Hydrogen Peroxide. Anal. Chem. 2016, 88 (11), 5865-5870. 39.

Yuan, L.; Lin, W. Y.; Xie, Y. N.; Chen, B.; Zhu, S. S., Single Fluorescent Probe Responds to

H2O2, NO, and H2O2/NO with Three Different Sets of Fluorescence Signals. J. Am. Chem. Soc. 2012, 134 (2), 1305-1315. 40.

Gao, C. C.; Tian, Y.; Zhang, R. B.; Jing, J.; Zhang, X. L., Endoplasmic Reticulum-Directed

Ratiometric Fluorescent Probe for Quantitive Detection of Basal H2O2. Anal. Chem. 2017, 89 (23), 12945-12950. 41.

Tsourkas, A.; Newton, G.; Perez, J. M.; Basilion, J. P.; Weissleder, R., Detection of

Peroxidase/H2O2-mediated oxidation with enhanced yellow fluorescent protein. Anal. Chem. 2005, 77 (9), 2862-2867. 42.

Dickinson, B. C.; Huynh, C.; Chang, C. J., A Palette of Fluorescent Probes with Varying

Emission Colors for Imaging Hydrogen Peroxide Signaling in Living Cells. J. Am. Chem. Soc. 2010, 132 (16), 5906-5915. 43.

Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J., Boronate-based fluorescent

probes for imaging cellular hydrogen peroxide. J. Am. Chem. Soc. 2005, 127 (47), 16652-16659. 44.

Albers, A. E.; Okreglak, V. S.; Chang, C. J., A FRET-based approach to ratiometric fluorescence

detection of hydrogen peroxide. J. Am. Chem. Soc. 2006, 128 (30), 9640-9641. 45.

Srikun, D.; Miller, E. W.; Dornaille, D. W.; Chang, C. J., An ICT-Based approach to ratiometric

fluorescence imaging of hydrogen peroxide produced in living cells. J. Am. Chem. Soc. 2008, 130 (14), 4596-4597. 46.

Miller, E. W.; Tulyanthan, O.; Isacoff, E. Y.; Chang, C. J., Molecular imaging of hydrogen

peroxide produced for cell signaling. Nat. Chem. Biol. 2007, 3 (5), 263-267. 47.

Liu, J.; Zhou, S. Q.; Ren, J.; Wu, C. L.; Zhao, Y. B., A lysosome-locating and acidic pH-

activatable fluorescent probe for visualizing endogenous H2O2 in lysosomes. Analyst 2017, 142 (23), 4522-4528. 48.

Murale, D. P.; Liew, H.; Suh, Y. H.; Churchill, D. G., Mercuric-triggered hydrogen peroxide

"turn-on" fluorescence detection in neuronal cells with novel fluorescein-based probe obtained in one pot. Anal. Methods 2013, 5 (11), 2650-2652. 49.

Li, S. Y.; Liu, L. H.; Rong, L.; Qiu, W. X.; Jia, H. Z.; Li, B.; Li, F.; Zhang, X. Z., A Dual-

FRET-Based Versatile Prodrug for Real-Time Drug Release Monitoring and In Situ Therapeutic Efficacy Evaluation. Adv. Funct. Mater. 2015, 25 (47), 7317-7326. 30 ACS Paragon Plus Environment

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

50.


Kempf, O.; Kempf, K.; Schobert, R.; Bombarda, E., Hydrodabcyl: A Superior Hydrophilic

Alternative to the Dark Fluorescence Quencher Dabcyl. Anal. Chem. 2017, 89 (22), 11893-11897. 51.

Kawaguchi, M.; Ikegawa, S.; Ieda, N.; Nakagawa, H., A Fluorescent Probe for Imaging Sirtuin

Activity in Living Cells, Based on One-Step Cleavage of the Dabcyl Quencher. ChemBioChem 2016, 17 (20), 1961-1967. 52.

Alouane, A.; Labruere, R.; Le Saux, T.; Schmidt, F.; Jullien, L., Self-Immolative Spacers:

Kinetic Aspects, Structure-Property Relationships, and Applications. Angew. Chem. Int. Ed. 2015, 54 (26), 7492-7509. 53.

Murphy, M. P.; Smith, R. A. J., Targeting antioxidants to mitochondria by conjugation to

lipophilic cations. Annu. Rev. Pharmacol. 2007, 47, 629-656. 54.

Han, Z. X.; Zhang, X. B.; Zhuo, L.; Gong, Y. J.; Wu, X. Y.; Zhen, J.; He, C. M.; Jian, L. X.;

Jing, Z.; Shen, G. L.; Yu, R. Q., Efficient Fluorescence Resonance Energy Transfer-Based Ratiometric Fluorescent Cellular Imaging Probe for Zn2+ Using a Rhodamine Spirolactam as a Trigger. Anal. Chem. 2010, 82 (8), 3108-3113. 55.

Chen, X. Q.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J., Fluorescent Chemosensors Based on

Spiroring-Opening of Xanthenes and Related Derivatives. Chem. Rev. 2012, 112 (3), 1910-1956. 56.

Zhao, Y.; Zhang, X. B.; Han, Z. X.; Qiao, L.; Li, C. Y.; Jian, L. X.; Shen, G. L.; Yu, R. Q.,

Highly Sensitive and Selective Colorimetric and Off-On Fluorescent Chemosensor for Cu2+ in Aqueous Solution and Living Cells. Anal. Chem. 2009, 81 (16), 7022-7030. 57.

Chen, Y. Q.; Yang, C. Q.; Li, T.; Zhang, M.; Liu, Y.; Gauthier, M. A.; Zhao, Y. B.; Wu, C. L.,

The Interplay of Disulfide Bonds, alpha-Helicity, and Hydrophobic Interactions Leads to Ultrahigh Proteolytic Stability of Peptides. Biomacromolecules 2015, 16 (8), 2347-2355. 58.

Donner, J. S.; Thompson, S. A.; Kreuzer, M. P.; Baffou, G.; Quidant, R., Mapping Intracellular

Temperature Using Green Fluorescent Protein. Nano Lett. 2012, 12 (4), 2107-2111. 59.

Huang, Z. L.; Li, N.; Zhang, X. F.; Wang, C.; Xiao, Y., Fixable Molecular Thermometer for

Real-Time Visualization and Quantification of Mitochondrial Temperature. Anal. Chem. 2018, 90 (23), 13953-13959. 60.

Spiess, C.; Beil, A.; Ehrmann, M., A temperature-dependent switch from chaperone to protease

in a widely conserved heat shock protein. Cell 1999, 97 (3), 339-347.



Page 32 of 32

TOC O B

Built-in self-immolative spacer

H2O2-responsive handle O N

N

O

O

N

Rhodamine fluorophore

O

Lactam

N

O

O

N

Eliminable quencher

P

Mitochondrion-targeting reagent


A Doubly-Quenched Fluorescent Probe for Low-Background Detection

Recommend Documents