Synthesis and application of a ratiometric probe for hydrogen peroxide

Article abstract of DOI:10.1016/bs.mie.2020.04.007

Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hinder

Full text of DOI:10.1016/bs.mie.2020.04.007

CHAPTER TWO
Synthesis and application of a
ratiometric probe for hydrogen
peroxide
a,†
a,†
b
a,
Ruwen Yin , Yuan Fang , Xinqi Zhou , Cliff I. Stains *
a
Department of Chemistry, University of Virginia, Charlottesville, VA, United States
b
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, United States
Corresponding author: e-mail address: cstains@virginia.edu
*
Contents
1
2
. Introduction
. Synthesis of RF620
24
25
26
26
27
28
29
30
30
32
32
32
35
35
35
2
2
2
2
.1 General procedures and instrumentation
.2 Synthesis of 3-bromo-N,N-dimethylaniline (1)
.3 Synthesis of 4,4 -(o-tolylmethylene)bis(3-bromo-N,N-dimethylaniline) (2)
.4 Synthesis of the borinic acid form of RF620 (3)
0
3
4
5
. In vitro detection of hydrogen peroxide
3
3
.1 General reagents and instrumentation
.2 In vitro H assay
2 2
O
. Ratiometric imaging of hydrogen peroxide accumulation in living cells
4
4
.1 General reagents and instrumentation
.2 Ratiometric detection of endogenous H
2 2
O production in living cells
. Summary and conclusions
Acknowledgments
References
Abstract
Molecular imaging of biological analytes provides detailed insights into signaling
processes. Ratiometric probes are particularly attractive due to the ability to quantify
analyte production. However, design strategies for ratiometric probes can be hindered
by spectral overlap of the product and reactant species. In this chapter, we provide pro-
2 2
tocols for the synthesis and application of RF620, a ratiometric probe for H O displaying
dramatic changes in both excitation and emission wavelengths, designed using an
approach we term chemoselective alteration of fluorophore scaffolds. The probe
contains a chemoselective functional group within a fluorescent xanthene scaffold,
resulting in the in situ synthesis of a new fluorophore upon reaction with H O .
2
2
†
These authors contributed equally.
Methods in Enzymology, Volume 639
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.007
# 2020 Elsevier Inc.
All rights reserved.
2
3

24
Ruwen Yin et al.
Under physiological conditions, RF620 exhibits far-red to near-infrared excitation and
emission, and upon reaction with H O , RF620 is chemically converted into
2 2
tetramethylrhodamine, producing a significant (ꢀ66nm) blue-shift in excitation and
2 2
emission. RF620 can be used for ratiometric, molecular imaging of endogenous H O
production in living cells.
1. Introduction
Ratiometric fluorescent probes allow for the quantitative analysis of
analytes in complex biological systems (Lee, Kim, & Sessler, 2015).
Common design strategies for ratiometric probes rely on intramolecular
charge transfer or fluorescence resonance energy transfer. Although power-
ful, these design approaches can suffer from spectral overlap of product and
reactant species, making quantification difficult. Our lab was intrigued by
the observations that: (1) reactions on functional groups placed at the
bridging position of the xanthene scaffold could be utilized to tune both
excitation and emission of the fluorophore (Zhou, Lai, Beck, Li, &
Stains, 2016) and (2) the pioneering work of the Chang lab in the develop-
ment of activity-based sensing (ABS) approaches for molecular imaging
(Bruemmer, Crossley, & Chang, in press; Chan, Dodani, & Chang,
2
012). Expanding upon this work, we envisioned an approach in which
the chemical reactivity of a functional group within a fluorescent scaffold
would be used to afford the in situ generation of a new fluorophore in the
presence of a target analyte, producing a robust change in ratiometric signal
(Fig. 1A) (Zhou et al., 2017). We termed this approach Chemoselective
Alteration of Fluorophore Scaffolds (CAFS).
As our first target for design of a CAFS probe, we chose hydrogen per-
oxide (H O ), now recognized as an important signaling molecule in living
2
2
systems (D’Autreaux & Toledano, 2007). The physiological role of H O2
2
has been illuminated by elegant efforts to generate ABS-based optical probes
for H O detection in living systems (Brewer, Garcia, Onak, Carroll, &
2
2
Chang, 2015; Lin, Dickinson, & Chang, 2013; Lippert, De Bittner, &
Chang, 2011). Indeed, this early ABS work demonstrated the selectivity
of boronates for H O in living systems. Building off of this work, we chose
2
2
to incorporate a borinate functionality at the bridging position of
tetramethylrhodamine (TMR). We hypothesized that the resulting TMR
derivative, termed RF620, would react selectively with H O to produce
2
2
TMR in situ (Fig. 1B). Indeed, reaction of H O with RF produces
620
2
2

Ratiometric probe for hydrogen peroxide
25
2 2
Fig. 1 The CAFS approach and its use to design a ratiometric probe for H O .
(
(
A) A schematic of the CAFS approach. Installation of a chemoselective functional group
FG) within a fluorescent scaffold results in in situ generation of a new fluorophore upon
reaction with a target analyte. Production of a new fluorophore enables ratiometric
detection of the target analyte. (B) RF620, a CAFS-based probe for detection of H
Oxidation by H O under physiological conditions leads to formation of TMR and a clear
2 2
O .
2
2
change in absorbance and fluorescence. Reprinted with permission from Zhou, X.,
Lesiak, L., Lai, R., Beck, J. R., Zhao, J., Elowsky, C. G., et al. (2017). Chemoselective alteration
of fluorophore scaffolds as a strategy for the development of ratiometric chemodosimeters,
Angewandte Chemie International Edition, 56 (15), 2017, 4197–4200, https://doi.org/
1
0.1002/anie.201612628.
an ꢀ66nm blue-shift in both excitation and emission, enabling sensitive
detection of H O with a 1720-fold change in ratiometric signal.
2
2
Moreover, RF620 is selective for H O and can be used to image endoge-
2
2
nous H O accumulation in living cells in response to biologically relevant
2
2
stimulation. Below we provide our optimized protocols for synthesis of
RF620 and its use for ratiometric imaging of H O accumulation. These pro-
2
2
cedures can be modified to detect H O production in applications defined
2
2
by the user.
2. Synthesis of RF620
In this section, we provide a detailed procedure for the synthesis of
RF620, a ratiometric probe for detection of H O (Fig. 2). This procedure
2
2
routinely yields ꢀ128mg of RF6 which can be used for ꢀ17,000 down-
20
stream assays, using the conditions described below. This procedure assumes
knowledge of standard organic chemistry techniques and the availability of
basic organic synthesis laboratory equipment.

26
Ruwen Yin et al.
Fig. 2 Synthetic scheme for RF620
.
2
.1 General procedures and instrumentation
All reactions were performed in oven-dried glassware under nitrogen unless
otherwise noted. All the reagents and solvents were used directly from
commercial suppliers without further purification. Anhydrous THF was
˚
prepared using 3A molecular sieves (Bradley, Williams, & Lawton, 2010).
Reaction progress was monitored by thin layer chromatography (TLC).
Products were purified by flash chromatography using Merck silica gel
6
0 (230–400 mesh). Prep HPLC purification was done with a semi-prep
column (YMC-Pack ODS-A, 5μm, 250ꢁ20mm).
2
.2 Synthesis of 3-bromo-N,N-dimethylaniline (1)
1
. Add aqueous formaldehyde (37%, 32.5mL, 435.9mmol) to a 1L round
bottom flask containing a magnetic stir bar.
2
3
. Add tetrahydrofuran (390mL) to the same flask.
. Immerse the solution in an ice bath (0°C) and stir continuously for
5
min.
4
5
. Slowly add aqueous H SO (3M, 87.5mL) using a graduated cylinder.
2
4
. Slowly add 3-bromoaniline (25.0g, 145.3mmol) to the flask using a
dropping funnel.
6
. Add sodium borohydride (22.0g, 581.3mmol) to the flask over 30min
while maintaining the reaction temperature at 0°C.
7
. Allow the reaction mixture to warm up to room temperature, then stir
at room temperature for an additional 1h.
8
. Immerse the reaction mixture into an ice bath (0°C) for 10min.
. Slowly pour 300mL of saturated sodium bicarbonate solution into the
reaction mixture while maintaining its temperature at 0°C.
9
1
0. Evaporate the tetrahydrofuran using a rotary evaporator.

Ratiometric probe for hydrogen peroxide
27
1
1
1
1
1. Transfer the reaction mixture to a 1L separatory funnel and extract
with dichloromethane (200mL, three times).
2. Combine the organic layers, transfer them into a 1L separatory funnel
and wash with saturated aqueous sodium chloride (200mL).
3. Transfer the organic layer to a 1L Erlenmeyer flask, dry the solution
with a bed of sodium sulfate (Na SO ), and filter the mixture.
2
4
4. Dry the filtrate using low-vacuum followed by high-vacuum to furnish
a pale-yellow oil (1), which is used in the next step without further
purification.
0
2
.3 Synthesis of 4,4 -(o-tolylmethylene)bis(3-bromo-N,N-
dimethylaniline) (2)
1
. Sequentially add 3-bromo-N,N-dimethylaniline (1) (8g, 40.2mmol),
-methylbenzaldehyde (1.2mL, 10.0mmol), and toluene (20mL) to
2
a 25mL round bottom flask containing a magnetic stir bar.
. Add p-Toluenesulfonic acid monohydrate (1.9g, 10.0mmol) to the
reaction mixture.
2
3
. Attach a Dean-Stark apparatus (10mL scale) to the round bottom flask
and add 10mL of toluene to the trap of the Dean-Stark apparatus.
. Reflux the reaction mixture at 130°C for 8h.
4
5
. Cool down the reaction mixture then evaporate the toluene by
rotovap.
6
. Dissolve the resulting residue in 20mL dichloromethane, transfer the
solution to a 250mL separatory funnel.
7
8
. Add 100mL dichloromethane into the separatory funnel.
. Slowly pour a solution of saturated aqueous sodium bicarbonate
(50mL) into the separatory funnel and shake vigorously with frequent
venting.
9
. Wash the organic phase with saturated aqueous sodium chloride solu-
tion (50mL).
1
1
1
0. Transfer the organic phase to a 250mL Erlenmeyer flask and dry the
solution with anhydrous sodium sulfate (Na SO ).
2
4
1. Filter the mixture and dry the filtrate using low vacuum rotovap to fur-
nish a brownish oil.
2. The resulting oil is further purified by flash chromatography (silica gel,
230–400 mesh, 15:1 hexanes/ethyl acetate) to afford a white solid (2)
(2.7g, 53%) after solvent evaporation.
1
3. The identity of the product should be confirmed using NMR and MS
by referring to the published spectra (Zhou et al., 2016).

28
Ruwen Yin et al.
2
.4 Synthesis of the borinic acid form of RF620 (3)
1
. Flame dry a 50mL round bottom flask for 10min, add a dry stir bar, and
cap the flask with a rubber septum.
2
3
. Charge the flask with nitrogen.
0
. Dissolve 4,4 -(o-tolylmethylene)bis(3-bromo-N,N-dimethylaniline) (2)
(200mg, 0.4mmol) in anhydrous tetrahydrofuran (10mL) and add the
solution into the flask using a 10mL syringe.
4
5
. Immerse the reaction mixture in an acetone/dry ice bath for 10min.
. Add sec-butyllithium (1.4M in cyclohexane, 626μL) dropwise
through a 1mL syringe to the reaction mixture.
6
7
. Stir the reaction in the acetone/dry ice bath for 1h.
. Add triisopropyl borate (101μL, 0.44mmol) to the reaction mixture
dropwise using a 500μL microscale syringe.
. Stir the reaction in the acetone/dry ice bath for 3h.
. Transfer the reaction to an ice bath and let it warm up gradually to room
temperature. Stir the reaction for another 8h.
8
9
1
0. Add a solution of saturated aqueous ammonium chloride (10mL) to the
reaction mixture to quench the reaction.
11. Transfer the reaction to a 50mL separatory funnel.
12. Add dichloromethane(20mL) andshake vigorously with frequent venting.
13. Dry the organic phase using anhydrous sodium sulfate.
14. Filter the mixture and dry the filtrate using low-vacuum followed by
high-vacuum rotovap to furnish a pale-yellow oil.
1
5. In a 25mL round bottom flask, dissolve the pale-yellow oil in anhy-
drous dichloromethane (10mL).
16. Immerse the solution into an ice bath for 5min.
17. To the solution, add p-chloranil (294mg, 1.2mmol).
18. Transfer the reaction mixture to room temperature and let it proceed
for 30min.
1
2
9. Place the reaction mixture in an ice bath for 10min.
0. Filter the reaction suspension with vacuum and wash the filter cake
with cool dichloromethane.
2
1. Evaporate the dichloromethane using a rotovap.
2. Dissolve the resulting dark blue solid in 6N hydrochloric acid (10mL)
and stir at room temperature for 45min.
2
2
2
3. Filter the reaction mixture and dry the filtrate by rotovap.
4. Dissolve the resulting residue in 8mL of HPLC solvent (50% acetoni-
trile in water with 0.1% trifluoroacetic acid).

Ratiometric probe for hydrogen peroxide
29
25. Filter the solution through a 0.22μm filter and then purify via HPLC
using a gradient from 5% acetonitrile in water (containing 0.1%
trifluoroacetic acid) to 95% acetonitrile in water (containing 0.1%
trifluoroacetic acid) over 40min. Monitor absorption at 280 and 365nm.
6. Lyophilize fractions containing the product to yield a dark blue solid
2
2
(128mg, 67%).
7. Verify the structure and purity of the product by NMR and MS by
referring to the published spectra (Zhou et al., 2017).
3. In vitro detection of hydrogen peroxide
With a purified sample of RF620 now in-hand, H O can be detected
2
2
in samples using a ratiometirc fluorescence assay (Zhou et al., 2017).
Exposure of RF6 to H O leads to formation of TMR with a rate constant
20
2
2
ꢂ1 ꢂ1
of 0.15M s . The resulting ꢀ66nm blue-shift in excitation and emission
relative to RF6 , allows for facile spectroscopic monitoring of reactant and
20
product (Fig. 3A and B). Additionally, previous work has also shown that
Fig. 3 Comparison of the photophysical properties of RF620 and TMR. (A) Normalized
absorption spectra. (B) Normalized emission spectra. (C) Summary of photophysical
parameters. Reprinted with permission from Zhou, X., Lesiak, L., Lai, R., Beck, J. R.,
Zhao, J., Elowsky, C. G., et al. (2017). Chemoselective alteration of fluorophore scaffolds
as a strategy for the development of ratiometric chemodosimeters, Angewandte
Chemie International Edition, 56 (15), 2017, 4197–4200, https://doi.org/10.1002/anie.
2
01612628.

30
Ruwen Yin et al.
RF620 and TMR display similar brightness (Fig. 3C), allowing for sensitive
detection of H O . Below we provide a protocol for validation of RF
620
2
2
activity and selectivity towards H O . This can be used to confirm the activ-
2
2
ity of RF620 as well as analyze in vitro samples.
3
.1 General reagents and instrumentation
DMSO (Sigma, D8418, for molecular biology)
PBS (Gibco, 10010023, 1ꢁ, pH 7.4)
H O (Sigma, 516813, 50wt% in H O)
2
2
2
Potential off-target reactive oxygen species (Srikun, Miller, Dornaille, &
Chang, 2008; Sun et al., 2014)
Vortex mixer (VWR, 97043-562)
Synergy H1 Hybrid Multi-Mode Plate Reader (BioTek Instruments) or
similar
Eppendorf tubes (1.5mL, Fisher Scientific, 05-402-27)
Nontreated clear flat bottom 96-well assay plates (Corning 3370)
3
1
.2 In vitro H O assay
2
2
. In a series of 1.5mL eppendorf tubes; add 999μL PBS (pH 7.4) con-
taining H O (200μM), superoxide (20μM), superoxide (20μM)+
2
2
ꢂ1
superoxide dismutase (SOD, 15U mL ), superoxide (20μM)+catalase
ꢂ1
(
15U mL ), tert-butyl hydrogen peroxide (TBHP) (200μM), HOCl
t
(
20μM), NO (200μM), ꢃO Bu (200μM), ꢃOH (200μM), or pure
PBS (blank).
Note: Superoxide dismutase and catalase are used to assess off-target
signal from superoxide decomposition.
2
. To each tube add 1μL of a 10mM 3 stock prepared in DMSO. Stock
concentrations can be determined using the extinction coefficient of
RF620 in PBS (Fig. 3C).
Note: Previous experiments have shown that 3 equilibrates to RF620
at pH 7.4 (Zhou et al., 2017).
3
4
. Vortex each sample.
. Transfer 200μL of each sample to one well of a 96-well plate. Repeat
three times for a total of three assays per sample.
5
. Overlay each well with 70μL mineral oil to prevent evaporation.
. Setup the fluorescence plate reader to detect the fluorescence of RF620
and TMR. RF620 signal is readout by 620 determined by excitation
6

Ratiometric probe for hydrogen peroxide
31
at 610 nm and monitoring emission at 636nm. For TMR, 544 nm exci-
tation is used, and emission is monitored at 570nm.
7
8
9
. Record the fluorescence data at intervals of 5, 15, 30, 60, 120, and
1
80min.
. Determine the ratio of TMR/RF620 fluorescence by dividing the emis-
sion of TMR by the emission of RF620
.
. The ratio of TMR/RF620 fluorescence is averaged for each sample and
normalized to the 5min time point. The normalized TMR/RF620 fluo-
rescence ratio is then plotted for each sample over time (Fig. 4).
Note: Using this procedure we routinely obtain ꢀ1700-fold increases
in TMR/RF6 fluorescence in the presence of H O .
20
2
2
Fig. 4 Selectivity of RF620 (10μM) toward different reactive oxygen species over time.
ꢂ1
(
(
a) H
2
O
2
, (b) superoxide, (c) superoxide with superoxide dismutase (SOD, 15U mL ),
ꢂ1
d) superoxide with catalase (15U mL ), (e) tert-Butyl hydrogen peroxide (TBHP), (f )
t
HOCl, (g) NO, (h) ꢃO Bu, (i) ꢃOH, and (j) blank. Reprinted with permission from Zhou, X.,
Lesiak, L., Lai, R., Beck, J. R., Zhao, J., Elowsky, C. G., et al. (2017). Chemoselective alteration
of fluorophore scaffolds as a strategy for the development of ratiometric chemodosimeters,
Angewandte Chemie International Edition, 56 (15), 2017, 4197–4200, https://doi.org/
1
0.1002/anie.201612628.

32
Ruwen Yin et al.
4
. Ratiometric imaging of hydrogen peroxide
accumulation in living cells
After validation, RF620 can be used to ratiometrically detect H O2
2
accumulation in mammalian cells. Phorbol 12-myristate 13-acetate
PMA) (Xu, Zhang, Yu, Gao, & Shao, 2016) and human epidermal growth
(
factor (EGF) (Ermakova et al., 2014) can be used as positive controls to stim-
ulate production of endogenous H O . Ebselen is used as a H O scavenger
2
2
2
2
to verify the selectivity of RF620 signal production in cells (Muller, Cadenas,
Graf, & Sies, 1984). This protocol can be adapted to investigate H O pro-
2
2
duction in response to user-defined conditions.
4
.1 General reagents and instrumentation
HeLa cells (ATTC, CCL-2)
Dulbecco’s Modified Eagle Medium (DMEM, high glucose, GlutaMAX
Supplement, pyruvate, Gibco, 10569010)
Imaging Media (DMEM, high glucose, HEPES, no phenol red, Gibco,
2
1063029)
Fetal Bovine Serum (FBS, Gibco, 16000044)
Antibiotic-Antimycotic (Anti-Anti, 100ꢁ, Gibco, 15240062)
DPBS (with calcium and magnesium, Gibco, 14040133)
Phorbol 12-myristate 13-acetate (PMA, Sigma, P1585)
Ebselen (Sigma, E3520)
Human epidermal growth factor (EGF, Sigma, E9644)
Hoechst 33342 (Invitrogen, H1399)
35mm tissue-culture treated culture dishes (Corning, CLS430165)
Confocal imaging system with the following lasers and emission filters:
blue (excitation: 405nm, emission filter: 425–475nm), green (excitation:
4
90nm, emission filter: 500–530nm), red (excitation: 560nm, emission
filter: 560–617nm), and deep red (excitation: 640nm, emission filter:
63–738nm)
6
ImageJ software (Schneider, Rasband, & Eliceiri, 2012)
4
.2 Ratiometric detection of endogenous H O production
2
2
in living cells
1
. Prepare cell culture media as follows. To 445mL of DMEM (Gibco,
0569010) add 50mL FBS and 5mL Anti-Anti. Sterile filter using a
1

Ratiometric probe for hydrogen peroxide
33
0
.22μm filter. This media contains 10% FBS and 1ꢁ Anti-Anti, store
at 4°C and warm to 37°C before use.
. Prepare the indicated reagents as follows:
2
ꢂ1
a. PMA is dissolved in ethanol to make a 1mg mL stock, store
at ꢂ20°C.
ꢂ1
b. Ebselen is dissolved in DMSO to make a 1mg mL stock, store
at ꢂ20°C.
ꢂ1
c. EGF is dissolved in 10mM acetic acid to make a 1mg mL stock,
store at ꢂ20°C.
ꢂ1
d. Hoechst 33342 is dissolved in DMSO to make a 10mg mL stock,
store at ꢂ20°C.
3
. Seed five cultures of HeLa cells on 35mm culture dishes and grow to
80% confluency in an incubator at 37°C with a humidified, 5% CO2
atmosphere.
4
5
. Remove the cell culture medium and wash with prewarmed DPBS
(37°C).
ꢂ1
. Add 2mL DPBS containing 0.5μg mL Hoechst 33342 and 10μM
RF620 to all the dishes.
6
7
8
. Place the dishes back in the CO incubator for 30min.
2
. Label the dishes as sample i, ii, iii, iv, and v.
. Remove the DPBS solution and wash the cells with prewarmed DPBS
three times.
9
. Add reagent to each dish as follows:
a. 2mL DPBS to dish i.
ꢂ1
b. 2mL DPBS containing 1μgmL PMA to dish ii.
ꢂ1
c. 2mL DPBS containing 1μgmL PMA and 5μM ebselen to
dish iii.
ꢂ1
d. 2mL DPBS containing 500ngmL EGF to dish iv.
ꢂ1
e. 2mL DPBS containing 500ngmL EGF and 5μM ebselen to
dish v.
1
0. Put all five dishes back in the CO incubator for 90min.
2
1
1. Aspirate off the media and wash the cells with prewarmed DPBS
three times.
1
2. Add 2mL prewarmed imaging media and image.
3. If using a confocal imaging setup as described above, use the blue chan-
nel for Hoechst 33342, the red channel for TMR, and the deep red
channel for RF620. Use a 60ꢁ objective to find three different places
on the dish to take images. The images should be taken with the same
parameters to allow for subsequent analysis.
1

2 2
Fig. 5 Ratiometric detection of endogenous production of H O in living cells by RF620.
(
A) Confocal fluorescence microscopy imaging of living HeLa cells incubated with 10μM
ꢂ1
RF620 for 30min, washed (3ꢁ), and incubated with (i) blank, (ii) PMA (1μgmL ), (iii) PMA
ꢂ1
ꢂ1
ꢂ1
(
1μgmL ) with ebselen (5μM), (iv) EGF (500ngmL ) or (v) EGF (500ngmL ) with
ebselen (5μM) for 90min. (B) Comparison of the normalized ratio between TMR and
RF620 emission. Scale bar: 25μm. Adapted with permission from Zhou, X., Lesiak, L.,
Lai, R., Beck, J. R., Zhao, J., Elowsky, C. G., et al. (2017). Chemoselective alteration of fluo-
rophore scaffolds as a strategy for the development of ratiometric chemodosimeters,
Angewandte Chemie International Edition, 56 (15), 2017, 4197–4200, https://doi.org/
1
0.1002/anie.201612628.

Ratiometric probe for hydrogen peroxide
35
14. Use ImageJ software to obtain quantified data for Hoechst 33342,
TMR, and RF620 channels.
a. Using these conditions we routinely obtain ꢀ16- and ꢀ14-fold
increases in the TMR/RF6 fluorescence ratio with PMA and
20
EGF stimulation, respectively (Fig. 5) (Zhou et al., 2017).
Samples containing ebselen, should display a TMR/RF620 fluores-
cence ratio similar to the blank (i), validating the selectivity of
the assay.
b. Previous work has demonstrated that TMR formation is primarily
observed in the mitochondria when using RF620 (Zhou
et al., 2017).
5. Summary and conclusions
We have developed a new approach for the design of ratiometric pro-
bes, termed CAFS. Utilizing this approach, we designed RF620 a ratiometric
fluorescent probe for H O . RF produces aꢀ66nm blue-shift in excita-
2
2
620
tion and emission upon reaction with H O , resulting in a 1720-fold
2
2
increase in ratiometric fluorescence signal. In this chapter, we provide pro-
tocols for the synthesis of RF620 and its application to imaging endogenous
H O production. This probe enables quantitative imaging of H O accu-
2
2
2
2
mulation in living systems. We envision that RF620 will provide new
insights into H O -mediated cellular signaling process.
2
2
Acknowledgments
This work was supported by the NIH (R35GM119751) and the University of Virginia. The
content of this work is solely the responsibility of the authors and does not necessarily
represent the official views of the NIH.
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