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perylene-3,4,9,10-tetracarboxyl bisimide and
2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)
(Maki et al ., 2009). This compound emits
fluorescence in the visible region and is
highly selective for the hydroxyl radical.
micromolar concentrations of H 2 O 2 in vivo .
Boronate-derived probe oxidation can be
used for studying localization, trafficking
and in vivo production of H 2 O 2 in various liv-
ing systems (Lippert et al. , 2011).
o r g a n e l l e - s p e c i f i c d e t e c t i o n o f h 2 o 2 u s i n g
s n a p - t a g p r o t e i n l a b e l l i n g . A refining of bor-
onate chemistry has been recently devel-
oped. Using SNAP-tag technology,
site-specific protein labelling can be done
in practically any cell compartment
(nucleus, mitochondria, plasma membrane
and endoplasmic reticulum). Hence, local-
ized H 2 O 2 production has been detected
with one of these fusion products named
SNAP-peroxy-Green (SNAP-PG). The spe-
cificity of this probe was evaluated using
scanning confocal microscopy (Dickinson
et al. , 2010; Srikun et al. , 2010).
r h o d a m i n e n i t r o x i d e p r o b e s . Most recently,
Yapici and coworkers developed a series of
rhodhamine nitroxide probes (I, II and III)
specific for OH that can be used for fluores-
cence and ESR detection (Yapici et al .,
2012). These molecules have been success-
fully assayed for OH detection in cell-free
systems (Fenton reagent), ARPE-19 cell
stimulated with PMA and tumour lines
such as HeLa, HepG2 and SW-620.
A new probe for superoxide
Circularly permuted yellow fluorescent
protein (cpYFP), previously used as the core
structure for the Ca 2+ indicator pericam
(Nagai et al. , 2001), is a novel biosensor for
O 2 , the primal ROS generated by the elec-
tron transfer chain. The fluorescence emis-
sion (515 nm) of purified cpYFP when
excited at 488 nm is five times brighter
under strong oxidizing conditions. Extensive
in vitro experiments revealed the superox-
ide selectivity of cpYFP over other physio-
logically relevant oxidants and metabolites.
The O 2 associated increase in cpYFP fluor-
escence is completely reversed by the sub-
sequent addition of Cu/Zn-superoxide
dismutase (SOD, 600 U/ml) or prevented by
prior addition of SOD. By contrast, cpYFP
emission is unchanged by H 2 O 2 and peroxy-
nitrite, and is decreased by HO and NO
(Wang et al. , 2008).
h y p e r . OxyR is a sensor and transcriptional
regulator that can detect H 2 O 2 through
domains that may sense this ROS. The sen-
sor domain is called OxyR-RD, and by
fusion with cpYFP gives rise to the Hyper.
This probe has two excitation peaks at 420
and 500 nm, with emission at 516 nm. When
exposed to H 2 O 2 , the 420 nm peak decreases
while the 500 nm increases. This is there-
fore a ratiometric sensor probe. Hyper is
highly selective for H 2 O 2 and cell transfec-
tion is required for its use. Hyper has been
demonstrated as a valuable tool to monitor
hydrogen peroxide generated in different
cellular compartments (Malinouski et al. ,
2011). The fusion Hyper-PTS1 has recently
been used for specific hydrogen peroxide
detection in peroxisomes as well (Gehrmann
and Elsner, 2011).
New probes for hydrogen peroxide
New probes for hypochlorous acid
p e r o x i f l u o r -1 . In order to improve the spe-
cific detection of H 2 O 2 , Chang and coworkers
developed the peroxifluor-1 probe (Chang
et al. , 2004). Indeed, their response to H 2 O 2 is
500 times greater than for other ROS. A fam-
ily of boronate probes has been synthesized,
such as peroxyresofurin-1 (PR-1), greenfluo-
rescent PF-1 and the blue-fluorescent
peroxyxanthone-1 (PX1). Interestingly, all
these probes are permeable and can detect
s u l f o n a p h t h o a m i n o p h e n y l f l u o r e s c e i n ( s n a p f ) .
SNAPF is a newly developed fluorescein-
derived probe for the specific detection of
intracellular levels of HOCl generated by
MPO activity. In the presence of HOCl, the
4-aminophenyl function suffers oxidative
cleavage to release fluorescein. This probe
can be used for the in vivo non-invasive
detection of HOCl (Freitas et al ., 2009).
 
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