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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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