Chemiluminescence
probes are considered to be among the most sensitive
diagnostic tools that provide high signal-to-noise ratio for various
applications such as DNA detection and immunoassays. We have developed
a new molecular methodology to design and foresee light-emission properties
of turn-ON chemiluminescence dioxetane probes suitable for use under
physiological conditions. The methodology is based on incorporation
of a substituent on the benzoate species obtained during the chemiexcitation
pathway of Schaap’s adamantylidene–dioxetane probe.
The substituent effect was initially evaluated on the fluorescence
emission generated by the benzoate species and then on the chemiluminescence
of the dioxetane luminophores. A striking substituent effect on the
chemiluminescence efficiency of the probes was obtained when acrylate
and acrylonitrile electron-withdrawing groups were installed. The
chemiluminescence quantum yield of the best probe was more than 3
orders of magnitude higher than that of a standard, commercially available
adamantylidene–dioxetane probe. These are the most powerful
chemiluminescence dioxetane probes synthesized to date that are suitable
for use under aqueous conditions. One of our probes was capable of
providing high-quality chemiluminescence cell images based on endogenous
activity of β-galactosidase. This is the first demonstration
of cell imaging achieved by a non-luciferin small-molecule probe with
direct chemiluminescence mode of emission. We anticipate that the
strategy presented here will lead to development of efficient chemiluminescence
probes for various applications in the field of sensing and imaging.
Chemiluminescence is among the most sensitive methods for achieving a high signal-to-noise ratio in various chemical and biological applications. We have developed a modular practical synthetic route for preparation of turn-ON fluorophore-tethered dioxetane chemiluminescent probes. The chemiluminescent emission of the probes was significantly amplified through an energy-transfer mechanism under physiological conditions. Two probes were composed with green and near-infrared (NIR) fluorescent dyes tethered to Schaap's dioxetane. While both probes were able to provide chemiluminescence in vivo images following subcutaneous injection, only the NIR probe could provide a chemiluminescence image following intraperitoneal injection. These are the first in vivo images produced by Schaap's dioxetane chemiluminescence probes with no need of an enhancer. Previously, chemiluminescence cell images could only be obtained with a luciferin-based probe. Our NIR probe was able to image cells transfected with β-galactosidase gene by chemiluminescence microscopy. We also report, for the first time, the instability of dioxetane-fluorophore conjugates to ambient light. Our synthetic route effectively overcomes this limitation through a late-stage functionalization of the dioxetane intermediate. We anticipate that our practical synthetic methodology will be useful for preparation of various chemiluminescent probes for numerous applications.
Bioluminescent and chemiluminescent probes are widely used for noninvasive imaging applications because of their high sensitivity and the simplicity of the equipment required to perform the measurement. Synthetic luciferin-analogue probes with in vivo imaging performance better than that of luciferin are now available. In addition, caged luciferin-based bioluminogenic probes have been emerged as a general tool for the visualization of different enzymes and analytes in vivo. Recent discoveries have led to development of highly efficient chemiluminescent probes that are extremely bright under physiological conditions. As discussed in this Minireview, chemiluminescence is ready to realize its potential as a valuable tool for imaging in living systems.
Chemiluminescence
is gradually being recognized as a powerful tool
for sensing and imaging. Most known light-emitting compounds undergo
chemiexcitation through spontaneous decomposition of cyclic peroxide
moieties. A ground-breaking milestone in the chemistry of such compounds
was achieved 30 years ago with the discovery of triggerable dioxetanes
by Schaap’s group. Our group has recently developed a distinct
methodology to significantly improve the light emission efficiency
of such phenoxy-dioxetane luminophores under physiological conditions.
Introduction of an electron-withdrawing substituent at the ortho position
of the phenoxy-dioxetane resulted in an approximately 3000-fold increase
of the chemiluminescence quantum yield in aqueous media. Furthermore,
we discovered that the emission wavelength and the kinetics of the
chemiexcitation could be determined by the electronic nature of the
substituent incorporated on the dioxetane luminophore. This recent
development has provided scientists with new powerful chemiluminophores
that can act as single-component probes for in vivo and in vitro detection
and imaging of various analytes and enzymes. This outlook describes
the recent progress toward applications of synthetic chemiluminescence
luminophores suitable for sensing and imaging in aqueous environments.
Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life-times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of O is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe (SOCL-CPP) for the detection of O in living cells. The probe reacts with O to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL-CPP demonstrated promising ability to detect and image intracellular O produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL-CPP the most effective known chemiluminescence probe for the detection of O . We anticipate that our chemiluminescence probe for O imaging would be useful in PDT-related applications and for monitoring O endogenously generated by cells in response to different stimuli.
Activatable (turn‐on) probes that permit the rapid, sensitive, selective, and accurate identification of cancer‐associated biomarkers can help drive advances in cancer research. Herein, a NAD(P)H:quinone oxidoreductase‐1 (NQO1)‐specific chemiluminescent probe 1 is reported that allows the differentiation between cancer subtypes. Probe 1 incorporates an NQO1‐specific trimethyl‐locked quinone trigger moiety covalently tethered to a phenoxy‐dioxetane moiety through a para‐aminobenzyl alcohol linker. Bio‐reduction of the quinone to the corresponding hydroquinone results in a chemiluminescent signal. As inferred from a combination of in vitro cell culture analyses and in vivo mice studies, the probe is safe, cell permeable, and capable of producing a “turn‐on” luminescence response in an NQO1‐positive A549 lung cancer model. On this basis, probe 1 can be used to identify cancerous cells and tissues characterized by elevated NQO1 levels.
Selective and sensitive molecular probes for hydrogen peroxide (H2O2), which plays diverse roles in oxidative stress and redox signaling, are urgently needed to investigate the physiological and pathological effects of H2O2. A lack of reliable tools for in vivo imaging has hampered the development of H2O2 mediated therapeutics. By combining a specific tandem Payne/Dakin reaction with a chemiluminescent scaffold, H2O2‐CL‐510 was developed as a highly selective and sensitive probe for detection of H2O2 both in vitro and in vivo. A rapid 430‐fold enhancement of chemiluminescence was triggered directly by H2O2 without any laser excitation. Arsenic trioxide induced oxidative damage in leukemia was successfully detected. In particular, cerebral ischemia‐reperfusion injury‐induced H2O2 fluxes were visualized in rat brains using H2O2‐CL‐510, providing a new chemical tool for real‐time monitoring of H2O2 dynamics in living animals.
Rational design of phenoxy-dioxetane luminophores with rapid chemiexcitation is described; these next generation luminophores yielded chemiluminescent probes with considerably increased sensitivity.
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