Apart from mice, Caenorhabditis elegans is a widely used animal model for
studying neurodegenerative disorders. Transgenic C. elegans strains expressing
green, yellow, or red fluorescent proteins in embryos were developed by Heppert
et al., to image embryos expressing fluorescent proteins under the same conditions
with probe mNeonGreen. Monomeric green (GFP, mNeonGreen [mNG]), yellow
(mNG, monomeric yellow fluorescent protein for energy transfer [mYPet]), and
red (TagRFP-T, mRuby2, mCherry, mKate2) fluorescent proteins were evaluated
for comparative in vivo experiments in C. elegans [179]. Since C. elegans is a
transparent small animal, fluorescent protein-based sensors provide sensitive imaging ability to track its biological events in a much better way compared to large
animals. Hirayama et al. designed a first-generation near-IR turn-on fluorescent
sensor, CS790AM, to report dynamic copper fluctuations in vivo and detected the
basal, endogenous levels of exchangeable copper in living mice platform to monitor
labile copper pools role in murine Wilson’s disease model [180].
5 Drawbacks Associated with the Use of Fluorescent
Proteins in Biosensors
Although fluorescent protein-based biosensors offer a realistic, cost-effective, and
high-throughput imaging approach for studying various biological processes of
cells, their application is also limited by the inherent problem of photobleaching,
phototoxicity, low quantum yield, high background signal, and especially tissue
attenuation in in vivo imaging applications. Upon repeated cycles of excitation, the
fluorophore of the sensor protein gets damaged and leads to loss of fluorescence
signal which can result in non-specific sensor signal. In addition, it severely limits
the application of fluorescent protein biosensors for real-time imaging where timedependent pattern of biosensor signal is important for achieving reliable result for the
studies [181, 182]. On the other hand, exposure to higher-energy photons tends to
generate reactive oxygen species (ROS) which are highly reactive species capable of
inflicting damage to cellular biomolecules like DNA, RNA, and proteins by oxidation, which in turn limits the possibilities of multiplexing the assays in in vitro and
in vivo imaging applications [183]. In the event of avoiding photobleaching and
phototoxicity, the narrow operation bandwidth and shorter imaging time leave us
with lower quantum yield of these fluorescent proteins with limited sensitivity. The
cells by itself possess biomolecules that prominently contribute to autofluorescence
and render high background while imaging such fluorescent proteins with low
quantum yield, which limits the spectral resolution and sensitivity of the biosensor
[184, 185]. To overcome the limitation of tissue attenuation, the development of
fluorescent proteins with high quantum yields and far-red or near-infrared (NIR)
shifted absorption and emission wavelengths are preferred. Currently there are
several fluorescent protein variants which emit light in the NIR range and have
been developed from bacterial phytochrome photoreceptors and are used in various
biosensor applications [186, 187].
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U. K. Sukumar et al.
studying neurodegenerative disorders. Transgenic C. elegans strains expressing
green, yellow, or red fluorescent proteins in embryos were developed by Heppert
et al., to image embryos expressing fluorescent proteins under the same conditions
with probe mNeonGreen. Monomeric green (GFP, mNeonGreen [mNG]), yellow
(mNG, monomeric yellow fluorescent protein for energy transfer [mYPet]), and
red (TagRFP-T, mRuby2, mCherry, mKate2) fluorescent proteins were evaluated
for comparative in vivo experiments in C. elegans [179]. Since C. elegans is a
transparent small animal, fluorescent protein-based sensors provide sensitive imaging ability to track its biological events in a much better way compared to large
animals. Hirayama et al. designed a first-generation near-IR turn-on fluorescent
sensor, CS790AM, to report dynamic copper fluctuations in vivo and detected the
basal, endogenous levels of exchangeable copper in living mice platform to monitor
labile copper pools role in murine Wilson’s disease model [180].
5 Drawbacks Associated with the Use of Fluorescent
Proteins in Biosensors
Although fluorescent protein-based biosensors offer a realistic, cost-effective, and
high-throughput imaging approach for studying various biological processes of
cells, their application is also limited by the inherent problem of photobleaching,
phototoxicity, low quantum yield, high background signal, and especially tissue
attenuation in in vivo imaging applications. Upon repeated cycles of excitation, the
fluorophore of the sensor protein gets damaged and leads to loss of fluorescence
signal which can result in non-specific sensor signal. In addition, it severely limits
the application of fluorescent protein biosensors for real-time imaging where timedependent pattern of biosensor signal is important for achieving reliable result for the
studies [181, 182]. On the other hand, exposure to higher-energy photons tends to
generate reactive oxygen species (ROS) which are highly reactive species capable of
inflicting damage to cellular biomolecules like DNA, RNA, and proteins by oxidation, which in turn limits the possibilities of multiplexing the assays in in vitro and
in vivo imaging applications [183]. In the event of avoiding photobleaching and
phototoxicity, the narrow operation bandwidth and shorter imaging time leave us
with lower quantum yield of these fluorescent proteins with limited sensitivity. The
cells by itself possess biomolecules that prominently contribute to autofluorescence
and render high background while imaging such fluorescent proteins with low
quantum yield, which limits the spectral resolution and sensitivity of the biosensor
[184, 185]. To overcome the limitation of tissue attenuation, the development of
fluorescent proteins with high quantum yields and far-red or near-infrared (NIR)
shifted absorption and emission wavelengths are preferred. Currently there are
several fluorescent protein variants which emit light in the NIR range and have
been developed from bacterial phytochrome photoreceptors and are used in various
biosensor applications [186, 187].
174
U. K. Sukumar et al.