Ratiometric biosensors are severely limited for quantitative estimations owing to
their lower sensitivity (i.e., smaller dynamic range), larger spectral bandwidth, and
the need to acquire images with two combinations of excitation and emission filters
for fluorescence measurement.
The sensitivity of biosensor for measuring ionic concentration has been
determined by its binding affinity for an ion and its dynamic range. Binding affinity
of an ion can be defined in terms of dissociation constant (Kd), which is the ion
concentration at which 50% of the sensor binding sites are occupied. This can be
experimentally determined by sensor titration experiments. Dynamic range is essentially an indicator of a sensor’s measurement sensitivity and its signal-to-noise ratio
(SNR). In order to monitor ion concentration changes, it is preferable to choose a
sensor that is 20% saturated at baseline, whereas a sensor that is ~50% saturated at
baseline is more suitable for comparing differences in resting ion concentrations in
different cells or different environmental conditions. For instance, Cameleon-Nano
sensors have lower Kd and are better for quantitative measurement of cytosolic Ca
2+
in some cell types, whereas D1ER is preferred for ER measurement because Ca
2+
levels are high in the ER and the Kd of D1ER is much higher than other Cameleons.
Tables 2 and 3 summarize Kd and DRs of some ratiometric ion sensors which are
designed for measuring ion concentrations in subcellular organelles.
3.2 Fluorescent Protein Complementation (Split Fluorescent
Proteins) Sensors
3.2.1 Sensors Fused with Intermediate Recognition Domains from
Target Proteins for Designing the Biosensors
To expand the scope of sensors specific to some analytes, an analyte-specific
extrinsic recognition domain has been inserted into FPs. In the conventional design
of bimolecular fluorescent complementation (BiFC) sensors, a FP is split into two
fragments and then fused to recognition domains that are associated with the
analytes of interest [86, 87]. The two halves of the FP do not emit fluorescence in
the state of dissociation since no intact chromophore will be reconstituted. Upon the
analyte-induced change in the recognition domains, the complementary fragments of
the FP are brought into close proximity and reconstitute the β-barrel chromophore
structure of the FP, resulting in the recovery of the fluorescence signal. In general,
split FP strategies have a much lower background so that it may produce a greater
dynamic range than those of FRET and single FP-based biosensors. On the other
hand, a major drawback of split FP-based biosensors is that they are not reversible.
While irreversibility provides a significant advantage for detecting transient and/or
weak interactions, it is not suitable for analyzing dynamics of an analyte in real time
[88]. Study of protein-protein interaction involves split FP fragments that do not
associate with each other spontaneously. In this strategy, the fluorescent protein
halves are fused to two different target proteins of interest. In the event of interaction
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U. K. Sukumar et al.
their lower sensitivity (i.e., smaller dynamic range), larger spectral bandwidth, and
the need to acquire images with two combinations of excitation and emission filters
for fluorescence measurement.
The sensitivity of biosensor for measuring ionic concentration has been
determined by its binding affinity for an ion and its dynamic range. Binding affinity
of an ion can be defined in terms of dissociation constant (Kd), which is the ion
concentration at which 50% of the sensor binding sites are occupied. This can be
experimentally determined by sensor titration experiments. Dynamic range is essentially an indicator of a sensor’s measurement sensitivity and its signal-to-noise ratio
(SNR). In order to monitor ion concentration changes, it is preferable to choose a
sensor that is 20% saturated at baseline, whereas a sensor that is ~50% saturated at
baseline is more suitable for comparing differences in resting ion concentrations in
different cells or different environmental conditions. For instance, Cameleon-Nano
sensors have lower Kd and are better for quantitative measurement of cytosolic Ca
2+
in some cell types, whereas D1ER is preferred for ER measurement because Ca
2+
levels are high in the ER and the Kd of D1ER is much higher than other Cameleons.
Tables 2 and 3 summarize Kd and DRs of some ratiometric ion sensors which are
designed for measuring ion concentrations in subcellular organelles.
3.2 Fluorescent Protein Complementation (Split Fluorescent
Proteins) Sensors
3.2.1 Sensors Fused with Intermediate Recognition Domains from
Target Proteins for Designing the Biosensors
To expand the scope of sensors specific to some analytes, an analyte-specific
extrinsic recognition domain has been inserted into FPs. In the conventional design
of bimolecular fluorescent complementation (BiFC) sensors, a FP is split into two
fragments and then fused to recognition domains that are associated with the
analytes of interest [86, 87]. The two halves of the FP do not emit fluorescence in
the state of dissociation since no intact chromophore will be reconstituted. Upon the
analyte-induced change in the recognition domains, the complementary fragments of
the FP are brought into close proximity and reconstitute the β-barrel chromophore
structure of the FP, resulting in the recovery of the fluorescence signal. In general,
split FP strategies have a much lower background so that it may produce a greater
dynamic range than those of FRET and single FP-based biosensors. On the other
hand, a major drawback of split FP-based biosensors is that they are not reversible.
While irreversibility provides a significant advantage for detecting transient and/or
weak interactions, it is not suitable for analyzing dynamics of an analyte in real time
[88]. Study of protein-protein interaction involves split FP fragments that do not
associate with each other spontaneously. In this strategy, the fluorescent protein
halves are fused to two different target proteins of interest. In the event of interaction
160
U. K. Sukumar et al.