spectral characteristics and act as biosensors (1) to measure target enzyme functions
(e.g., protein kinases and proteases); (2) to measure the concentration of intracellular
ions, metabolites, and messengers (H
+
, Ca
2+ , Cl
À
, H 2 O 2 , cAMP, etc.); (3) to monitor
cellular physicochemical parameters (i.e. specific analyte, covalent modification,
mechanical influence, redox potential, membrane potential); and (4) for highthroughput screening of drug candidates and their evaluations in preclinical studies.
One of the earliest fluorescent proteins to be discovered was green fluorescent
protein (GFP), when, in 1962, a Japanese organic chemist and marine biologist
Osamu Shimomura stumbled upon this remarkable protein in the jellyfish Aequorea
victoria [5]. At present this discovery has reached beyond the realm of science and
our homes with the development of transgenic fluorescent fishes and green fluorescent pigs and cats [6, 7]. The first use of GFP as a fluorescent tag for in vivo labeling
was demonstrated in 1995 by Dhandayuthapani et al., where they reported the
application of GFP-engineered mycobacteria (M. smegmatis and M. bovis BCG)
for analysis of fundamental biological and pathogenesis related to mycobacteria
[8]. It was rather serendipitous that GFP turned out to be a natural monomer which
enabled its wide use for labeling of various proteins of interest by simple in-frame
fusion to the –COOH or –NH 2 terminus or even as an insert within a flexible loop of
a protein [9].
Biosensing encompasses a diverse array of techniques for the generation of
an experimentally accessible “readout” of a molecular interaction between a
biomolecule-derived molecular recognition element (MRE) (e.g., a protein domain)
and an analyte of interest (e.g., a small molecule, another protein, or an enzymatic
activity) [10, 11]. Molecular entities or devices that enable biosensing are generally
referred to as biosensors. The primary challenge of creating biosensors is transducing the nanometer-scale event of a biorecognition process into an observable
change in a macroscopic property such as color or fluorescence hue [12]. One of
the nanometer-scale changes that typically accompany biorecognition events is the
change in molecular “geometry” of the MRE. This change could be a distance
between the MRE and its analyte, as in the case of a protein-protein interaction, or
a conformational change of the MRE, as in the case of allosteric proteins [13–15]. As
we will discuss in this chapter, researchers have now devised a variety of strategies
to develop fluorescent protein-based biosensors for many applications [16].
The protein-based fluorescent biosensors can be broadly categorized into two
classes based on the construction method: the first class are genetically encoded
fluorescent proteins such as GFP and its variants, whereas the second class comprises of chemically constructed biosensors made of natural protein scaffolds and
artificial fluorescent molecules [17]. We will primarily discuss protein-based fluorescent biosensors in this chapter. In the case of genetically encoded biosensors
(GFP-based), the GFP protein acts as a signal transducer that manifests change in
fluorescence intensity or wavelength shift in response to triggered stimuli. Different
versions of such biosensors have been established in the past, including single
FP-based biosensors, split GFP-based biosensors, and dual FP-fused FRET-based
biosensors [18]. Such biosensors are a powerful tool for in-cell imaging and/or
elucidating biological events of cells in normal and pathological processes.
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U. K. Sukumar et al.
(e.g., protein kinases and proteases); (2) to measure the concentration of intracellular
ions, metabolites, and messengers (H
+
, Ca
2+ , Cl
À
, H 2 O 2 , cAMP, etc.); (3) to monitor
cellular physicochemical parameters (i.e. specific analyte, covalent modification,
mechanical influence, redox potential, membrane potential); and (4) for highthroughput screening of drug candidates and their evaluations in preclinical studies.
One of the earliest fluorescent proteins to be discovered was green fluorescent
protein (GFP), when, in 1962, a Japanese organic chemist and marine biologist
Osamu Shimomura stumbled upon this remarkable protein in the jellyfish Aequorea
victoria [5]. At present this discovery has reached beyond the realm of science and
our homes with the development of transgenic fluorescent fishes and green fluorescent pigs and cats [6, 7]. The first use of GFP as a fluorescent tag for in vivo labeling
was demonstrated in 1995 by Dhandayuthapani et al., where they reported the
application of GFP-engineered mycobacteria (M. smegmatis and M. bovis BCG)
for analysis of fundamental biological and pathogenesis related to mycobacteria
[8]. It was rather serendipitous that GFP turned out to be a natural monomer which
enabled its wide use for labeling of various proteins of interest by simple in-frame
fusion to the –COOH or –NH 2 terminus or even as an insert within a flexible loop of
a protein [9].
Biosensing encompasses a diverse array of techniques for the generation of
an experimentally accessible “readout” of a molecular interaction between a
biomolecule-derived molecular recognition element (MRE) (e.g., a protein domain)
and an analyte of interest (e.g., a small molecule, another protein, or an enzymatic
activity) [10, 11]. Molecular entities or devices that enable biosensing are generally
referred to as biosensors. The primary challenge of creating biosensors is transducing the nanometer-scale event of a biorecognition process into an observable
change in a macroscopic property such as color or fluorescence hue [12]. One of
the nanometer-scale changes that typically accompany biorecognition events is the
change in molecular “geometry” of the MRE. This change could be a distance
between the MRE and its analyte, as in the case of a protein-protein interaction, or
a conformational change of the MRE, as in the case of allosteric proteins [13–15]. As
we will discuss in this chapter, researchers have now devised a variety of strategies
to develop fluorescent protein-based biosensors for many applications [16].
The protein-based fluorescent biosensors can be broadly categorized into two
classes based on the construction method: the first class are genetically encoded
fluorescent proteins such as GFP and its variants, whereas the second class comprises of chemically constructed biosensors made of natural protein scaffolds and
artificial fluorescent molecules [17]. We will primarily discuss protein-based fluorescent biosensors in this chapter. In the case of genetically encoded biosensors
(GFP-based), the GFP protein acts as a signal transducer that manifests change in
fluorescence intensity or wavelength shift in response to triggered stimuli. Different
versions of such biosensors have been established in the past, including single
FP-based biosensors, split GFP-based biosensors, and dual FP-fused FRET-based
biosensors [18]. Such biosensors are a powerful tool for in-cell imaging and/or
elucidating biological events of cells in normal and pathological processes.
152
U. K. Sukumar et al.