REV

Biotechnology
Journal
DOI 10.1002/biot.201300001 Biotechnol. J. 2013, 8, 1280–1291
1280 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1 Introduction
Cell metabolism boasts a complicated network. Exploring
the tremendous amount of information contained within
this network is of great significance to related studies. To
be able to visualize and monitor cell activities in vivo with
high spatial and temporal resolution is attractive because
traditional analysis methods destroy living cells to extract
metabolites of interest. Since the discovery of green fluo-
rescent protein (GFP), scientists have used site-directed
and random mutagenesis approaches to develop fluores-
cent protein (FP) mutants, and have created a family of
proteins that almost span the fluorescence spectrum. This
family has enlightened efforts to construct genetically
encoded fluorescent biosensors based on protein/protein
interactions and intramolecular conformational changes
targeted to living cells or tissues. Recently, another sort of
genetically encoded biosensor, the RNA-based biosensor,
has been developed for tracking small-molecule metabo-
lites such as adenosine, ADP, S-adenosyl methionine
(SAM), guanine, and GTP, and shows more advantages
than FP-based biosensors. Here, we describe different
modes of constructing fluorescent biosensors and review
recent advances in the use of genetically encoded fluo-
rescent biosensors for tracing intracellular metabolism.
2 Fluorescent proteins: Gifts from the ocean
When a bright, greenish protein in jellyfish extracts was
observed by Shimomura in the 1960s [1], the glow of FPs
Review
Imaging and tracing of intracellular metabolites utilizing
genetically encoded fluorescent biosensors
Chang Zhang*, Zi-Han Wei* and Bang-Ce Ye
Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science
and Technology, Shanghai, China
Intracellular metabolites play a crucial role in characterizing and regulating corresponding cellular
activities. Tracking intracellular metabolites in real time by traditional means was difficult until the
powerful toolkit of genetically encoded biosensors was developed. Over the past few decades, iter-
ative improvements of these biosensors have been made, resulting in the effective monitoring of
metabolites. In this review, we introduce and discuss the recent advances in the use of genetical-
ly encoded biosensors for tracking some key metabolites, such as ATP, cAMP, cGMP, NADH, reac-
tive oxygen species, sugar, carbon monoxide, and nitric oxide. A brief phylogeny of fluorescent pro-
teins and several typical construction modes for genetically encoded biosensors are also
described. We also discuss the development of novel RNA-based sensors, which are genetically
encoded biosensors active at the transcriptional level.
Keywords: Genetically encoded fluorescent biosensors · In vivo imaging · Metabolite
Correspondence: Prof. Bang-Ce Ye, Lab of Biosystems and Microanalysis,
State Key Laboratory of Bioreactor Engineering, East China University of
Science and Technology, Meilong RD 130, Shanghai 200237, China
E-mail: bcye@ecust.edu.cn
Abbreviations: 2OG, 2-Oxogluatarate; BRET, bioluminescence resonance
energy transfer; cAMP, cyclic adenosine monophosphate; cGMP, cyclic
guanosine 5’-monophosphate; CFP, cyan fluorescent protein; cpFP, circu-
lar permutation fluorescent protein; DFHBI, 3,5-difluoro-4-hydroxybenzyli-
dene imidazolinone; FLIP, fluorescence indicator protein; FP, fluorescent
protein; FRET, fluorescence resonance energy transfer; GFP, green fluores-
cent protein; GPCR, G-protein coupled receptor; PBP, periplasmic binding
protein; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent pro-
tein kinase; OHP, organic hydroperoxide; RFP, red fluorescent protein;
ROS, reactive oxygen species; SAM, S-adenosyl methionine; YFP, yellow
fluorescent protein
Received 31 MAR 2013
Revised 02 AUG 2013
Accepted 26 AUG 2013
Supporting information
available online
* These authors contributed equally to this work.
Color online: refer to online PDF file for figures in color.

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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
provided the ability to light up the invisible metabolism in
a living life form. In 1992, Prasher and colleagues [2] used
a cDNA library from Aequorea victoria to clone the gene
encoding the aequorin companion protein, GFP. They
demonstrated GFP’s promising future, a gift for cellular
biologists: genetically encoded GFP could be expressed in
a variety of biological systems without additional factors
or external enzyme components. Stable chromophore for-
mation within a cylinder structure makes GFP a powerful
cellular reporter [3]. A hexapeptide sequence starting at
amino acid 64 (number indicates the position in the intact
peptide sequence) determines the light absorption prop-
erties of GFP. The adjacent Ser65-Tyr66-Gly67 sequence
forms the chromophore, and fluorescence results from the
oxidization of the Tyr66 a–b carbon bond [4]. The crystal
structure for GFP shows the cyclic tripeptide chro-
mophore buried in the center of an 11-stranded β-barrel
cylinder structure [5]. In further studies of the cylinder
structure, mutagenesis of the amino acid residues sur-
rounding the chromophore significantly affected the
spectral properties of the protein. A series of FPs, with
emission wavelengths ranging from the blue to yellow
regions of the visible spectrum, and “enhanced” (E) FPs,
with increased protein folding efficiency and maturation
at physiological temperature (Supporting information,
Table S1), were produced [6].
Over the past 15 years, Anthozoa-based FPs spanning
the visible spectrum have been characterized and opti-
mized for imaging applications [6]. DsRed-based variants
and the mFruit (“m” for monomer) series are two repre-
sentative FP families with longer wavelength emissions
[7]. Members of the mFruit family, including mHoneydew,
mBanana, mOrange, mTangerine, mStrawberry, mCherry,
mApple, mPlum, and dTomato (“d” for dimer) derive from
monomeric red FP (RFP) 1, a mutant of DsRed (33 amino
acid substitutions) [8]. They have been useful in multi-
color imaging experiments. EosFP, another type of pho-
toactivatable FP, from the reef coral Lobophyllia hem-
prichii can be photoconverted from green to red fluores-
cence by near-ultraviolet light [9, 10]. Mutagenesis was
used to develop a monomeric protein named mEosFP. An
improved form, mIrisFP, enables pulse-chase experiments
with superb resolution and provides an excellent fusion
marker for imaging in living cells.
Figure 1. Design methods for construct-
ing single fluorophore sensors. The units
of a dimeric binding domain can be
directly attached to the original N and
C termini of an FP (A) or to novel N and
C termini of a cpFP (C). A monomeric
binding domain can be inserted into an
FP (B) or a cpFP (D). The binding or
effective domain undergoes a structural
change upon substrate (red star) bind-
ing or in response to other influencing
factors, such as ROS, leading to a
change in the fluorescent signal of the
sensor.

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3 Design modes for genetically encoded
fluorescent biosensors
3.1 Single fluorophore sensors
Proteins can tolerate circular permutation, which pro-
duces novel N- and C-termini from different portions of
the protein, while maintaining a stable structure. Tsien
and coworkers [11], researching circular permutation and
receptor insertion within GFPs, showed that GFP is sur-
prisingly robust for circular permutation and insertion,
and offered a new strategy for creating genetically encod-
ed indicators [12–14]. Circular permutation FP (cpFP) is
especially attractive because the binding domain of inter-
est undergoes a weak structural change upon substrate
binding, and the signal change is amplified when the
domain is placed in the sensitive region of the chro-
mophore. Depending on the structure of the receptor,
such as the distance between N and C termini, oligomer-
ization, etc., researchers can choose a proper design
method for constructing cpFP-based sensors (Fig. 1). Giv-
en the structural similarity between GFP and its mutants,
many active cpFP have been produced using yellow FP
(YFP) [15–17], Venus [18], and RFP [19, 20]. Recently,
cpYFP-based sensors for NADH were developed by
inserting cpYFP into dimeric NADH binding domain Rex
as pictured in Fig. 1C.
In addition to circular permutation, GFP family mem-
bers are also amenable to protein fragmentation [21]. When
the fragments of an FP are brought into proximity with
each other, they can reunite to form a functional protein
(Fig. 2). This method, termed bimolecular fluorescence
complementation, has been exploited to detect protein-
protein interactions in living cells and plants [22–25].
A great advantage of this strategy is its high sensitivity,
since GFP fragments in the separated state only display a
modest background fluorescence until they are brought
closer by protein-protein interactions [26].
3.2 Fluorescence resonance energy transfer-based
reporter systems
Fluorescence resonance energy transfer (FRET) is used in
the design of indicators for optically imaging biochemical
and physiological functions in living cells [27, 28].
A genetically encoded FRET biosensor consists of a
recognition module, which specifically binds a ligand,
sandwiched between two variants of GFP – typically cyan
FP (CFP) and YFP [29, 30]. The efficiency of fluorescence
energy transfer between the two fluorophores is highly
dependent on their distance and orientation [31–33]. Tra-
ditional FRET sensors can be divided into two classes, as
described below.
Bimolecular probes are designed based on protein
interaction and are suitable for studying the dissociation
or association of a protein upon ligand binding [34, 35].
Donor and acceptor FPs are fused to interacting proteins
separately to form a pair of FRET sensors. When the inter-
action of proteins X and Y brings the FRET sensors clos-
er, the intermolecular FRET signal efficiency between
donor and acceptor FPs is elevated (Fig. 3A). This mech-
anism has been used to construct probes for measuring
the cytosolic Ca2+ concentration and protein interactions
within the same cell, using Fura-2 with super-enhanced
CFP and YFP as a FRET pair [36]. Single-chain probes are
designed based on a protein conformational change that
occurs upon ligand binding or in response to another
effector. Kolossov et al. [37] inserted redox-sensitive link-
ers between CFP and YFP to engineer single-chain FRET
sensors for reactive oxygen species (ROS) detection.
FRET-based sensors are also suitable for detecting lig-
ands of interest in vivo [38]. The selected binding domain
is sandwiched between FP pairs, usually CFP and YFP.
A conformational change in the domain upon ligand bind-
ing likely leads to a change in the distance or orientation
between the CFP and YFP chromophores, resulting in a
FRET efficiency change (Fig. 3B). Optimization is impor-
tant to obtain sensors with obvious FRET ratio changes or
Figure 2. When the interacting proteins
X and Y bind to each other, the seg-
ments of a fluorescent protein are
brought into proximity to form a func-
tional protein with recovered fluorescent
signal.

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a dynamic range consistent with concentration level of
the target molecules of interest in cell metabolism [31, 32].
Traditional bioluminescence resonance energy trans-
fer (BRET)-based assays have also been used for detect-
ing protein interactions [39–41]. In a BRET-based sensor,
luciferase is usually used as the BRET donor and an FP as
the BRET acceptor. However, using BRET to monitor pro-
tein complexes at the subcellular level has been limited by
the amount of light emission intrinsic to the luciferase
donor [42]. Recently, Dragulescu-Andrasi et al. [43]
demonstrated the use of red light-emitting BRET systems
for investigating protein interactions in deep tissue and in
small animal tumor models, which may help to eliminate
this drawback. Binkowski et al. [44] employed a circularly
permuted form of firefly luciferase to construct a lumines-
cent biosensor for intracellular cyclic adenosine mono-
phosphate (cAMP) detection. They studied signal trans-
duction mediated by G-protein coupled receptors
(GPCRs), and showed that luciferase is suitable for the
construction of genetically encoded biosensors as cpFPs.
4 Biosensors for tracing metabolism
4.1 Visualization of ATP levels inside cells
ATP is the major energy currency in cellular processes.
Real-time monitoring of ATP levels inside individual living
cells would help elucidate the compartmentation of ATP
and its role in modulating ion channels and signaling cas-
cades [45]. Imamura et al. [46] generated a series of FRET-
based indicators for ATP, and showed that the ATP levels
differed between the mitochondrial matrix and cyto-
plasm/nucleus in HeLa cells. Additional research demon-
strated that the ATP-generating pathway changed in
response to nutritional changes in the environment. The
probes used in these studies show high selectivity for
ATP over other nucleotides. By modulating the affinity
through substitution of different effector domains, or
mutation of residues at the binding interface, the probes
can measure ATP levels ranging from 2 μM to 8 mM. On
attaching a proper signal sequence, these sensors could
monitor any intracellular compartment of interest and
facilitate biological research in other fields.
Since the absolute levels of ATP, ADP, and AMP can
fluctuate, the ratio of [ATP]/[ADP] is thought to be a more
reliable indicator of energy status in cells. Berg et al. [47]
presented a novel cpFP-based fluorescent probe for meas-
uring the cellular ATP/ADP ratio. They measured the
energy level by competitive binding of ATP and ADP in a
cell without perturbing the cellular energy balance, in
contrast to sensors based on luciferase [48]. The regula-
tion of the metabolic machinery in cancer is complicated.
A better understanding of differential cancer cell metab-
olism would greatly benefit therapeutics. Zadran et al. [49]
monitored cytosolic ATP in tumor cells with a sensor that
could also be used in mitochondria, nuclei, and the endo-
plasmic reticulum. These efforts will improve our under-
standing of cellular and subcellular energy variations in
normal and abnormal cells [50, 51].
4.2 Fluorescent indicators of cAMP
cAMP is a second messenger of many GPCRs and regu-
lates cAMP-dependent protein kinase (PKA) and
exchange proteins activated by cAMP (Epacs) to mediate
cellular functions. Spatial and temporal readouts of cAMP
levels are vital to the comprehension of network regula-
tion in various signaling cascades. DiPilato et al. [52] con-
structed fluorescent indicators to report intracellular
Figure 3. Schematic presentation of
(A) a bimolecular FRET probe and (B) a
single-chain FRET probe. (A) When the
interacting proteins X and Y bind to each
other, the fluorescent pair is brought
into proximity and a FRET phenomenon
appears. (B) When the binding domain
in a single-chain FRET probe undergoes
conformational changes upon ligand
binding, the distance or orientation
between the fluorescent pair changes
and then alters the FRET efficiency.

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cAMP dynamics and Epac activation. They revealed that
the cAMP response at the membrane was faster than that
in the cytoplasm and mitochondria. In later work that has
drawn much attention, they directed an improved sensor
to membrane rafts and caveolae and successfully investi-
gated the role of membrane raft integrity via cholesterol
depletion. This promising indicator could provide insight
into differences between membrane microdomains in
healthy and diseased states with good spatiotemporal
resolution [53]. Nikolaev et al. [54] exploited the cAMP-
binding domains of Epac and PKA to construct FRET-
based indicators without cooperativity, catalytic proper-
ties, of interactions with other proteins. The investigators
used these indicators to trace intracellular cAMP, and
found that cAMP signals traveled very fast throughout
hippocampal neurons and peritoneal macrophages. They
also used cAMP sensors to directly monitor the spatial
and temporal distribution of cAMP in adult cardiomy-
ocytes, and distinguished the cAMP signal diffusion
mediated by different adrenergic receptors.
Recently, Fan and colleagues [44, 55] optimized a nov-
el biosensor based on PKA and a circularly permuted form
of luciferase, and demonstrated its suitability for high-
throughput screening applications. Mazina et al. [56] con-
structed a simple and robust system for ligand screening
in a variety of mammalian cell lines utilizing viral infec-
tion. This system is applicable to high-throughput screen-
ing as a fast and dependable platform for drug discovery.
4.3 Visualization of intracellular cGMP signaling
The second messenger cyclic guanosine 5’-monophos-
phate (cGMP) participates in a variety of physiological
processes in mammals, especially in the nervous and vas-
cular systems [57–59]. These processes include mediation
of smooth muscle relaxation and modulation of synaptic
plasticity. cGMP functions by regulating effectors such as
cGMP-specific phosphodiesterases [60], cGMP-depend-
ent protein kinases (PKGs), and cyclic nucleotide-activat-
ed ion channels [61]. Understanding of the cGMP signal-
ing pathway has triggered great interest. Sato et al. [62]
described fluorescent indicators for cGMP in single living
cells that contained PKG I fused to FPs. Modified PKG I
(some amino acid residues were deleted in a dimerization
domain) was found to increase affinity upon cGMP bind-
ing and to respond reversibly to cGMP in nitric oxide
stimulated HEK293 cells. Honda et al. [63] increased the
selectivity for cGMP and eliminated the constitutive
kinase activity of the binding domain to diminish inter-
ference from the sensor. Using transfected Purkinje neu-
rons, they also confirmed that cGMP increases in cells
under conditions that induce synaptic plasticity.
To investigate the dynamics of cGMP in living cells,
Russwurm et al. [64] systematically studied the design of
FRET-based cGMP indicators and created indicators with
excellent specificity and rapid kinetics. The binding
affinities of these indicators ranged from 500 nM to 6 μM,
and radioimmunoassays were applied to validate their
performance. Nausch et al. [65] reported the design of a
cpFP-based cGMP biosensor to assess the dynamics of
intracellular cGMP synthesis, showing that such sensors
could act as innovative tools in elucidating the cGMP
dynamics. All indicators using different fragments of PKG
I displayed good selectivity, with high dynamic ranges
and apparent KDs.
4.4 Sensors for intracellular NADH detection
Reduced NADH and its oxidized form, NAD+, are the most
important cofactors in electron transfer metabolism. They
participate in multiple biological and pathological
processes, such as ischemia [66], energy metabolism [67],
tumor cell migration [68], and epilepsy [69]. Zhao et al. [70]
recently developed cpYFP-based sensors for NADH and
monitored the dynamic changes in NADH levels in the
organelles of mammalian cells. The NADH-binding
domain was mutated to expand the dynamic range, to
achieve high sensitivity with minimal perturbation, and
to target the sensor to subcellular organelles. When this
probe was used to trace the transport of exogenous NADH
across the plasma membrane in different cell lines,
researchers found that the inhibitor of P2X7R did not play
a role in the transport of NADH across the plasma mem-
brane. Zhao et al. [70] also investigated the differences in
NADH concentration in response to environmental
changes in different subcellular compartments. Mito-
chondria tended to maintain NADH at a stable level. To
evaluate the cellular NADH-NAD+ redox state, Hung et al.
[71] used a circularly permuted GFP T-Sapphire-based
sensor to monitor NADH responses to Phosphatidylinosi-
tide 3-kinase pathway inhibition. The fluorescence signal
of the sensor was calibrated with exogenous lactate and
pyruvate to measure cytosolic NADH:NAD+ ratios in vivo
and in vitro.
4.5 Optical measurement of amino acids
Glutamate plays a vital role in amino acid metabolism
[72], regulating not only nitrogen circulation together with
glutamine and 2-oxoglutarate [73], but also affecting sig-
nal transduction [74, 75]. Glutamate is the predominant
excitatory neurotransmitter in the mammalian brain.
Okumoto and colleagues [76, 77] developed a FRET-based
indicator for the detection of glutamate release from neu-
rons and glutamate imaging in brain slices. Site-directed
mutagenesis of the binding pocket was used to generate
mutants with binding affinities covering physiological
glutamate concentrations. Hires et al. [78] also quantita-
tively measured synaptic glutamate release with centi-
second temporal and dendritic spine-sized spatial resolu-
tion. With systematic optimization of linkers and gluta-
mate binding affinities, the improved sensor exhibits a

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6.2-fold increase in response magnitude over that of the
original. Thus, the sensor may be useful as a calibration
tool in tracing the propagation of glutamate, mapping the
functional connectivity of the brain, or in drug screening
for cerebral ischemia. Gruenwald et al. [79] studied anoth-
er critical amino acid, glutamine, and estimated the glut-
amine concentration in cells using sensors with different
affinities. Site-directed mutagenesis was performed to
create sensors with different affinities to facilitate physi-
ological glutamine analysis. These sensors can be easily
used to confirm the properties of glutamine transporters.
Many efforts have been made to enrich the family of sen-
sors for other amino acids. Okada et al. [80] used bacteri-
al periplasmic binding proteins (PBPs) to construct FRET-
based sensors, taking advantage of the structure of PBPs
to expand the dynamic range of the sensors by circular
permutation of the PBP module.
4.6 Fluorescent sensors for sugar detection
Sugars involved in key metabolic reactions need to be
monitored in real time to determine their subcellular dis-
tribution [81]. PBPs were utilized to develop the substrate-
binding element of sensors by linking an FP. These were
defined as fluorescence indicator protein (FLIP) family
sensors (also FRET-based sensors) because their hinge-
bend movement leads to highly responsive FRET. From-
mer and colleagues [32, 82–85] used PBPs to design a
series of FLIP sensors, and monitored the cytosolic distri-
bution of glucose, galactose, maltose, ribose, arabinose,
and sucrose in vivo. Glucose/galactose-binding protein
for glucose and galactose detection [82], maltose-binding
protein (for maltose detection [83], ribose-binding protein
for ribose detection [84], sucrose-binding protein for
sucrose detection [85], and L-arabinose-binding protein
for arabinose detection have been used as PBPs to devel-
op FLIP biosensors in which the bacterial PBPs are
flanked with GFP variants [32]. These FLIP sensors have
been improved and optimized to yield large dynamic
ranges when binding to target sugar molecules [83].
Effectively monitoring the cytosolic and intracellular dis-
tribution and concentration of sugars in real time will
shed light on their flux during metabolism. For example,
the FLIPGlu detection experiments showed that the con-
centration of cytosolic glucose fluctuates by several
orders of magnitude depending on the external glucose
supply [82]. FLIPmal, on the other hand, paved a way for
a better understanding of the transport processes within
and between cells. The determination of sucrose (as an
energy and carbon skeleton supplier in non-photosyn-
thetic organs of plants) and maltose concentrations by
this sensor in organelles such as chloroplasts character-
ized the actual function of transporters from correspon-
ding compartments [85]. Since the cytosolic and intercel-
lular sugar concentration and distribution directly relate
to carbohydrate metabolism, FLIP sensors additionally
have potential applications for fermentation processes in
the food, pharmaceutical, and biofuel industries. Sensors
for arabinose and maltose were used to determine the
concentrations of intracellular ligands in bacterial cul-
tures, making it possible to calculate accumulation rates
after the addition of the metabolite [86]. The use of PBP-
based sensors in metabolite flux research and bioprocess
visualization has progressed, but novel and optimized
sensors for other important sugar metabolites remain to
be developed.
4.7 Fluorescent sensors for redox state
Changes in ROS and, by extension, in cellular or intercel-
lular redox equilibrium are important for regulating a wide
range of physiological and pathological functions. Oster-
gaard and colleagues developed a redox-sensitive GFP
with two surface-exposed cysteines close to the chro-
mophore for real-time visualization of the molecule’s own
redox state [87–89]. Subsequently, a high oxidative
response sensor based on a redox-sensitive mutant of YFP
(rxYFP) was developed [90]. In other cases, sensors were
designed by flanking a polypeptide, acting as redox-sen-
sitive linker or redox-sensitive switch, with CFP/YFP,
leading to a FRET signal change in response to a different
redox environment [37]. These sensors depend on ROS-
induced intramolecular formation of disulfide bonds,
which lead to reversible conformational changes and
enable the imaging of ROS-induced oxidoreductase reac-
tion processes and signal transduction. HyPer, developed
by Belousov et al. [91], is an example of an ROS (mainly
H2O2) response domain-based sensor. HyPer consists of a
sensing domain, derived from Escherichia coli OxyR, with
two redox-active cysteines and cpYFP. The single-site
mutation A406V in HyPer (HyPer-2, A233V in OxyR)
expands the dynamic range of the probe up to
6-fold, thereby improving the detection of H2O2 levels in
the cytosol and peroxisome of tobacco and Arabidopsis
[92]. Sakai and colleagues [93, 94] engineered a novel
FRET-based redox sensor, named Redoxfluor, by fusing
the GFP variants Cerulean and Citrine to the N terminus
and C terminus, respectively, of the cysteine-rich domain
(I601 to N650) of sensory Yap1. This multifunctional sen-
sor has been used to detect peroxisomal and cytosolic
redox states in wild-type and mutant cells, screen for
drugs that modulate abnormal cytosol redox state in CHO
cells, and investigate redox state maintenance via cyto-
plasmic thioredoxin in the yeast Saccharomyces cerevisi-
ae. Similarly, Chen’s group designed a highly selective
and sensitive FRET-based sensor for organic hydroperox-
ides (OHPs), which constantly generate cellular stress, by
combining the responsive domain of the transcriptional
regulatory protein OhrR with an environmentally sensi-
tive FP, a modified Venus [15]. Moreover, ROS or redox
state probes could be designed based on redox regulato-
ry systems from different species to monitor the response

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processes of redox systems per se, and further define the
mechanisms of these regulatory networks. More conven-
ient and effective probes still need to be mined for com-
plex mechanism research.
4.8 Other significant fluorescent sensors
Traditionally, carbon monoxide (CO) is viewed as a toxi-
cant or a pollutant. However, a recent study revealed that
CO, like NO, functions as an essential second messenger
[95]. Wang et al. [17] constructed the fluorescent probe
COSer for CO by fusing cpYFP to a dimeric CO-sensitive
heme protein from Rhodospirillum rubrum, CooA, as a CO
recognition site. However, further studies are needed to
improve the properties of CO probes used in biological
systems and other settings, such as industrial facilities.
Sato et al. [96] have reported a novel cell-based indicator
to visualize NO release from living cells, made by com-
bining endogenously expressed guanylate cyclase with a
FRET-based cGMP indicator. The indicator was used to
visualize NO diffusion from single vascular endothelial
cells. It showed good sensitivity, selectivity, and potential
for deciphering NO dynamics in biological systems.
2-Oxogluatarate (2OG), a metabolite from the highly
conserved Krebs cycle, not only plays a critical role in
metabolism, but also acts as a signaling molecule in a
Table 1. A list of fluorescent biosensors for studying metabolism in vivo
Name Applied for Based on Sensory domain Donor/receptor Ref
ATeams ATP Single-chain FRET FoF1-ATP synthase MseCFP/ cpmVenus [46]
EAF Modified FoF1 synthase GFP/YFP [49]
Perceval [ATP]/[ADP] cpFP GlnK1 cpmVenus [47]
ICUE cAMP Single-chain FRET Epac1 ECFP/Citrine [52]
HCN2-camps HCN2 channel EYFP/ECFP [54]
22F BRET PKA /RIIβB Luciferase [44]
RII-CFP/C-YFP Bimolecular FRET PKA CFP/YFP [102]
cGMP Single-chain FRET cNMP-BD/GAF EYFP/ECFP [64]
FlincGs cpFP PKG I cpEGFP [65]
CGY Single-chain FRET PKG I ECFP/EYFP [62]
Cygnet-2.1 cNMP-BD ECFP/Citrine [63, 103, 104]
Frex/ FrexH NADH cpFP Rex cpYFP [70]
Peredox [NADH]/[NAD+] cpFP Rex cpT-Sapphire [71]
GluSnFR Glu Single-chain FRET Gltl ECFP/Citrine [78]
FLIPQ-TV3.0 Gln GlnH mTFP1/Venus [79]
OGsor 2OG GAF ECFP/EYFP [97]
Arg QBP ECFP/Citrine [105]
Laconic Lactate LldR mTFP/Venus [98]
FLIPsuc-4mu Sucrose Sugar-binding protein ECFP/EYFP [85]
FILP-HisJ His HisJ ECFP/Venus [80]
FLIPGlu Glucose/galactose GGBP CFP/YFP [82]
FLIPmal Maltose MBP ECFP/EYFP [83]
FLIPrib Ribose RBP ECFP/EYFP [84]
FLIPara Arabinose L-Arabinose-binding protein ECFP/EYFP [86]
roGFP1/roGFP2 ROS roFP Surface-exposed cysteine roGFP [89]
HyPer H2O2 cpFP OxyR cpYFP [91]
Hyper-2 Mutant OxyR [92]
CY-RL5 Redox states Single-chain FRET RL5 ECFP/EYFP [37]
Redoxfluor Yap1 c-CRD Cerulean/Citrine [93, 94]
OHSer OHPs cpFP Xc-OhrR cpVenus [15]
CLPY BAI-2 Single-chain FRET LuxP CFP/YFP [106, 107]
FLIP-CIT Citrate CitA Venus/CFP [29]
COSer CO cpFP CooA cpYFP [95]
FRET-MT NO Single-chain FRET hMTIIa ECFP/EYFP [108]
Piccell Cell-based indicator sGC CFP/YFP [96]
RNA-based ADP RNA-based ADP binding aptamer DFHBI [99, 100]
Adenosine fluorescence Ade binding aptamer
Guanine enlargement Gua binding aptamer
GTP GTP binding aptamer
SAM SAM binding aptamer

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selecting the targeted binding domains for signaling mol-
ecules, especially in small molecules, is difficult. Recent-
ly, fluorescent RNA has been generated as a novel genet-
ically encoded biosensor that mimics GFP. It is increas-
ingly used in various applications [99]. GFP contains a
three-residue fluorophore formation that produces the
green fluorescence, whereas native RNA lacks a fluo-
rophore element. Therefore, to confer a GFP-like ability to
RNA, Paige et al. [100] engineered a fluorophore-binding
RNA aptamer, termed Spinach, containing 3,5-difluoro-
4-hydroxybenzylidene imidazolinone (DFHBI), which
resembles the fluorophore in GFP, thereby yielding a dif-
ferent type of genetically encoded biosensor with GFP-
like brightness, photostability, and cellular compatibility.
Moreover, RNA-fluorophore complexes with emission
wavelengths spanning the entire visible spectrum have
been created using different optimized aptamers. These
RNA-based probes can be used widely to detect a variety
of small molecules, such as adenosine, ADP, SAM, gua-
nine, and GTP. DFHBI fluorescence is activated when the
targeted small molecule binds to a specific aptamer con-
nected to Spinach via an optimized stem (Fig. 4). RNA-
based sensors, unlike FP-based FRET sensors, produce
approximately 20-fold increases in fluorescence upon
metabolite binding. Thus, they are promising tools for
imaging various metabolites in vivo.
6 Conclusions
The discovery and artificial evolution of FPs has been a
prominent contribution to biology [101], making it possi-
ble to directly observe biochemical processes inside liv-
ing cells and organisms [6]. With developments in FPs and
variety of organisms. Environmental inorganic nitrogen is
reduced to ammonium by microorganisms, which boast
the metabolic pathways via conversion of 2OG to gluta-
mate and glutamine. Recently, Zhang et al. [97] developed
FRET-based biosensors for tracking 2OG in real-time; the
dynamic ranges of the sensors appeared identical to the
physiological range observed in E. coli. Citrate is another
important intermediate in catabolic pathways involving
glycolysis and lipid metabolism. Ewald et al. [29] have
described FRET-based citrate sensors. In an optimization
process, different peptide linkers were screened to
achieve a maximal change ratio, and residues in the cit-
rate-binding pocket were modified to construct sensors
with the proper affinity for the application. When
expressed in E. coli, the indicator showed that cells could
respond to a carbon source even after long-term starva-
tion. Lactate also plays significant roles in healthy tissues.
As an abnormal lactate level is associated with inflamma-
tion, hypoxia, ischemia, neurodegeneration, and cancer,
tracing lactate levels in cells has diagnostic and thera-
peutic applications. San Martin et al. [98] recently devel-
oped a lactate sensor that discriminates lactate flux in dif-
ferent cells and found that T98G glioma cells have a three
to fivefold higher rate of lactate production than normal
astrocytes, which is consistent with tumor metabolism.
All genetically encoded fluorescent biosensors construct-
ed to trace intracellular metabolites are listed in Table 1.
5 RNA-based fluorescent sensors
FP-based biosensors are powerful tools for visualizing cel-
lular activities, but the fusion of FPs may potentially inter-
fere with the function of sensory domains. Moreover,
Figure 4. RNA sensor structure and
imaging of SAM in E. coli. (A) The sensor
consists of Spinach (black), a transducer
(orange), and a target-binding aptamer
(blue). DFHBI (green) fluorescence is
activated when the sensor binds to the
target metabolite (purple). (B) Emission
spectra of the SAM sensor. (C) Imaging
shows distinct SAM accumulation pat-
terns. Some cells exhibit higher than
average (arrow) or slow (arrowhead)
increases in SAM, and one cell shows
increased and then decreased SAM
levels (double arrow).

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Journal
Biotechnol. J. 2013, 8, 1280–1291
design methods, genetically encoded fluorescent probes
have enabled scientists to decode the mechanisms of
intracellular signal transduction pathways and gather a
large amount of biological information from cellular sys-
tems. Genetically encoded fluorescent indicators present
a popular way to study metabolic processes, take advan-
tage of FRET technology, and allow noninvasive, spa-
tiotemporal monitoring of signaling molecules in vivo.
Future work will expand the range of detection ligands to
include molecules such as 2OG, ADP, NAD, and others,
and will emphasize the optimization of existing biosen-
sors [33]. Moreover, RNA-based fluorescent sensors will
provide a new platform for exploration in cells.
This study was supported by the China NSF (21276079,
21335003), SRFDP (no. 20120074110009), the Key Grant
Project (no. 313019) of the Chinese Ministry of Education,
and the Fundamental Research Funds for the Central Uni-
versities.
The authors declare no commercial or financial conflict of
interest.
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Bang-Ce Ye is Professor of biology and
chemistry in the East China University
of Science and Technology in China,
and Director of the Laboratory of
Biosystems and Microanalysis in State
Key Laboratory of Bioreactor Engineer-
ing. He obtained his PhD degree in
1998. His research now focuses on
analytical biotechnology and systems
biotechnology.
Chang Zhang received his BSc in Life
Science in 2009 from the East China
University of Science and Technology
in China. He is pursuing a PhD in bio-
chemistry and molecular biology in the
Laboratory of Biosystems and Micro-
analysis group at the same institution.
His research is centered on tracking
intracellular metabolites in real time by
developing genetically encoded biosensors. His present work focuses
on the study of metabolites such as 2-oxogluatarate in tumor cells to
provide a better understanding of the regulatory pathways and signal-
ing networks for cancer.
Zihan Wei majored in biotechnology at
Jiangsu University (B.S. 2007) before
pursuing graduate work in the Labora-
tory of Biosystems and Microanalysis,
State Key Laboratory of Bioreactor Engi-
neering at the East China University of
Science and Technology. Under the
direction of Prof. Bang-Ce Ye, Zihan
is currently engineering genetically
encoded FRET-based biosensors for detecting metabolites such as
ROS and 2-oxoglutarate in Saccharopolyspora erythraea and characteriz-
ing its related metabolic pathways.

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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com
Editorial: Biotechnology Journal in Asia –
the first AFOB special issue
Tai Hyun Park and George G.Q. Chen
http://dx.doi.org/10.1002/biot.201300415
Review
Organ-on-a-chip technology and microfluidic whole-body
models for pharmacokinetic drug toxicity screening
Jong Bum Lee and Jong Hwan Sung
Review
Microfluidic-integrated biosensors: Prospects for point-of-care
diagnostics
Suveen Kumar, Saurabh Kumar, Md. Azahar Ali,
Pinki Anand,Ved Varun Agrawal, Renu John, Sagar Maji
and Bansi D. Malhotra
Review
Imaging and tracing of intracellular metabolites utilizing
genetically encoded fluorescent biosensors
Chang Zhang, Zi-Han Wei and Bang-Ce Ye
Review
Applications of cell-free protein synthesis in synthetic biology:
Interfacing bio-machinery with synthetic environments
Kyung-Ho Lee and Dong-Myung Kim
Review
Current developments in high-throughput analysis
for microalgae cellular contents
Tsung-Hua Lee, Jo-Shu Chang and Hsiang-Yu Wang
Research Article
Heterologous prime-boost immunization regimens using
adenovirus vector and virus-like particles induce broadly
neutralizing antibodies against H5N1 avian influenza viruses
Shih-Chang Lin, Wen-Chun Liu,Yu-Fen Lin,
Yu-Hsuan Huang, Jin-Hwang Liu and Suh-Chin Wu
Research Article
Repetitive Arg-Gly-Asp peptide as a cell-stimulating agent
on electrospun poly(ε-caprolactone) scaffold for tissue
engineering
Pacharaporn Chaisri, Artit Chingsungnoen
and Sineenat Siri
Research Article
Global gene expression analysis of Saccharomyces cerevisiae
grown under redox potential-controlled very-high-gravity
conditions
Chen-Guang Liu,Yen-Han Lin and Feng-Wu Bai
Research Article
Automated formation of multicomponent-encapuslating
vesosomes using continuous flow microcentrifugation
Huisoo Jang, Peichi C. Hu, Sungho Jung, Won Young Kim,
Sun Min Kim, Noah Malmstadt and Tae-Joon Jeon
Research Article
Size and CT density of iodine-containing ethosomal vesicles
obtained by membrane extrusion: Potential for use as CT
contrast agents
Bomin Na, Byoung Wook Choi and Bumsang Kim
Research Article
Site-targeted non-viral gene delivery by direct DNA injection
into the pancreatic parenchyma and subsequent in vivo
electroporation in mice
Masahiro Sato, Emi Inada, Issei Saitoh, Masato Ohtsuka,
Shingo Nakamura,Takayuki Sakurai and
Satoshi Watanabe
Biotechnology Journal – list of articles published in the November 2013 issue.
This issue is the first official special issue of Biotechnology Journal with the Asian Federation of
Biotechnology (AFOB). In the cover, we use the grain as a symbol and analogy for biotechnology,
as both the grain and biotechnology are common and uniting factors that link the member
countries of the AFOB. Gran image credit: © Myimagine – Fotolia.com.
Systems & Synthetic Biology ·
Nanobiotech · Medicine
ISSN 1860-6768 · BJIOAM 8 (11) 1245–1364 (2013) · Vol. 8 · November 2013
11/2013
Microfluidics
Biosensors
Organ-on-chip

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