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\DOI{10.5802/crchim.365}
\datereceived{2024-04-02}
\daterevised{2024-10-16}
\dateaccepted{2024-11-21}
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\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Locally activated semisynthetic fluorescent biosensors for
imaging cellular biochemistry}

\alttitle{Biosenseurs fluorescents semi-synth\'{e}tiques localement
activ\'{e}s pour imager la biochimie cellulaire}

\author{\firstname{Justine} \lastname{Co\"{i}s}}
\address{Laboratoire des biomol\'{e}cules, LBM, D\'{e}partement de
chimie, \'{E}cole normale sup\'{e}rieure, PSL University, Sorbonne
Universit\'{e}, CNRS, 75005 Paris, France}
\address{Laboratoire Neurosciences Paris Seine, Sorbonne
Universit\'{e}, CNRS, INSERM, 75005 Paris, France}

\author{\firstname{Blaise} \lastname{Dumat}\CDRorcid{0000-0002-0455-7282}\IsCorresp}
\addressSameAs{1}{Laboratoire des biomol\'{e}cules, LBM,
D\'{e}partement de chimie, \'{E}cole normale sup\'{e}rieure, PSL
University, Sorbonne Universit\'{e}, CNRS, 75005 Paris, France}
\email[B. Dumat]{blaise.dumat@ens.psl.eu}

\keywords{\kwd{Biosensors}\kwd{Chemogenetic probes}\kwd{Fluorogenic
probes}\kwd{Fluorescence imaging}}

\altkeywords{\kwd{Biosenseurs}\kwd{Sondes
chimiog\'{e}n\'{e}tiques}\kwd{Sondes fluorog\'{e}niques}\kwd{Imagerie
de fluorescence}}

\thanks{``Interface pour le vivant'' (IPV) initiative of Sorbonne
Universit\'e}

\begin{abstract}
Biosensors based on fluorescent proteins are widely used as genetically
encoded indicators due to their capacity to target various biological
analytes (metal ions, reactive oxygen species, biomolecules, etc.) within
cells with precise localization. However, their complex development
associated with the lack of photophysical versatility constrains the
scope of their application in biosensing. Alternatively, semisynthetic
fluorescent biosensors that combine a small chemical indicator with a
self-labeling protein tag benefit from the versatility of molecular
engineering and from the selectivity of genetic encoding of the
recombinant protein. The variations in photophysical properties of
the chemical indicator upon analyte recognition provide high
sensitivity and rapid response time, making them attractive alternatives
for biosensing. Fluorogenic semisynthetic biosensors that are
fluorescent only upon local activation by reaction with a
genetically encoded self-labeling protein tag provide an additional
level of selectivity, allowing wash-free imaging experiments. This
minireview focuses on the latter class of hybrid sensors and provides an
outlook on the different small molecular probe design strategies and
self-labeling protein tag combinations (mostly SNAP-tag and HaloTag) for
their construction. The authors expect to present new clues and ideas to
researchers for further advances in this field.
\vspace*{-2pt}
\end{abstract}

\begin{altabstract}
Les biosenseurs bas\'{e}s sur les prot\'{e}ines fluorescentes sont
largement utilis\'{e}s comme indicateurs g\'{e}n\'{e}tiquement
encod\'{e}s en raison de leur capacit\'{e} \`{a} cibler divers analytes
biologiques (ions m\'{e}talliques, esp\`{e}ces r\'{e}actives de
l'oxyg\`{e}ne, biomol\'{e}cules, etc.) dans les cellules avec une
localisation pr\'{e}cise. Cependant, leur d\'{e}veloppement complexe,
associ\'{e} \`{a} des propri\'{e}t\'{e}s photophysiques peu versatile,
limite l'\'{e}tendue de leur application en imagerie. En revanche, les
biosenseurs fluorescents semi-synth\'{e}tiques, qui combinent un
indicateur chimique avec une \'{e}tiquette de marquage de prot\'{e}ine,
b\'{e}n\'{e}ficient de la polyvalence de l'ing\'{e}nierie des petites
mol\'{e}cules organiques et de la s\'{e}lectivit\'{e} de l'encodage
g\'{e}n\'{e}tique des prot\'{e}ines recombinantes. La variation des
propri\'{e}t\'{e}s photophysiques de l'indicateur chimique lors de la
reconnaissance de l'analyte offrent une grande sensibilit\'{e} et un
temps de r\'{e}ponse rapide, ce qui en fait une alternative
int\'{e}ressante pour la bioimagerie. Les biosenseurs
semi-synth\'{e}tiques fluorog\'{e}niques, qui ne deviennent
fluorescents que lorsqu'ils sont activ\'{e}s localement par la
r\'{e}action avec une \'{e}tiquette de marquage de prot\'{e}ine
g\'{e}n\'{e}tiquement encod\'{e}e, offrent un niveau suppl\'{e}mentaire
de s\'{e}lectivit\'{e}, permettant des exp\'{e}riences d'imagerie sans
\'{e}tape de lavage. Cette mini-revue se concentre sur cette
derni\`{e}re classe de senseurs hybrides et donne un aper\c{c}u des
diff\'{e}rentes strat\'{e}gies de conception de senseurs
mol\'{e}culaires et de leur combinaison avec des \'{e}tiquette de
marquage de prot\'{e}ine tels que HaloTag et SNAP-tag pour leur
construction. Nous esp\'{e}rons apporter de nouvelles pistes et
id\'{e}es aux chercheurs pour de futures avanc\'{e}es dans ce domaine.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}
\subsection{Definitions}

This review refers to several types of fluorescent probes and sensors.\
To avoid any confusion, we define a few terms that can sometimes be
found with different meanings in articles.\ A fluorescent
\textbf{chemosensor} refers to a synthetic small molecule for sensing
ions or other chemical species with a change in their fluorescent
properties (e.g., intensity, wavelength, lifetime)~\cite{1}.\ 
\textbf{Fluorogenic probes} or \mbox{\textbf{fluorogens}} are small
molecular fluorophores that bind biomolecules (herein proteins) with an
increase in their fluorescence~\cite{2}. \textbf{Biosensors} refer to
fluorescent protein (FP) based sensors of biological activity.
\textbf{Semisynthetic} biosensors are fluorescent reporters based on
the combination of a small molecular fluorescent indicator and a
genetically encoded nonfluorescent protein; they are also called
\textbf{chemogenetic}~\cite{3} or \textbf{chemigenetic}
sensors~\cite{4}.\vspace*{-1.5pt}

\subsection{Fluorescent sensors for bioimaging} \label{sec1.2}

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}General principle of the different classes of
sensors for bioimaging: (A)~chemosensors, (B)~semisynthetic biosensors,
and (C)~locally activated semisynthetic biosensors.}
{\vspace*{-.3pc}}
\end{figure*}

\begin{figure*}
{\vspace*{-.2pc}}
\includegraphics{fig02}
{\vspace*{-.3pc}}
\caption{\label{fig2}Comparison of HaloTag and SNAP-tag strategies for
subcellular bioimaging.}
{\vspace*{-.2pc}}
\end{figure*}

\looseness=-1
Genetically encoded FPs have revolutionized~bioimaging in living
systems by providing{\break} unparalleled selectivity with cell- or
organelle-specific expression~\cite{5}. Significant efforts have also
been put into engineering FPs to develop biosensors with diverse
photophysical properties and target analytes using evolved recognition
motifs~\cite{6,7}. However, the development and optimization of
FP-based biosensors is complex especially for long wavelength emitting ones,
and numerous biological substances remain challenging or even
impossible to detect using protein-based recognition motifs.
Alternatively, fluorescent chemosensors based on organic fluorophores
benefit from a long research history and offer virtually endless
versatility thanks to the power of modern synthetic organic chemistry
and well-identified sensing mechanisms and recognition groups
(Figure~\ref{fig1}A)~\cite{8,9}. However, the precise control of the
intracellular localization of chemosensors in cells to visualize
biological processes with high spatial selectivity remains an issue and
explains the success of FPs and biosensors. Small organic molecules
usually accumulate in a given organelle or cellular compartment
depending on their charge and lipophilicity (e.g., cationic rhodamines
in mitochondria), but it is hard to predict and modulate. Chemical
targeting strategies have been put in place with functional groups that
can direct chemosensors towards specific cellular locations, but again
these strategies are not completely general and can fail due to
competition with spontaneous localization of the parent
molecule~\cite{10,11}. Recently, hybrid chemical and genetic systems
have emerged as an alternative that combines the best of both
approaches. They are based on the targeting of small chemical compounds
towards genetically encoded proteins called self-labeling protein (SLP)
tags and benefit from both the targeting selectivity of genetically
encoded proteins and from the diversity and versatility of chemical
probes and chemosensors (Figure~\ref{fig1}B). Efforts in the
development of SLP-tags have led to two categories of covalent
(SNAP-tag, HaloTag, CLIP-tag, PYP-tag, BL-tag, and TMP-tag) and
noncovalent (FAP and FAST) binding systems~\cite{12}. Semisynthetic
biosensors can be obtained by conjugating chemosensors to an
SLP-tag~\cite{13}. Two different possibilities exist depending on
whether the chemosensor is fluorogenic (i.e., activated by reaction
with the protein, Figure~\ref{fig1}C) or not (Figure~\ref{fig1}B).
Numerous examples of the latter class have been reported for the
genetically targeted sensing of Zn$^{2+}$~\cite{14,15,16,17},
Ca$^{2+}$~\cite{18,19,20}, K$^{+}$~\cite{21}, pH~\cite{22,23}, nitric
oxide~\cite{24,25}, viscosity~\cite{26}, membrane tension~\cite{27},
and voltage~\cite{28}. In all these examples however, the chemosensor
is sensitive to its analyte or to the biological process whether or not
it is linked to its target protein, which requires extensive washing
steps to remove the excess of unbound molecules and ensure the
selectivity of the signal (Figure~\ref{fig1}B).

To provide an alternative to FP-based biosensors with comparable
selectivity, semisynthetic biosensors should be built on fluorogenic
probes that only become fluorescent when they are bound to the target
protein (Figure~\ref{fig1}C). To achieve such fluorogenic semisynthetic
sensors, two strategies have been used. The first one consists in using
fluorogenic probes targeted at modified SLP-tags that incorporate
a sensing moiety~\cite{4,29,30,31}. In that case, the detection is
performed by the protein moiety, and it does not avoid some drawbacks
associated with FP-based biosensors such as complex protein evolution
to ensure efficient detection and slower detection kinetics for fast
biological processes such as calcium oscillations compared to synthetic
chemosensors~\cite{32}. This approach has been recently reviewed by
Broch {et~al.}~\cite{3}. This minireview focuses on the few reported
examples of the second strategy, which is all-molecular and
consists in incorporating two fluorescence control mechanisms within a
single molecule. The first ensures the pre-activation of the
fluorescence upon binding to the cognate SLP-tag so that it is able to
become fluorescent upon detection of the target analyte
(Figure~\ref{fig1}C). The most commonly encountered SLP-tags are
SNAP-tag and HaloTag, which enable highly selective and rapid protein
labeling (Figure~\ref{fig2}). HaloTag in particular has established
itself as the most promising technology with a chemically simple linker
that facilitates the synthesis of conjugates as well as their cell
penetration and a very fast reaction rate. For a detailed review of
HaloTag applications in bioimaging, the readers are referred to a
recent review by Cook {et~al.}~\cite{33}.

In practice, these locally activatable chemosensors that we also refer
to as dual-input probes due to the dual control of the fluorescence are
based on the incorporation of an analyte-sensing group on a fluorogenic
protein probe sometimes called fluorogen.\ This approach has the
advantage of using well-established
SLP-tags and is based on 
\mbox{small-molecule} engineering.\ The different fluorogenic
chemosensors are classified depending on the type of fluorogens on
which they are built rather than on the target analytes, considering
that the sensing strategies are relatively well known and that the
originality of these research works lies in the efficient combination
of the two fluorescence control mechanisms within the same molecule.
The common theme of the works covered by this minireview is thus the
local activation of fluorescent chemosensors by genetically encoded
proteins.\ It encompasses diverse structures with different target
analytes and includes some articles where the local activation was not
necessarily underlined, especially for early works. We thus aim to show
the common character of all these molecules and identify some
guidelines for the design of future fluorogenic semisynthetic
biosensors. In doing so, we also recall some generalities on the
{design} of fluorescent chemosensors and fluorogenic\break
probes.

\subsection{Analyte detection mechanisms} \label{sec1.3}

To develop dual-input probes for genetically targeted analyte sensing,
the protein fluorogen targeting a specific SLP-tag must be combined
with an analyte-sensing mechanism. As previously stated, the field of
chemosensors benefits from a very large body of work, with numerous
well-identified analyte receptors (e.g., ion chelators, pH- or
ROS-sensitive groups) that can be coupled to a fluorophore to control
its fluorescence with different photophysical
mechanisms~\cite{9,34,35,36,37}. The mechanisms that have been
implemented in the design of locally \mbox{activated} semisynthetic sensors
are the three most commonly encountered:\ photo-induced \mbox{electron}
transfer (PeT), intramolecular charge transfer (ICT),{\break} and
F\"{o}rster resonance energy transfer (FRET) (Figure~\ref{fig3}).
Chemosensors based on PeT or ICT are composed of three distinct parts:
a receptor for analyte detection, a fluorophore, and a linker to couple
them. Depending on the nature of this linker, the chemosensors operate
via PeT or ICT~\cite{38}.

\begin{figure*}
{\vspace*{-4pt}}
\includegraphics{fig03}
{\vspace*{-4pt}}
\caption{\label{fig3}Schematic illustration of the principle of
(A)~photo-induced electron transfer (PeT), (B)~intramolecular charge
transfer (ICT; EWG: electron-withdrawing group, EDG:
electron-donating group separated by a $\uppi$-conjugated linker), and
(C)~FRET (D:~FRET donor, A:~FRET acceptor) detection mechanisms used in
the design of chemosensors.}
{\vspace*{-4pt}}
\end{figure*}

If the linker is not conjugated, the receptor and fluorophore can
essentially be considered electronically independent and the receptor
can transfer an electron to the excited state fluorophore, thus
effectively quenching the fluorophore. Upon analyte binding, the energy
of the electron donor (the analyte receptor) is lowered, thus
inhibiting the PeT process and restoring the fluorescence
(Figure~\ref{fig3}A). Although the opposite is possible, reductive PeT
is the most common mechanism found in the literature~\cite{39}. A
variety of fluorescent chemosensors based on PeT have been developed
for targeting a variety of biological analytes~\cite{40,41}. The
difficulty in the design of PeT sensors lies in the necessity of having
\mbox{properly} positioned orbital energy levels to enable electron
transfer. The probability of PeT between an electron donor and acceptor
is governed by the Rehm--Weller equation~\cite{39}.

If the receptor and fluorophore are electronically conjugated, they
cannot be considered independent and PeT will be replaced by ICT. Upon
analyte binding, the change in the electron density of the receptor
will cause an ICT within the molecule that can affect the absorption
properties and/or the fluorescence emission and result in a
fluorescence turn-on or in a bathochromic or hypsochromic shift
depending on whether the receptor is the electron donor or the electron
acceptor (Figure~\ref{fig3}B). ICT is another classical mechanism for
the development of chemosensors, with a more complex response than the
off/on PeT sensors that can be exploited to develop ratiometric
sensors~\cite{42}. 

FRET-based fluorescent chemosensors rely on two
distinct fluorophores, a donor and an acceptor in close proximity
(10--100~{\AA}) (Figure~\ref{fig3}C) \cite{37}.\ FRET requires a
spectral overlap between the{\break} emission of the donor and the
absorption of the acceptor and is highly sensitive to the distance
between the two. In FP-based FRET sensors, the most usual sensing
mechanism is based on a change in the distance between the donor and
the acceptor~\cite{6}. \mbox{However}, for chemosensors, it is very
common that the analyte binding will induce a change in the
photophysical properties of the donor or the acceptor, usually via one
of the two previous photophysical mechanisms, PeT or ICT
(Figure~\ref{fig3}C)~\cite{37}.

\begin{scheme*}
\includegraphics{sc01}
{\vspace*{.6pc}}
\caption{\label{sch1}Structure of dual-input calcium and magnesium
sensors based on the FlAsH fluorogenic probe.}
{\vspace*{-.5pc}}
\end{scheme*}

\section{Design of locally activated semisynthetic{\hfill\break}
biosensors} \label{sec2}
\subsection{Early examples based on the biarsenical{\hfill\break}
FlAsH/tetracysteine tag system} \label{sec2.1}

In 2007, the group of Roger Tsien reported on the first example of a
locally activated semisynthetic fluorogenic biosensor for Ca$^{2+}$
CaGF (Scheme~\ref{sch1})~\cite{43}. It is based on the fluorescein
derivative FlAsH, which reacts selectively in cells with a
tetracysteine tag \mbox{(TC-tag)}, coupled to a BAPTA-derived calcium
chelator with a low affinity~\cite{44}. 
The chelator controls the fluorescence by PeT, and the fluorescein
becomes fluorescent in the presence of calcium. Although the dual
activation properties were not discussed in detail in this paper, the
reaction of FlAsH with the TC-tag is intrinsically fluorogenic, and so
CaGF can be considered a locally activated semisynthetic biosensor. It
was targeted at the connexin 43 protein in HeLa cells and shown to
report on depolarization-evoked calcium waves at gap junctions. More
recently, Fujii \mbox{et~al.}\ reported another example of a dual-input
sensor for magnesium ions based on FlAsH (Scheme~\ref{sch1})~\cite{45}.
KMG-104-AsH is based on a biarsenical difluorofluorescein, for lower pH
sensitivity, combined with a magnesium chelator selective for Mg$^{2+}$
over Ca$^{2+}$. As for the previous example, this semisynthetic
fluorogenic biosensor only becomes fluorescent when bound to both the
TC-tag and Mg$^{2+}$ with more than 10-fold fluorescence activation due
to fluorogenic binding with the TC-tag and PeT quenching of the
chelating moiety. It was used to visualize intracellular magnesium by
targeting the mitochondria or actin. The TC-tag combined with FlAsH was
a pioneering approach opening the era of hybrid chemogenetic labeling
strategies. It has the advantage of being intrinsically fluorogenic and
based on a small nonperturbing peptide tag, but it suffers from poor
selectivity due to the competition with other endogenous dithiols. The
excess of probes must thus be quenched with 1,2-ethanedithiol to avoid
nonspecific signals. Moreover, the TC-tag cannot be easily adapted to
other fluorophores in order to tune photophysical properties. Due to
these limitations, the TC-tag was outperformed by the development of
SLP-tags, such as SNAP-tag or HaloTag, which enable rapid and selective
labeling of proteins with virtually any organic dye. However, the
reaction of usual fluorophores such as rhodamines and fluoresceins with
SLP-tags is not fluorogenic, and the development of locally activated
semisynthetic biosensors first required the development of adequate
fluorogens targeted at these SLP-tags. Such fluorogens have been
developed based mostly on rhodamines and small viscosity-sensitive
molecular rotors. In the following sections of this review, we describe
these two classes of fluorogenic probes and how they can be combined
with analyte-sensing\break groups.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Structure of fluorogenic rhodamine derivatives.}
\end{figure*}

\subsection{Fluorogenic semisynthetic biosensors based on rhodamines}
\label{sec2.2}
\subsubsection{Fluorogenic rhodamine probes} \label{sec2.2.1}

Rhodamines are a class of xanthene-derived dyes with excellent
photophysical properties, such as high brightness and good
photostability (Figure~\ref{fig4}). \mbox{Rhodamines} can undergo 
polarity-sensitive equilibrium between a closed nonfluorescent and
colorless spirocyclic form and an open fluorescent quinoid red-emitting
form. This equilibrium has not only been exploited to develop
chemosensors but also to fluorogenically target
biomolecules~\cite{46,47,48,49,50,51}.
The Johnsson group pioneered this strategy by developing fluorogenic
silarhodamines (SiRs), which transformed from a closed form in water to
an open fluorescent form in the presence of a biomolecular
target~\cite{52,53}. It is assumed that the SiR fluorogens form
hydrophobic aggregates in their closed form in water and that upon
reaction with a biomolecule, they move
from a hydrophobic environment to a polar aqueous medium in which they
are open and fluorescent~\cite{54}. It is worth noting that this
cyclization is also sensitive to pH. By using electron-poor amides such
as cyanamide and sulfonamide, it has been possible to tune the
spirocyclization reaction and adapt it to rhodamine or carborhodamine,
leading to a palette of bright fluorogenic rhodamine dyes
(Figure~\ref{fig4})~\cite{48,55}. Given the excellent properties of the
rhodamine scaffold, it has become a platform of choice to develop
fluorogenic probes for bioimaging. The Lavis group has for instance
played around with aniline auxochrome groups to improve brightness and
photostability~\cite{46,50}. Since the HaloTag protein was optimized
with the tetramethylrhodamine dye, it has been proved to be excellent
in reacting rapidly and activating the fluorescence of fluorogenic
rhodamines (HaloTag7 version, $k_{2} = 1.9 \times
10^{7}~\text{M}^{-1}{\cdot}\text{s}^{-1}$ for TMR-Halo at
25~{\textdegree}C)~\cite{56,57}.

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Fluorogenic calcium probes targeted at
HaloTag. (A)~First design of rhodamine-based fluorogenic and
chemogenetic Ca$^{2+}$ reported in Ref.~\cite{58}. (B)~Structures of
MaPCa indicators. (C)~Calcium titrations of MaPCa$_{\mathrm{high}}$
and MaPCa$_{\mathrm{low}}$. (D)~Fluorescence microscopy images of
HEK293 cells expressing and nonexpressing HaloTag--NLS incubated with
1~$\upmu$M of MaPCa-558$_{\mathrm{high}}$ (left),
MaPCa-619$_{\mathrm{high}}$ (middle), and MaPCa-656$_{\mathrm{high}}$
(right) for 2~h imaged under wash-free conditions. Turn-on numbers
indicate average of $n = 200$ cells. Scale bar ${=}$ 20~$\upmu$m.
Images~(B), (C), and (D) adapted from Ref.~\cite{60} under the
\href{https://creativecommons.org/licenses/by/4.0/}{CC BY 4.0
license}.}
{\vspace*{-.35pc}}
\end{figure*}

\subsubsection{Combination of fluorogenic rhodamines with 
analyte-sensing modalities} \label{sec2.2.2}

Fluorogenic rhodamines in combination with HaloTag have been used to
develop semisynthetic biosensors. The first attempt was made by the
Lavis group that combined a fluorogenic rhodamine with the calcium
chelator BAPTA, yielding molecule~\textbf{13}
(Figure~\ref{fig5}A)~\cite{58}. BAPTA was discovered by Roger Tsien and
has become the gold standard for building up fluorescent calcium
chemosensors since it has suitable micromolar affinity and good
selectivity for calcium over magnesium~\cite{59}. As discussed above,
it can control the fluorescence of an associated fluorophore via PeT or
ICT depending on the conjugation. In that case, it is a PeT process
that creates an off/on trigger actuated by calcium. The resulting probe
has good calcium sensitivity, but it is poorly fluorogenic upon
reaction with HaloTag with only a 2.4-fold increase in fluorescence,
which does not allow performing wash-free imaging. This first example
testifies to the difficulty in combining efficiently several
fluorescent control mechanisms within the same molecular scaffold.

\begin{figure*}
{\vspace*{-.2pc}}
\includegraphics{fig06}
\caption{\label{fig6}TRaQ-G dual-input glutathione sensor.
(A)~Principle of the TRaQ-G sensing platform. (B)~Design of the
ratiometric GSH sensor using TRaQ-G combined to mGold fluorescent
protein. (C)~Fluorescence intensity spectra of mGold--TRaQ-G ratiometric
probe with varying GSH concentration. (D)~Visualization of nuclear GSH
concentrations during cell proliferation with H2B--TRaQ-G--mGold:
images of a single HeLa nucleus showing a decrease in GSH concentration
from S~phase to mitosis. Image adapted from Ref.~\cite{62} under the
\href{https://creativecommons.org/licenses/by/4.0/}{CC BY 4.0
license}.}
{\vspace*{-.2pc}}
\end{figure*}

A few years later, the Johnsson group developed another series of
locally activated fluorescent semisynthetic calcium indicators called
``MaPCa indicators'' with good fluorogenicity and tunable emission
wavelength and affinity ($K_{D}$ from 0.41 to 457~$\upmu$M)
(Figure~\ref{fig5}B)~\cite{60}. MaPCa-656$_{\mathrm{high}}$
($\lambda_{\mathrm{exc}} = 656$~nm) was identified as the best
candidate with the highest fluorogenicity in the presence of calcium
(6-fold increase) and HaloTag (120-fold increase) and the highest
contrast in wash-free live-cell imaging (Figure~\ref{fig5}B--D). It
exhibits calcium affinity in a suitable range for cytosolic
measurement ($K_{D} = 580$~nM). A cell-permeant
MaPCa-656$_{\mathrm{high}}$ acetoxymethyl ester derivative was
incubated on HEK293 cells expressing nuclear localized HaloTag and,
after 2~h of incubation, fluorescence microscopy images highlighted
efficient HaloTag labeling of the probe under no-wash conditions with
a signal-to-background ratio of 9 (Figure~\ref{fig5}D). Moreover, the
performance of the MaPCa-656$_{\mathrm{high}}$ calcium indicator was
evaluated in rat primary hippocampal neurons and showed efficient and
homogeneous HaloTag labeling without any significant off-target signal.
This work thus provides a very solid basis for the development of
locally activated semisynthetic probes based on fluorogenic rhodamines,
and it is possible to envision replacing BAPTA with a different
receptor to target other analytes. It is worth noting that the calcium
sensitivity of the MaPCa sensors (F($+$Ca$^{2+}$)/F($-$Ca$^{2+}$)
between~6 and~11) is much lower than what can be attained with
comparable rhodamine-based molecular calcium indicators~\cite{61}.
This is another indication of the difficulty in combining the two
fluorescence control mechanisms on a single molecule while maintaining
their full efficiency.

\looseness=-1
The combination of an SiR fluorogen with the HaloTag protein has also
been used by Emmert \mbox{et~al.}\ to develop a glutathione (GSH) sensor
named ``TRaQ-G'' based on the ring-opening process upon HaloTag
binding, resulting in a bright fluorescence emission, and on the
nucleophilic attack of the xanthene $\uppi$-system of the silarhodamine core by
the nucleophilic GSH, which quenches the fluorescence
\mbox{(Figure~\ref{fig6}A)~\cite{62}.}\ The structure containing a cyanamide
nucleophile, TRaQ-G, was found to act as a GSH sensor locally activated
by HaloTag. This example is particularly interesting since the unbound
form of TRaQ-G is unable to react with GSH, in contrast to the previous
calcium sensors where unbound indicators can bind their target but
without a detectable associated signal. However TRaQ-G is based on a
decrease in fluorescence, which is less practical than a turn-on effect
as an absence of signal is always harder to detect without bias. The
combination of HaloTag with mGold, a photostable redox-insensitive
protein, was used to generate a ratiometric GSH sensor
(Figure~\ref{fig6}B). A calibration curve of the ratio of mGold/TRaQ-G
fluorescence intensities to GSH concentrations confirmed the linear
response of this ratiometric fluorogenic semisynthetic GSH sensor in
the biologically relevant range (1--20~mM) (Figure~\ref{fig6}C).
Live-cell imaging experiments using multiple organelle-targeting
sequences confirmed the efficiency and accuracy of the TRaQ-G sensor
for precise detection of GSH in cells (Figure~\ref{fig6}D). Since there
is no need to wash the excess ligand, the newly expressed protein can
undergo continuous labeling in prolonged experiments.

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig07}
\caption{\label{fig7}(A)~Principle of fluorescence activation mechanism
of FMR-based semisynthetic probes upon SLP binding. An EDG is
$\uppi$-conjugated to an EWG. Examples of some FMRs for~(B) SNAP-tag
labeling and~(C) HaloTag labeling.}
{\vspace*{3pt}}
\end{figure*}

These three examples show the early developments of rhodamine-based
fluorogenic and chemogenetic biosensors. Although this approach benefits
from the excellent photophysical properties of the rhodamine scaffold,
the synthesis of the most efficient rhodamine analogues is relatively
complex, especially when it comes to combining several sensing groups
on the same molecule. More chemically accessible alternatives have been
developed based on small viscosity-sensitive molecular rotors.

\subsection{Fluorogenic semisynthetic sensors based on fluorescent
molecular rotors} \label{sec2.3}

\subsubsection{Fluorogenic molecular rotors targeting{\hfill\break}
SLP tags} \label{sec2.3.1}

\begin{figure*}
{\vspace*{-.1pc}}
\includegraphics{fig08}
{\vspace*{-.1pc}}
\caption{\label{fig8}(A)~Structure of the FRET-based locally activated
pH indicator targeting FAP. (B)~Fluorescence emission of the tandem,
showing the shift in emission of the Cy3 moiety due to pH and the FRET
with MG. (C)~Ratiometric pH calibration curve. Adapted with permission
from Ref.~\cite{79}. Copyright 2018 American Chemical Society.}
{\vspace*{-.5pc}}
\end{figure*}

Another class of fluorogens are fluorescent molecular rotors (FMRs)
based on fluorescent structures displaying rotation around an
intramolecular single bond (Figure~\ref{fig7})~\cite{63,64,65}. These
molecules exhibit viscosity-sensitive fluorescence emission related to
the degree of intramolecular motion upon photoexcitation. In
low-viscous media, the fast {intramolecular} rotation of FMRs induces
\mbox{nonradiative} thermal \mbox{relaxation} of the photoexcited state, but when the
viscosity of the environment increases, the immobilization of the
molecule leads to a restoration of the fluorescence emission. This is a
very general mechanism, and FMRs encompass a wide variety of
structures, but the most commonly found are ``push--pull'' structures
composed of electron-withdrawing and electron-donating groups
conjugated through a flexible bond (Figure~\ref{fig7}A)~\cite{64,65}.\ 
These small fluorogens are particularly valuable tools as they are
chemically accessible, combining straightforward synthesis and easily
tunable photophysical properties~\cite{66,67}. FMRs have been widely
used as fluorogens to monitor viscosity in cellular compartments, but
the viscosity-sensitive emission can also be used for the fluorogenic
targeting of biomolecules and in particular SLP-tags.\ Yu and colleagues
have generated a fluorescence-switchable probe using BG-CCVJ
\mbox{fluorogen} targeting SNAP-tag with a fluorescence activation up
to 170-fold (Figure~\ref{fig7}B)~\cite{68}. In order to red-shift the
emission wavelength, Zhang and colleagues developed a red Kaede-like
chromophore activated by SNAP-tag binding ($\lambda_{\mathrm{em}} =
620$~nm and 90-fold enhancement upon SNAP-tag binding)
(Figure~\ref{fig7}B)~\cite{69}.\ The most comprehensive work on SNAP-tag
fluorogens based on dipolar FMRs has led to the development of a
palette of probes with emissions ranging from 485 to 700~nm by varying
the electron-donating and electron-withdrawing groups~\cite{66}. As for
HaloTag, Kool and coworkers developed dimethylaminostyrylpyridinium
fluorogens exhibiting a viscosity-sensitive emission that is activated
upon reaction with HaloTag with a 27-fold fluorescence increase
(Figure~\ref{fig7}C)~\cite{70}. Our group also strongly focuses on the
development of semisynthetic fluorescent \mbox{reporters} based on a palette
of FMRs covering the green to far-red emission range and targeted at
HaloTag (Figure~\ref{fig7}C)~\cite{67,71}.\looseness=1

Noncovalent semisynthetic fluorogenic probes involving FMRs have also
been reported for noncovalent genetically encoded strategies such as
4-hydroxybenzylidene rhodanine derivatives combined to the FAST
protein~\cite{72,73} or 3,5-difluoro-4-hydroxybenzylidene imidazolinone
(DFHBI) derivatives that have been extensively engineered as
fluorogens, enabling fluorescence emission upon binding to RNA
aptamers~\cite{3,74,75}.
Noncovalent fluorogen-activating protein (FAP) labeling has also been
used to selectively bind FMR-based fluorogens such as malachite green
and thiazole orange, and activate their fluorescence for wash-free
bioimaging of subcellular organelles~\cite{76,77}.

{\vspace*{-2pt}}

\subsubsection{FRET-based semisynthetic pH sensors} \label{sec2.3.2}

\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig09}
\vspace*{-4pt}
\caption{\label{fig9}Structure and properties of the Ca-DIP sensor.
(A)~Structure of Ca-DIP. (B)~Absorption and emission properties in the
four possible states of the system with and without calcium and
HaloTag. (C)~Fluorimetric titration. (D)~Dual-input fluorescence
activation. (E)~Genetically targeted calcium imaging in HeLa cells
expressing HaloTag--NLS protein: a selective signal sensitive to calcium
can be recorded in cell nuclei under wash-free conditions. Adapted
with permission from Ref.~\cite{80}. Copyright 2022 Royal Society of
Chemistry.}
\vspace*{-5pt}
\end{figure*}

\begin{figure*}
\vspace*{-.2pc}
\includegraphics{fig10}
\vspace*{-3pt}
\caption{\label{fig10}Semisynthetic fluorogenic sensor to monitor
protein exocytosis. (A)~Structure and dual-input activation of
pHluo-Halo-1. (B)~Absorption and emission spectra at various pH values.
(C)~Labeling selectivity of pHluo-Halo-1 in HeLa cells expressing a
nuclear HaloTag--NLS under wash-free conditions. Turn-on number represents
the median value for 130 cells. (D)~General principle of exocytosis
imaging with pHluo-Halo-1. (E)~TIRF microscopy images of HeLa cell
expressing CD63-HaloTag labeled with pHluo-Halo-1 undergoing fusion:
normalized intensity trace for a selected fusion event corresponding to
the orange arrow on the image. Adapted with permission from
Ref.~\cite{81}. Copyright 2024 American Chemical Society.}
\vspace*{-3pt}
\end{figure*}

Using FAP labeling, Bruchez and coworkers reported semisynthetic
fluorogenic pH probes based on FRET
ratiometric fluorescence activation~\cite{79,78}. These pH probes rely
on a tandem of a pH-sensitive cyanine dye and a molecular rotor 
activated by a FAP that is either the donor~\cite{78} or the acceptor
\mbox{(Figure~\ref{fig8}A)~\cite{79}.}
The variation in pH modulates the ICT in the cyanine structures and
thus the emission properties of the donor cyanine3 (Cy3) dye,
which in turn impacts the energy transfer (Figures~\ref{fig3}C
and~\ref{fig8}A,B). Local activation of the fluorogen moiety by FAP
binding thus creates a ratiometric pH sensor that can be used under
wash-free conditions. The second design, with a fluorogenic acceptor,
ensures better selectivity since the nonemissive acceptor also quenches
the fluorescence of the pH-sensitive donor by energy transfer for
unbound molecules (Figure~\ref{fig8})~\cite{79}. The resulting
cell-impermeant probe was used to monitor membrane GPCR trafficking but
cannot be used to image intracellular events. 
Additionally, the dual probe design uses most of the visible spectrum
and precludes the use of other fluorescent probes for
multiplexing.

\subsubsection{PeT- and ICT-based fluorogenic sensors} \label{sec2.3.3}

More versatile semisynthetic sensors based on a single fluorogen
scaffold have been recently developed.\ Our group reported a dual-input
calcium probe by combining a viscosity-sensitive red-emitting molecular
rotor targeting HaloTag with a BAPTA group that controls the
fluorescence by PeT (Ca-DIP, Figure~\ref{fig9})~\cite{80}. Similarly to
previous MaPCa indicators based on SiR fluorogens, Ca-DIP exhibits a
locally activated red-emitting fluorescence signal upon both HaloTag
binding and Ca$^{2+}$ chelation. More precisely, in the absence of
HaloTag protein, the intramolecular rotation of Ca-DIP quenches the
fluorescence emission, resulting in a very weak signal even in the presence
of calcium. When bound to HaloTag, Ca-DIP becomes fluorescent (25-fold
fluorescence activation) with good calcium sensitivity ($\rmDelta
F/F_{0} = 5.4$). Although the calcium sensitivity is on par with that
of the MaPCa indicators, its brightness is much lower owing to a
partial activation of the fluorescence by HaloTag.

\begin{figure*}
\includegraphics{fig11}
\vspace*{-.25pc}
\caption{\label{fig11}Fluorogenic chemogenetic pH sensor based on the
de novo design of mFAPs. (A)~Principle of DFHBI--mFAP\_pH complex
formation for pH monitoring. (B)~Normalized fluorescence excitation
spectra of the mFAP\_pH--DFHBI complex for a pH titration between
pH~3.6 and pH~8.4 (left) and normalized emission spectra at pH~3.6 and
8.4 (right). Image adapted from Ref.~\cite{83} under the
\href{https://creativecommons.org/licenses/by/4.0/}{CC BY 4.0
license}.}
\vspace*{-.45pc}
\end{figure*}

Following this work on a calcium probe, we have used a similar idea to
design a dual-input pH probe and develop a locally activated
chemogenetic sensor to image protein exocytosis~\cite{81}.\ By using a
\mbox{phenol} electron-donating group, we obtained the pH-sensitive
HaloTag ligand pHluo-Halo-1 (Figure~\ref{fig10}A).
Thanks to an adjacent amide function, the $\text{p}K_{\mathrm{a}}$ of
the phenol was lowered to~6.3, a value adapted to measure the
transition from acidic vesicles (pH~5.5) to the neutral environment
(pH~7.4) during protein exocytosis (Figure~\ref{fig10}D). As for
\mbox{Ca-DIP}, only the HaloTag-bound form is fluorescent with
\mbox{21-fold} activation due to the protein, and pH \mbox{modulates} the ICT
from the phenol with a ratiometric absorption and only one fluorescence
emission originating from the phenolate form (Figure~\ref{fig10}B). In
addition to tuning the $\text{p}K_{\mathrm{a}}$, the amide function was
used to introduce a hydrophilic diethylene glycol chain in order to
limit the nonspecific activation in lipophilic organelles. In HeLa
cells, pHluo-Halo-1 indeed exhibits remarkable labeling selectivity,
with virtually no nonspecific signal under wash-free conditions
(Figure~\ref{fig10}C). It was applied to image the exocytosis of a
CD63-HaloTag protein using TIRF microscopy. Moreover, comparison with
the pH biosensors super-ecliptic pHluorin and pHuji showed that our
hybrid sensor reported correctly on single fusion events
(Figure~\ref{fig10}D)~\cite{81}.

As for Ca-DIP, the fluorescence brightness of pHluo-Halo-1 remains
low.\ Dipolar FMRs are \mbox{inherently} less bright than symmetrical
fluorophores like rhodamines and cyanines,
but they still can attain much better properties and HaloTag does not
fully activate the fluorogen.
HaloTag was indeed optimized for the rhodamine scaffold and does not
necessarily activate efficiently the fluorescence of FMRs. Recent
findings however suggests that protein engineering can improve this
aspect~\cite{82}.

\begin{scheme*}
\includegraphics{sc02}
{\vspace*{.5pc}}
\caption{\label{sch2}Structure of the naphthalimide-based dual-input pH
probe BG-NDM.}
{\vspace*{-.5pc}}
\end{scheme*}

The Baker group used existing molecular rotor DFHBI derivatives to
develop a dual-input fluorogenic semisynthetic platform for
intracellular pH monitoring~\cite{83}.\ This strategy is based on the
binding of pH-sensitive DFHBI to mini fluorogen-activating proteins
(mFAPs; Figure~\ref{fig11}A). The moderate size, photostability, and
fast binding kinetics of mFAPs make them attractive candidates for the
{development} of hybrid chemogenetic biosensors~\cite{84}. The
challenge was about the ability of the mFAP to bind both phenolic and
phenolate states of DFHBI. Hence, to generate an effective
pH-responsive semisynthetic probe using a DFHBI--mFAP labeling
strategy, the authors successfully designed with a de novo
computational strategy an mFAP mutant, mFAP\_pH, capable of binding
these two states with equivalent affinities (Figure~\ref{fig11}A).
Fluorimetric pH titration showed a suitable $\text{p}K_{\mathrm{a}}$
of~5.4, corresponding to the $\text{p}K_{\mathrm{a}}$ of free, unbound
DFHBI in solution~\cite{85}. Upon stabilization of the
\textit{cis}-planar conformation by mFAP\_pH, the deprotonation of the
phenol moiety at neutral pH increases the ICT, which creates a
bathochromic shift of the excitation spectra and activates the
fluorescence, thus creating interesting ratiometric properties for
quantitative pH sensing in the biological range
(\mbox{Figure}~\ref{fig11}B). It is worth \mbox{noting} that in this
\mbox{paper}, the authors also report split reporters for protein/protein
interactions and an mFAP-based calcium sensor, two features that are
slightly out of the scope of this study. Although it reports on a
similar type of chemogenetic sensor with a molecule combining
sensitivity to pH and fluorogenic activation by a genetically encoded
protein, this work presents a key difference compared to the other
articles reviewed herein. It is indeed not based on small-molecule
engineering, since they use a previously reported pH-sensitive
fluorogenic probe, but solely on protein engineering to adapt the
binding \mbox{properties} of mFAP to this molecule. While conventional
protein engineering methods rely on the mutation of existing proteins,
the de novo design used in this example consists in the generation of
entirely new protein structures and functions~\cite{86}.\looseness=-1

\subsection{Another example of locally activated{\hfill\break}
fluorogenic semisynthetic biosensor for pH} \label{sec2.4}

Xu and coworkers developed a semisynthetic pH probe based on the
combination of 1,8-naphthalimide fluorogens (BG-NDM) targeted at
SNAP-tag and incorporating an amine group that quenches the
fluorescence via PeT (Scheme~\ref{sch2})~\cite{87}. In this design, the
SNAP-tag protein activates \mbox{BG-NDM} by preventing fluorophore
aggregation, and protonation of the amine at low pH inhibits the PeT.
It is worth noting that the residual fluorescence of BG-NDM is high,
and consequently, the fluorescence enhancement due to the reaction with
SNAP-tag is moderate.\ This dual-input locally \mbox{activated} pH probe
displays a $\text{p}K_{\mathrm{a}}$ of 8.0, which is slightly high for
the range of biological pH values albeit with a good dynamic response.
So far this is the only example of a dual-input probe with an
activation based on the aggregation ability of naphthalimide. Although
it is not mentioned in this study, PeT between the benzylguanine ligand
of SNAP-tag and a naphthalimide has been reported and may also
contribute to the fluorogenicity of this probe and constitutes a
possible strategy to design dual-input{\break} probes~\cite{88}.

\section{Conclusion} \label{sec3}

There have been so far only few examples of locally activated
semisynthetic biosensors, likely owing not only to the recent nature of
the field but also to the design and synthetic challenges of
efficiently combining two fluorescence-activating mechanisms on a
single molecule. Nonetheless, this approach certainly holds great
potential for developing efficient sensors that benefit not only from
the high versatility of small molecular fluorophores and sensing groups
but also from the selectivity of genetic encoding of the recombinant
protein. Although FRET-based examples have interesting properties such
as ratiometric emissions that enable quantitative measurements, the
most versatile design is the one based on a single fluorophore. The
development of biocompatible fluorogens targeting SLP-tags with low
cytotoxicity, cell permeability, high photostability, and a wide range
of emission wavelengths provides molecular
platforms that can be combined with a variety of sensing groups to
engineer dual-input probes. Due to the small number of examples, only a
few analytes have been targeted (pH, Ca$^{2+}$ and Mg$^{2+}$), but the
dual-input probes described herein should be adjustable to other
targets using the wide repertoire of chemical receptors as has been
done with the FlAsH derivative to shift from a calcium to a magnesium
indicator (Scheme~\ref{sch1}). Measurements of the redox potential of
fluorogens and receptors or density functional theory calculations can
help predict the PeT
process between two moieties to help design dual-input probes targeting
new analytes with good sensitivity. 
Although remarkable results have been achieved, researchers have also
encountered limitations such as limited fluorogenicity and brightness.
Even though it may be convenient and versatile to use well-established
SLP-tags that can bind a variety of small molecular probes, the future
of the field will likely rely on the combination of molecular and
protein engineering to ensure the adequation between the two partners and in
particular the efficient activation of fluorescence. Protein
engineering can be performed by rational design, directed evolution, or
more recent computational de novo design methods. A recent example of
the incorporation of a molecular calcium chelator (BAPTA) in a fusion
between a GFP and HaloTag also underlines other possibilities that
combine chemical and genetic approaches to developing hybrid sensors
with unique properties~\cite{89}.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}

JC is supported by a PhD fellowship funded by the ``interface pour le
vivant'' (IPV) initiative of Sorbonne Universit\'{e}.

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