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Recent advances in small-molecule fluorescent

probes for studying ferroptosis†
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Cite this: DOI: 10.1039/d1cs01167g

Ya-Lin Qi,‡abcd Hai-Rong Wang,‡c Li-Li Chen,c Yong-Tao Duan, *ab

Sheng-Yu Yang*d and Hai-Liang Zhu *abc

Ferroptosis is an iron-dependent, non-apoptotic form of programmed cell death driven by excessive

Received 20th May 2022
DOI: 10.1039/d1cs01167g
rsc.li/chem-soc-rev
lipid peroxidation (LPO). Mounting evidence suggests that the unique modality of cell death is involved
in the development and progression of several diseases including cancer, cardiovascular diseases
(CVDs), neurodegenerative disorders, etc. However, the pathogenesis and signalling pathways of
ferroptosis are not fully understood, possibly due to the lack of robust tools for the highly selective and
sensitive imaging of ferroptosis analytes in complex living systems. Up to now, various small-molecule
fluorescent probes have been applied as promising chemosensors for studying ferroptosis through
tracking the biomolecules or microenvironment-related parameters in vitro and in vivo. In this review,
we comprehensively reviewed the recent development of small-molecule fluorescent probes for
studying ferroptosis, with a focus on the analytes, design strategies and bioimaging applications. We also
provided new insights to overcome the major challenges in this emerging field.

Key learning points

(1) Brief introduction to the history of the discovery of ferroptosis.
(2) The main approaches to study ferroptosis.
(3) Applications of small-molecule fluorescent probes for monitoring ferroptosis.
(4) Summary of the findings from the use of small-molecule fluorescent probes in the study of ferroptosis.
(5) The challenges and opportunities that remain in the field of small-molecule fluorescent probes for tracking ferroptosis.

1. Introduction under the regulation of genetically defined effector molecules

Cell death plays a vital role in human health. It is well known
that cell death can be categorized as accidental cell death (ACD)
and regulated cell death (RCD).1 As a biologically uncontrolled
and unavoidable passive process, ACD is caused by chemical,
physical, or mechanical severe stress.2 By contrast, RCD is
a
Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases,
Children’s Hospital Affiliated to Zhengzhou University, Zhengzhou University,
Zhengzhou 450018, China. E-mail: duanyongtao860409@163.com,
zhuhl@nju.edu.cn
b
Henan Provincial Key Laboratory of Pediatric Hematology, Children’s Hospital
Affiliated to Zhengzhou University, Zhengzhou University, Zhengzhou 450018,
China
c
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing, 210023, China
d
Department of Cellular and Molecular Physiology, Penn State College of Medicine,
Hershey, PA, USA. E-mail: syang2@pennstatehealth.psu.edu
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d1cs01167g
‡ These authors contributed equally to this work.
and tightly structured signaling cascades.3 In the past decades,
several non-apoptotic forms of RCD have been identified, such as
necroptosis, pyroptosis and ferroptosis.4,5 In particular, ferropto-
sis, as a young and fast-growing research field, has attracted great
attention in recent years due to its unique pathological features.
A better understanding of this cell death mechanism could
inspire new insights for clinical diagnosis and treatment.6
Ferroptosis is a non-apoptotic form of RCD characterized by
excessive iron-dependent lipid peroxidation (LPO).7 In 2003,
Brent R. Stockwell’s group found that the oncogenic RAS-
selective lethal small molecule erastin could selectively kill
genetically-engineered human foreskin fibroblasts expressing
an oncogenic mutant HRAS (HRas proto-oncogene, GTPase).8
His group later identified that RAS-selective-lethal-3 (RSL3)
could also induce nonapoptotic and iron-dependent oxidative
cell death in 2008.9 In 2012, they proposed the name ferroptosis
for the erastin-induced iron-dependent cell death, because
this kind of cell death is morphologically, biochemically
and genetically distinct from apoptosis, necrosis and other

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well-characterized kinds of RCD.10 It should be pointed out that the normal development of certain fungal species as well as the
ferroptosis was not only discovered in mammalian systems, but developmental ageing of nematodes.13,14
also in other species including plants, protozoa and fungi.11–13 As described above, ferroptotic cell death shows specific
Of note, recent findings have indicated that ferroptosis modulates characteristics distinct from other types of RCD. Morphologically,

Ya-Lin Qi obtained his PhD from Hai-Rong Wang is a Masters

the School of Life Science, student at the School of Life
Nanjing University under the Science, Nanjing University under
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supervision of Prof. Hai-Liang the supervision of Prof. Hai-Liang

Zhu in 2022. Then, he joined the Zhu. Her research interests are
Department of Cellular & Mole- focused on multifunctional fluor-
cular Physiology, Penn State escent probes for bioimaging and
College of Medicine, where he disease diagnosis.
was a postdoctoral fellow in the
group of Prof. Sheng-Yu Yang. His
research interests are focused on
multifunctional fluorescent probes
Ya-Lin Qi for bioimaging and disease Hai-Rong Wang
diagnosis.

Li-Li Chen is a Masters student Yong-Tao Duan is an associate

at the School of Life Science, research fellow at Children’s
Nanjing University under the Hospital Affiliated to Zhengzhou
supervision of Prof. Hai-Liang University, China. He received his
Zhu. Her current research focu- PhD (2016) from Nanjing
ses on small-molecule fluorescent University. His interests are
probes and functional nano- focused on tumours, disease
probes for bioimaging appli- mechanisms and treatment.
cations.

Li-Li Chen Yong-Tao Duan

Sheng-Yu Yang is an Associate Hai-Liang Zhu is a professor

Professor in the Department of in the School of Life Science,
Cellular & Molecular Physiology, Nanjing University. He received
Penn State College of Medicine. his PhD (1998) from Nanjing
He received his PhD (2011) from University. Then, he moved to
the Graduate School and Institute Sun Yat-sen University and
of Biophysics, Chinese Academy University of Marburg for his
of Sciences and conducted his postdoctoral study. He was selec-
postdoctoral work at Cornell Uni- ted as a scholar of the ‘‘100-
versity Weill Medical College. His person plan’’ of the Chinese
research interests are focused on academy of sciences in 2008,
cancer metastasis, especially the and he is listed as a ‘‘highly
Sheng-Yu Yang mechanisms of ferroptosis. Hai-Liang Zhu cited Chinese researcher’’ in
biology by Elsevier. His research
interests include fluorescent probes, functional materials, and the
design and synthesis of small molecules with structural diversity
for pharmaceutical chemistry and chemical biology.

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this cell death process exhibits typical necrosis-like changes,
including cell swelling, plasma membrane rupture, normal
nuclei, shrinking mitochondria with increased membrane den-
sity as reduction in or vanishing of mitochondrial cristae, and
outer mitochondrial membrane rupture.8,10,15,16 Biochemically,
the most significant features of ferroptosis are the overproduc-
tion of lipid hydroperoxides (LOOH) and ferrous (Fe2+).17 As for
the genes, a few specific protein encoding genes that regulate
ferroptosis, such as genes encoding ribosomal protein L8
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Ferroptosis has been implicated in inflammation, immune
surveillance and tumour suppression.25 Furthermore, increasing
evidence showed that ferroptosis is tightly linked to neurologic
diseases,19 intestinal disease,26 cardiovascular diseases (CVDs),27
diabetes28 and cancer.18 Notably, compelling discoveries have
shown that therapy-resistant cancer cells are vulnerable to ferrop-
tosis, and thus it would be a promising way for overcoming the
‘Achille’s heel’ of drug-resistant cancer through promoting
ferroptosis.25 Additionally, ferroptosis plays underling roles in

(RPL8), ATP synthase F0 complex subunit C3 (ATP5G3) and
tetratricopeptide repeat domain 35 (TTC35), are different from
those that control other forms of RCD.18 In addition, recent
reports have indicated that ferroptosis presents specific immu-
nological features including inducing the death of leukocyte
subsets and the loss of immune function, as well as modulating
how immune system deals with dying cells or the resulting
corpses when it affects non-leukocytic cells.3 However, recent
studies have uncovered that, except for a few differences in
protein-signaling pathways, a calcium (Ca2+)-dependent non-
apoptotic form of oxidative cell death induced by glutamate
toxicity in neuronal cells, oxytosis, shares various similarities
with ferroptosis, such as the roles of lipoxygenases (LOX),
metal dependency (iron, copper (Cu2+) and Ca2+), ultrastruc-
tural features (e.g., mitochondria abnormality) and gene
expression.19–22 Thus, whether these two types of RCD should
be considered as the same or two highly overlapping cell death
pathway still remains to be investigated.21 On the other hand,
there is a crosstalk between ferroptosis and several other kinds of
RCD pathways including apoptosis, necroptosis and autophagy.23
For example, autophagy can contribute to ferroptosis-like cell
death through producing lysosomal reactive oxygen species
(ROS) as well as providing labile iron through NCOA4-induced
ferritinophagy,24 although ferroptosis is initially thought to be
autophagy-independent.10 Therefore, the complex interplay and
crosstalk between ferroptosis and other types of cell death path-
way needs to be further investigated.
systemic therapy, radiotherapy and immunotherapy.29 Therefore,
deep insights into ferroptosis might offer an alternative way for
disease diagnosis and therapy.
There have been many breakthroughs in this emerging field
over the past few years. For instance, Tang et al. recently
demonstrated that NUPR1 participated in the transactivation
of the gene encoding lipocalin 2 (LCN2), which alleviated iron-
induced oxidative damage and caused ferroptosis resistance.30
Gan and co-workers disclosed that dihydroorotate dehydro-
genase (DHODH) and mitochondrial glutathione peroxidase 4
(GPX4) constitute two major defensive systems for lipid per-
oxides detoxification in mitochondria.31 Indeed, with the devel-
opment of new technologies, researchers have gained a deeper
understanding of ferroptosis. Generally, several clinical techni-
ques, such as immunofluorescence (IF), transmission electron
microscopy (TEM), magnetic resonance imaging (MRI) and
Western blotting (WB), have been widely applied in the ferro-
ptosis studies.32–34 However, these classic modalities are
unable to realize non-invasive and real time-imaging of the
variations of biomolecules, pathophysiological microenviron-
ments or biological events to unravel ferroptosis pathogenesis
(Table 1).35,36 Furthermore, most of these traditional clinical
diagnostic techniques are time-consuming, expensive, low sen-
sitivity and poor selectivity, and limited by complicated sample
preparations.37,38 Moreover, some of the biological detection
technologies are incapable of in vivo imaging in animal
models.35 Fluorescence imaging (FI) based on small-molecule

Table 1 Comparison of typical modalities for ferroptosis studies

Methods Functions/targets Advantages Disadvantages

IF Tracking the key proteins in High sensitivity and selectivity Complicated sample preparations; unable
tissues to image in vivo
Liquid chromato- Tracking the biomolecules Quantitative analysis; high sensitivity and Unable to image in vivo
graphy mass selectivity
spectrometry
(LC-MS)
TEM Imaging of morphological Directly imaging of cellular morphological Cost; complicated sample preparations;
changes of mitochondria and the changes poor spatial resolution; unable to image
integrity of cell membrane in vivo
Positron emission Imaging of the organs and High sensitivity and tomographic capability Poor spatial and temporal resolution; cost;
tomography (PET) tumors hazardous ionizing radiation.
MRI Imaging of iron in the tissues High spatial resolution; in situ imaging Cost; poor sensitivity and specificity
WB Tracking the key proteins High sensitivity and selectivity; in situ Complicated sample preparations; unable
imaging; real-time tracking to image in vivo
Quantitative-PCR Tracking the mutation gens High accuracy Complicated sample preparations; unable
(q-PCR) to image in vivo
FI Monitoring the biomolecules or Real-time imaging; noninvasive; excellent Affected by tissue heterogeneities, depth
microenvironmental parameters sensitivity and selectivity; high-resolution; location and the animals’ own state;
easy operation; in vivo imaging relative short optimal imaging duration

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probes has many attractive attributes, such as real-time non-
invasive visualization, easy processability, superior biocompati-
bility and high signal-to-background ratios (SBR).39–44 These
probes has been used to monitor the diverse bioanalytes and
biological processes in vitro and in vivo to tackle mechanisms
underlying various diseases including drug-induced acute liver
injury,45 drug-induced acute kidney injury,46 depression47 and
Alzheimer’s disease (AD).48,49 In particular, the emergence of
organic probes in near-infrared (NIR) window (700–900 nm)
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and the two-photon microscopy (TPM) offer deep penetration
and high spatial resolution, which greatly promote the bioap-
plication of fluorescence-based imaging.42,50 More importantly,
fluorescence imaging locates in second near-infrared (NIR-II,
1000–1700 nm) features micron-scale resolution and outstanding
penetration (approximately 5–20 mm).51 These functionalities
cannot be achieved by other imaging modalities such as PET or
single-photon emission computed tomography (SPECT).52
In addition, FI based on small-molecule fluorescent probes can
be combined with other imaging techniques, such as photoacous-
tic imaging (PAI), MRI or PET, to construct multimodality imaging
systems, which are capable of avoiding false signals and mini-
mizing the interface induced by the animals’ own state.53–56 Other
advances in fluorescence imaging modalities including fluores-
cence lifetime imaging microscopy (FLIM), time-resolved fluores-
cence imaging (TRFI) and super-resolution imaging microscopy
remarkably boost the bioimaging in this field.57–60 Last but not
least, small-molecule fluorescent probes with specific scaffolds
are not only able to be used as imaging agents for image-guided
surgery,61,62 but also to integrate to other functions including
phototherapy,63 chemotherapy,64,65 radiotherapy,66 and anti-
bacterial therapeutics.67,68 Therefore, small-molecule fluorescent
probes have exhibited great potential for medical diagnosis,
preclinical research and clinical practice.
Here, we would provide an overview of the small-molecule
fluorescent probes for the study of ferroptosis, including the
design strategies, targeted analytes and the bioapplications of
these probes. We also briefly introduced the history of the
discovery, the basic concepts, the physiological and patho-
logical characteristics of ferroptosis. Furthermore, we discussed
the unresolved challenges and current issues in this rapidly
developing field. Finally, we discussed future directions for the
small-molecule fluorescent probes for studying ferroptosis, in
hope that our review would help to facilitate additional in-depth
investigation in this field in the near future.
2. Overview of small-molecule
fluorescent probes for studying
ferroptosis
Ferroptosis is activated by inactivation of cellular glutathione
(GSH)-dependent antioxidant system, which leads to the over-
production of iron-induced toxic lipid ROS.69 The key regula-
tors of ferroptosis mainly include iron metabolic pathway,
amino acid metabolism and lipid metabolism.17 Additionally,
mitochondrial activity, sugars and some related signalling
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pathways also affect ferroptosis sensitivity.25 The cystine
(Cys2)/glutamate antiporter system XC-imports cystine into cells
while exporting glutamate out of cells.70 Cys2 can be reduced to
cysteine (Cys) which is used for the biosynthesis of GSH.71
GPX4 can use GSH as reducing agent to reduce lipid peroxides
(LOOH) to lipid hydroxy derivative (L-OH), which prevents
ferroptosis by inhibiting the accumulation of LOOH.72 Iron
has been shown to regulate ferroptosis pathway through at least
by three mechanisms: (1) iron is a key component of various
metabolic enzymes and energy-generating protein complexes,
which generates cellular ROS through oxidation-based meta-
bolic processes, (2) iron is regarded as a cofactor of LOX and
cytochrome P450 oxidoreductase (POR) which are crucial in the
generation of phospholipid peroxides, and (3) Iron promotes
LPO by catalyzing Fenton reaction or Haber–Weiss reaction to
produce lipid ROS.73,74 Moreover, LPO is initiated by which
oxidants (e.g., free radicals or non-free radical substances)
attack the carbon–carbon double bonds of lipids (especially
in polyunsaturated fatty acids (PUFAs)) to form oxidized
lipid membranes via non-enzymatic Fenton reaction or enzy-
matic reaction pathways.75 Eventually, this process results in
membrane destruction and cell death.76 Importantly, ferropto-
sis is morphologically different from other RCD, and possibly
the former is accompanied by abnormal microenvironment.
Thus, these intracellular metabolites or parameters as well as
their related biomolecules involving in oxidative homeostasis
and energy metabolism can be recognized as important bio-
markers in ferroptosis.
Most of the emerging small-molecule fluorescent probes for
ferroptosis focus on the detection of ROS, reactive sulfur
species (RSS), iron ions and pathophysiological microenviron-
ments (Scheme 1). Notably, many fluorescent probes which
refers to multifunctional fluorescent probes (MFP) have been
applied for monitoring ferroptosis. These versatile probes,
Scheme 1 Small-molecule fluorescent probe for studying ferroptosis.
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Table 2 Classic inducers and inhibitors of ferroptosis
Classification Target Regent
Inducers System Erastin, sulfasalazine, sorafenib
XC-
GPX4 RSL3, FIN56, DPI7, withaferin A
GSH Buthioninesulphoximine (BSO), cisplatin
ROS and FINO2, artesunate (ART)
iron
Inhibitors Iron Deferoxamine (DFO), 2,2 0 -bipyridyl (Bpy)
LPO Ferrostatin-1 (Fer-1), liproxstain-1
(Lip-1), vitamin E
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which can detect multiple biomolecules at one time, have
higher detection efficiency than the traditional or single func-
tion sensors,50,77 and have great potential in exploring the
crosstalk and interplay of the biomolecules during ferroptosis.
Several inducers and inhibitors with different target of
ferroptosis have been proposed (Table 2). Of note, some of
these reagents, such as cisplatin, sorafenib and lapatinib, were
approved by FDA.4 Additionally, statins show good potential for
clinical trials through targeting the ferroptotic pathway.29
On the other hand, ionizing radiation and cytokines are also
able to cause ferroptosis.4,78 In the current research of small-
molecule fluorescent probes for studying ferroptosis, erastin is
commonly used as the classic activator by inhibiting the activity
of system XC-,71 and ferrostatin-1 (Fer-1) could serve as the
inhibitor through trapping lipid radicals.27
Although great achievements have been achieved in this
field over the past few years, three major unresolved issues
need to be tackled: (1) previous reports indicated that cells with
mitochondrial DNA depletion or without mitochondria would
still undergo ferroptosis, which suggest that mitochondria
and mitochondria-mediated ROS production may not be neces-
sary for ferroptosis,10,79 (2) iron has been shown to play
complex roles in ferroptosis, but recent evidences have dis-
closed that Cu2+ has been implicated in redox metabolism, iron
deposition, glutamate-induced oxidation and erastin-mediated
ferroptosis, thus posing a question regarding the exact role of
iron in ferroptosis,5,80 and (3) why and how LPO results in cell
death has not been fully elucidated.81 Thus, the regulation
mechanisms of the ferroptotic death pathway need to be
further explored.
3. Small-molecule fluorescent probes
for monitoring ROS under ferroptosis
Generally, ROS consists of hydrogen peroxide (H2O2), hydroxyl
radical (OH), superoxide anion radical (O2 ), and hypochloric
acid/hypochlorite (HClO/ClO).82 ROS are mainly generated
in the process of mitochondrial oxidative phosphorylation.3
Peroxisomes are another generator of cytosolic H2O2.83
Early studies have shown that the mitochondria-mediated
ROS production was not required for ferroptosis. However,
more recent data suggested mitochondrial ROS could still
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promote ferroptosis.3 Thus, there is an urgent need to provide
valid evidences to address these issues.
3.1. Small-molecule fluorescent probes for monitoring HClO
under ferroptosis
HClO (pKa 7.6) is endogenously generated from the peroxida-
tion reaction between H2O2 and chloride ions catalyzed by
myeloperoxidase (MPO).84 HClO at normal concentrations is
crucial in the immunity against pathogens owing to its robust
oxidizing and chlorinating ability.85,86 However, aberrant pro-
duction of HClO causes oxidative stress to damage nucleic
acids, proteins, and lipids, which contributes to various dis-
eases ranging from atherosclerosis to neurodegeneration.87
Thus, HClO shows double-edged sword effects in the metabolic
process, while the specific roles of HClO in ferroptosis are not
fully understood.
Two small-molecule fluorescent probes (1 and 2) have been
proposed for exploring the links between HClO and ferroptosis
by our and Huo’s group, respectively (Fig. 1).88,89 Previously
published fluorescent sensors for HClO imaging were mainly
based on oxidation reactions.87,90 By adopting the chlorination
reaction mechanism, our team designed and synthesized a
novel two-photon (TP) fluorescent probe 1. We chose quinoline
skeleton as the fluorescent platform not only because of its
excellent blood-brain barrier (BBB) cross ability, but also
because of its eminent TP excitation properties. This fluores-
cent chemosensor presented outstanding characteristics such
as fast response (o5 s), low limit of detection (LOD = 104 nM),
and high selectivity. Furthermore, the maximum two-photon
absorption cross-section (d2) of the free probe was determined
to be 1.6 GM, whereas the value increased to 13.4 GM in the
presence of HClO, which confirmed that it was suitable for TP
imaging. Our work demonstrated the overproduction of HClO
in kainic acid (KA)-induced epilepsy in vitro and in vivo. More-
over, the apigenin screened by high-throughput screening
(HTS) system, which constructed by facilitating high-content
assays (HCA) with probe 1, showed excellent neuroprotective
functions through regulating ferroptosis. Notably, our studies
indicated that apigenin administration caused the overexpres-
sion of SIRT1, GPX4, TrxR and GSH, as well as the down-
Fig. 1 The structures of probes 1 and 2, and their responsive mechanisms
for HClO.
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Fig. 2 (A) In vivo fluorescence imaging with probe 1. (B) and (C) The
examination of MPO, SIRT1, Ac-p53, p53, and GPX4 expressed levels of
brain tissues by IF and WB. Reprinted with permission from ref. 88.
Copyright (2020) Published by National Academy of Sciences.

regulated MPO in mouse brains (Fig. 2). Furthermore, the

participation of GPx4 and p53 in this KA-induced ferroptosis
process was verified. However, the precise role played by Fig. 3 The structures of probes 3–5, and their responsive mechanisms for
apigenin as the inhibitor of ferroptosis needed to be further H2O2.

investigated. It was worth mentioning that abnormal levels of

HClO inhibited the activities of SIRT1 increased the LPO, and
group, respectively (Fig. 3).96–98 The boronic ester was selected
promoted ferroptosis, which could be a pathological mecha-
as the recognition moiety in all of these probes. In probe 3,
nism for epileptogenesis.
dicyanoisophorone (DCI) served as the chromophore. For the
The mitochondrion-targetable probe 2 was designed by incor-
TP probe 4, naphthalimide part was chose as the fluorophore.
poration of phenothiazine into the cationic benzopyrylium moiety.
And biotin, taken as the tumor targeting moiety, was connected to
Notably, Huo’s work revealed that the methoxy group on the
the platform bearing the boronic ester to construct an ‘‘A–p–A’’
phenothiazine part endowed the probe with a low oxidation
system. In addition, the boronic ester unit quenched the fluores-
potential, which thus dramatically promoted its high sensitivity
cence of the free probe. However, in the presence of H2O2, probe 4
to HClO. By taking advantage of photo-induced electron transfer
show strong green emission with a band at 550 nm upon the
(PET) theory, the ‘‘turn-on’’ fluorescent probe 2 initially emitted
excitation at 450 nm, owing to the cleavage of the boronic acid
weak fluorescence. However, upon addition of HClO, 55.7-fold
ester moiety to the formation of the typical ‘‘D–p–A’’ structure,
fluorescence enhancement at 627 nm was obtained within 5 s due
which promoted the intramolecular charge transfer (ICT) process.
to the inhibition of the PET process, which resulted from the
As for probe 5, they chose 2-(2 0 -hydroxyphenyl) benzothiazole
formation of the sulfoxide. Their work indicated that erastin could
(HBT) as the platform, which offered the excited-state intra-
induce the overproduction of HClO in HeLa and RAW 264.7 cells,
molecular proton transfer (ESIPT)-based probe with a large Stokes
which suggested that the overproduction of mitochondrial HClO
shift (162 nm). It was noteworthy that probe 4 had great ability for
during ferroptosis. Furthermore, this probe was able to detect the
making a distinction between normal cells and cancer cells.
exogenous and endogenous HClO in zebrafish.
Furthermore, probe 4 was successfully applied for detecting
3.2. Small-molecule fluorescent probes for monitoring H2O2 exogenous H2O2 in tumor tissue slices under 710 nm excitation,
under ferroptosis with the deep penetration up to a thickness 90 mm. All of their
work suggested that the upregulation of H2O2 in ferroptosis
H2O2 is a second messenger in cells essential in regulating the
process induced by erastin, while the inhibitors (DFO or Fer-1)
activities of phosphatases,91 kinases,92 ion channels,93 etc.
of ferroptosis would hijack the process. Unfortunately, as the
Furthermore, it is involved in the regulation of several physiolo-
three probes lacked suborganelle localization function, the loca-
gical and pathological processes in vivo, including the activation
lization of the alterations of H2O2 under ferroptosis was not
of immune cells and remodeling blood vessels.94 Excessive accu-
accurately examined.
mulation of H2O2 induces oxidative stress, which results in several
diseases, such as cancer, diabetes, and neurodegeneration.95
Notably, H2O2 mainly participates in ferroptosis through Fenton 3.3. Small-molecule fluorescent probes for monitoring OH
reaction. under ferroptosis
Three fluorescent probes (3–5) for tracing H2O2 during the LPO is an important chain reaction during ferroptosis. OH is
ferroptosis process were reported by Li’s, Kong’s and Zhou’s generated from Fenton reaction using iron as the catalyst and is

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one of the most reactive ROS.99,100 OH can be transformed into
other form of ROS.101 Importantly, OH can initiate the non-
enzymatic LPO by directly reacting with PUFA in the lipid
bilayer.25 OH is able to abstract a hydrogen from PUFAs to
produce carbon-centered lipid radical (L ), which considered
the first step of the chain reaction.76 Therefore, OH is tightly
correlated with ferroptosis.
Two versatile fluorescent probes 6 and 7, for simultaneously
detecting OH/viscosity and sequentially visualizing OH/Cys
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under ferroptosis, respectively, were developed by Li’s group
(Fig. 4).102,103 These probes employed OR logic gate and sequen-
tial logic gate separately, which outputted different signals in
response to different target of interests. In probe 6, two indolium
moieties were connected to the anisole unit via the vinyl bond,
which also served as the rotor, to yield the probe with D–p–A
platform. With the viscosity increased from 0.59 to 945 cP, the
green emission band with a peak at 520 nm was gradually
developed when excited at 400 nm. The probe itself show weak
red signals at 652 nm upon excitation at 590 nm. However,
B450 fold fluorescence enhancement was examined in the
presence of OH due to the OH triggered the hydroxylation led
to the formation of phenolic hydroxyl, followed by the transfor-
mation into the phenol intermediate, then underwent deproto-
nation and electron rearrangement, and finally a larger
polymethine p-conjugation skeleton produced. Application of
the probe 6 to cell imaging indicated that the overproduction of
OH was found in both apoptosis and ferroptosis, and DFO
could effectively inhibited the generation of OH in the latter
Fig. 4 The structures of probes 6 and 7, and their responsive mechanisms
for OH/viscosity and OH/Cys, respectively.
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Fig. 5 (A) Fluorescence images of HT-1080 cells under different condi-
tions. (B) Relative fluorescence intensity in (A). Reprinted with permission
from ref. 103. Copyright (2019) American Chemical Society.
process, but not in the former process. Furthermore, the
percentage of OH in the overall ROS from ferroptosis was
significantly higher compared to that of apoptosis. Addition-
ally, their work verified that ferroptosis was with the character-
istics of the elevated levels of OH, viscosity and lipid droplets
(LDs) (Fig. 5). In particular, their work also indicated that both
Fer-1 and Lip-1 could block the production of OH and the
increase of viscosity in ferroptosis, but their effects seemed weaker
than that of DFO.
The versatile fluorescent probe 7 could be used for sequen-
tially tracking OH and Cys with red and green channels
separately. This probe was fabricated through connecting cou-
marin to hydrocyanine. The latter was exploited as the recogni-
tion site for OH, and in particular, it would get hydrogen
abstracted by OH to induce the formation of coumarin-
cyanine with a larger p-conjugation. Subsequently, Cys would
attack the chloride atom and the double bond between the
coumarin and the cyanine through Michael addition, which
destroyed the newly generated p-system and lighted up the
skeleton with green signals. Correspondingly, the free probe
showed strong fluorescence emission with a band at 525 nm
when excited at 400 nm, whereas treatment of probe 7 with OH
resulted in about 15-fold fluorescence increase at 650 nm
under excitation at 580 nm. Subsequently, addition of Cys to
the above solution led to 40-fold fluorescence enhancement at
453 nm when excited at 400 nm. This probe was utilized for the
sequentially and independently monitoring OH and Cys in
living cells. Moreover, their work demonstrated that the
reduction of Cys and overproduction of OH in HT-1080 cells
treated with erastin, and the elevation of OH as well as no
significant changes of Cys in HT-1080 cells incubated with
RSL3. Thus, this probe was able to explore the ferroptosis
in two initiating pathways through sequentially sensing OH
and Cys.
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4. Small-molecule fluorescent probes

for monitoring RSS under ferroptosis
RSS, such as hydrogen polysulfide (H2Sn, n 4 1), hydrogen
sulfide (H2S), Cys and GSH, play an important roles as anti-
oxidants and signaling agents in various physiological
responses to maintain cellular health.104,105 It is now believed
that GSH and Cys are directly associated with ferroptosis, while
the links between ferroptosis and other RSS are still poorly
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understood.

4.1. Small-molecule fluorescent probes for monitoring H2Sn

under ferroptosis
The endogenous H2Sn is produced from persulfides catalyzed
by cystathionine b-synthase (CBS) and cystathionine g-lyase
(CSE),106,107 as well as from the oxidation reaction between
endogenous H2S and ROS.108,109 As a highly reducing and
nucleophilic substance, H2Sn is responsible for scavenging
oxidants and intracellular electrophiles.110–113 Besides, H2Sn
has been implicated in the activation of ion channels, tumor
suppressors, and transcription factors.114 Thus, H2Sn is a
potential factor driving the intracellular redox homeostasis
and could be used to track ferroptosis.
Recently, our group developed a TP fluorescent sensor 8 for
detecting H2Sn during ferroptosis (Fig. 6).115 In this probe, the
DCI unit linked to naphthalene part served as the platform and
2-fluoro-5-nitroben-zoate was used as the H2Sn recognition
moiety. The d2 value of probe 8 was calculated to be 134 GM
under TP excitation at 880 nm. The probe itself was almost
quenched due to the reactive part inhibited the ICT process,
whereas strong NIR signal was observed when the solution of
Fig. 6 (A) Schematic illustration of probe 8 (PSP) toward H2Sn. (B) Fluores-
probe 8 was in the presence of H2Sn. This probe was capable of
cence images of HeLa MCTSs by one-photon and two-photon excitation.
tracking H2Sn in 3D HeLa multicellular tumor spheroids (C) Reconstructed 3D fluorescent microscopic images in (B). Reprinted with
(MCTSs) at a depth of 120 nm under TP excitation (Fig. 6). permission from ref. 115. Copyright (2021) Elsevier B.V.
Notably, our work indicated that the levels of H2Sn remarkably
decreased in the HeLa cells under ferroptosis induced by
erastin. By contrast, both DFO and Fer-1 could mitigate the
process as expected. Thus, these results indicated that H2Sn
participated in the ferroptotic cell death and may be considered
as a key ferroptosis marker.

4.2. Small-molecule fluorescent probes for monitoring H2S

under ferroptosis
H2S is synthesized endogenously from Cys catalyzed by CBS
and CSE.116 As the third gaseous signaling molecule and
endogenous neuroprotective substance, H2S play significant
roles in anti-oxidation, anti-inflammation, and central nervous
regulation.117,118 Of note, H2S can be recognized as a key
reducing agent for monitoring ferroptosis process.
James et al. reported a robust NIR fluorescent probe 9 for
H2S detection in ferroptosis in a stroke model in vivo (Fig. 7).119
In probe 9, the benzyl thiocarbamate unit bearing an azide
moiety and the quinolinemalonitrile part linked to the thio-
phene through d-bonds were expected to act as the H2S reporter Fig. 7 (A) The structure of probe 9, and the responsive mechanism for
and fluorophore, respectively. Importantly, the d-bonds enabled H2S. (B) Visual imaging MCAO in living mice model. Reprinted with
the probe 9 to track viscosity at the same time. Furthermore, the permission from ref. 119. Copyright (2022) The Royal Society of Chemistry.

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thiocarbamate part also served as the H2S precursor. H2S reduced
the azido benzene unit to aniline group, followed by 1,6-
rearrangement-elimination reaction, and concomitant ejected
the platform and carbonyl sulfide (COS) which could be further
catalyzed by carbonic anhydrase (CA) to generate H2S. They used
this H2S triggered and H2S releasing fluorescent probe to monitor
H2S and viscosity in PC12 cells under ferroptosis induced
by erastin or RSL3, respectively. The results implied the down-
regulation of H2S in this process. Meanwhile, acetazolamide
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(AZ, the inhibitor of CA) could preclude the production of H2S.
The examination of Fe2+, MDA, and GSH levels verified the probe
itself exhibited no effect on ferroptosis due to the fact that this
novel probe consumed H2S and later generated H2S. Thus, the
results suggested that their design strategy realized high-fidelity
in situ imaging of H2S during ferroptosis. Interestingly, Fer-1
could effectively block the ferroptosis induced by erastin, while
Lip-1 exhibited significantly inhibition on the ferroptosis induced
by RSL3. Moreover, probe 9 was applied for exploring the relation-
ship between ferroptosis and ischemia-reperfusion by oxygen
glucose deprivation/re-oxygenation (OGD/R) model. Their work
suggested that ischemia-reperfusion resulted in the inhibition of
GPX4, which promoted the p62-Keap1-nuclear factor erythroid2-
related factor2 (Nrf2) signaling pathway and culminated in the
induction of ferroptosis. Finally, they used the probe to uncover
the relationship between cerebral apoplexy and ferroptosis by a
middle cerebral artery occlusion (MCAO) model. Interestingly,
they demonstrated that the pathological mechanisms of cerebral
apoplexy were similar to ischemia-reperfusion as stated above.
4.3. Small-molecule fluorescent probes for monitoring Cys
under ferroptosis
Recent studies indicated that Cys toxicity is a main cause of
age-related mitochondrial deterioration.120 Additionally, Cys
plays significant roles for protein and peptide construction,
enzyme active sites, and cofactors.121–124 Furthermore, Cys is
also important for maintaining biological nitrogen balance and
redox homeostasis.125,126 More importantly, Cys is the pre-
cursor of GSH which is rate limiting for GSH biosynthesis as
mentioned above. Thereby, it is an important metabolite for
monitoring ferroptosis.
Our group reported two fluorescent probes (10 and 11) to
study the variations of Cys under ferroptosis in vitro and in vivo
(Fig. 8).127 Like probes 3 and 8, DCI was selected as the part
of the backbone in this work. The DCI group was conjugated
to 4-aminobenzaldehyde or 4-hydroxybenzaldehyde to yield
probes 10 and 11, respectively. Probe 10 exhibited no signifi-
cantly fluorescence changes in response to Cys, whereas probe
11 generated remarkable fluorescence response with a band at
568 nm after incubated with Cys. The latter was successfully
applied for monitoring the exogenous and endogenous Cys in
living HeLa cells. HeLa cells incubated with erastin or sorafenib
produced weaker green signals in contrast with the control
groups, which directly suggested that the downregulation of
Cys in the ferroptosis process. Meanwhile, our work also
uncovered that external glutamate led to the same results.
Treatment of different cancer cell lines with various inhibitors
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Fig. 8 (A) The structures of probes 10 and 11, and their responsive
mechanisms for Cys. (B) Fluorescent images of living larval zebrafishes.
Reprinted with permission from ref. 127. Copyright (2020) Elsevier B.V.
of system XC- or blockers of cysteine biosynthesis could
improve the anticancer efficacy. Finally, probe 11 was used to
monitor the dynamic changes of Cys in hepatocellular carci-
noma (HCC) zebrafish and AB-wild-type zebrafish under fer-
roptosis, which implicated that this probe was able to visualize
system XC- regulating endogenous cysteine/cystine pool during
ferroptosis process in vivo.
4.4. Small-molecule fluorescent probes for monitoring GSH
under ferroptosis
As we mentioned above, the importance of GSH, which is the
most abundant thiol in cells,121 in ferroptosis is self-evident.
On the basis of Michael addition reaction, a reversible
ratiometric fluorescent probe 12 for monitoring GSH during
ferroptosis was proposed by Wang’s group in 2017 (Fig. 9).128
They selected coumarin derivative as the chromophore, and the
azetidine unit improved the quantum yield and photostability.
Additionally, the cyano group endowed the probe with rapid
reaction kinetics. Furthermore, the carboxylic acid parts were
expected to promote the water solubility of the probe and avoid
the unexpected binding. In particular, the acetoxymethyl (AM)
esters in the precursor of probe 12 was able to enhance its cell
permeability. Probe 12 alone displayed strong green fluores-
cence emission at 565 nm when excited at 488 nm. Upon
continuous additions of GSH, the fluorescence emission with
a band at 565 nm gradually faded when excited at 488 nm, and
concomitantly, the short emission with a peak at 488 nm
significantly developed under the excitation at 405 nm. Using
the AM form of probe 12, they realized the GSH detection in
living cells in reversible manner. Moreover, they confirmed that
NMDA-receptors (NMDAR) mediated the GSH levels in human
neurons. Importantly, with flow cytometry analysis, their work
further verified that the GSH levels decreased in the process of
ferroptosis. Besides, cell imaging results indicated that there
was no significant changes of GSH levels after HT1080 cells
were treated with erastin within 3 minutes, although remark-
able cellular morphology changes were observed.
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Fig. 9 (A) The structure of probe 12, and the responsive mechanisms for
GSH. (B) Representative images of HT1080 cells treated with 10 mM erastin.
Reprinted with permission from ref. 128. Copyright (2017) by the authors
(X. Jiang, J. Chen, A. Bajić, C. Zhang, X. Song, S. L. Carroll, Z.-L. Cai,
M. Tang, M. Xue, N. Cheng, C. P. Schaaf, F. Li, K. R. MacKenzie, A. C. M.
Ferreon, F. Xia, M. C. Wang, M. Maletić-Savatić and J. Wang). Published by
Springer Nature.
4.5. Small-molecule fluorescent probes for monitoring thiol
under ferroptosis
Based on thiol-chromene ‘‘click’’ nucleophilic chemistry, Yin
and co-workers tactfully designed and constructed a smart
fluorescent probe 13 for detecting thiol starvation in mitochon-
dria under ferroptosis (Fig. 10).129 In this probe, chromene was
selected as the reaction site for thiols, and carbonylpyridinium
cation not only enabled the probe to localize in mitochondria,
but also promote the recognition reaction between the probe
Fig. 10 (A) The structure of probe 13, and the responsive mechanisms for
thiols. (B) Screening of stemona alkaloids and their derivatives for control-
ling the thiol level. (C) The structure of SA-11. Reprinted with permission
from ref. 129. Copyright (2022) The Royal Society of Chemistry.
Chem. Soc. Rev.
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and thiols. Moreover, the o-quinone methide generated after
the reaction could label the proteins in the mitochondria,
which further facilitated the probe with high mitochondrial
targeting ability. The free probe initially emitted low fluores-
cence emission. However, remarkably enhanced fluorescence
signals were obtained in the presence of Cys, Hcy or GSH within
200 s, owing to the fact that these biological thiols attacked the
probe to induce a ring-opening reaction and suppressed the
intramolecular rotations, and thus lighted up the coumarin
derivative skeleton. Probe 13 was utilized for tracking the thiols
in living HeLa cells upon erastin or RSL3-induced ferroptosis.
The results indicated that ferroptosis caused the down-
regulation of the thiols in mitochondria. Notably, they screened
a range of stemona alkaloid derivatives and found that one of
these compounds (SA-11) could induce ferroptosis in cancer
cells and even in drug-resistant ones.
5. Small-molecule fluorescent probes
for monitoring Iron ions under
ferroptosis
Iron, which was recognized as the most abundant transition
metal in organisms, mainly existing in two forms in biological
systems: non-heme Fe3+ (ferric) and heme Fe2+.23 The cycle
between multiple oxidation states allows its involvement in
various biological events ranging from oxygen binding and
delivery to enzymatic reactions.130,131 The term ferroptosis is
derived from the Greek roots ‘‘ptosis’’, which means falling,
and the Latin word ‘‘ferrum’’, which means ferrous ion (Fe2+),
implying an essential role of cellular iron in this cell death
pathway.78 Thus, iron can be utilized as a hallmark for visualizing
ferroptosis. Besides, overload iron drives cell to go through
ferroptosis, whereas iron depletion leads to the reduction of
haemoglobin in the blood and causes iron-deficiency anemia.132
The study of iron ions will remain the focus of physiology and
pathology in the next decades.
5.1. Small-molecule fluorescent probes for monitoring
Fe2+under ferroptosis
A majority of iron exists in tightly protein-combined form,
while minor portion of free iron distributes in labile iron
pool (LIP),133 which refers to Fe2+ weakly bound to cellular
ligands.134,135 Taking into consideration its vital role in ferrop-
tosis, it is feasible to monitor ferroptosis by detecting the Fe2+
variations in living system.
In 2016, Chang’s group developed a ratiometric trioxolane
chemistry-based fluorescent probe 14 for studying Fe2+ under
ferroptosis (Fig. 11).136 The 5-aminomethyl fluorescein (5-AMF)
unit and cyanine 3 (Cy3) part was connected via endoperoxide
segment, which also served as the responsive site for Fe2+, to
form the fluorescence resonance energy transfer (FRET) plat-
form. The free probe exhibited dual absorption (labs1 = 495 nm,
labs2 = 545 nm) and emission (lem1 = 515 nm, lem2 = 556 nm),
which was consistent with the optical characteristics of the two
dyes, respectively. The addition of Fe2+ to the solution of probe
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Fig. 11 (A) The structures of probe 14, and the responsive mechanism for
Fe2+. (B) Detection of changes in LIP upon induction of ferroptosis. (C) The
structure of 35MEW28. Reprinted with permission from ref. 136. Copyright
(2016) American Chemical Society.
14 induced a distinct green emission increase at 515 nm, which
resulted from Fe2+ destroyed the endoperoxide linker and
released the two fragments, in the meanwhile, the FRET
process collapsed. Importantly, these features enabled the
probe to sense intracellular Fe2+ with ratiometric fluorescence
imaging. They facilitated this probe to detect Fe2+ in different
cell lines including HEK 293T cells, MCF10A, MDA-MB-231,
and U2OS cells, which suggested that probe 14 was capable of
tracking the variations of Fe2+ levels in living cells. Moreover,
their work indicated that the original level of Fe2+ in cancer cell
was higher than that of the normal cells. Furthermore, their
work disclosed the overproduction of Fe2+ in MDA-MB-231 cells
induced by 35MEW28 through ferroptosis pathway, and impor-
tantly, they found that DFO rather than Fer-1 exerted remark-
able effect on the inhibition of the mobilization of Fe2+.
Unlike probe 14, Hirayama et al. applied N-oxide chemistry
to design and synthesize a class of fluorescent probes (15–17)
for exploring the fluctuations of Fe2+in different organelles
during ferroptosis (Fig. 12).137 Rhodol and tetramethyl hydro-
xymethylrhodamine were chosen as the scaffolds of probes
15 and 16 separately, while probe 17 was based on silicon-
incorporated rhodamine. In particular, the triphenylphosphine
(TPP) unit endowed the probe 15 with mitochondrial localiza-
tion ability. Probes 16 and 17 selectively localized in lysosomes
and endoplasmic reticulum (ER), respectively, without specific
targeting moieties. The N-oxide unit in all of these probes
served as the reporter group for Fe2+. These three free probes
displayed weak fluorescence emissions. However, strong green,
yellow and NIR signals were clearly observed separately after
incubated with Fe2+ within 2000 s. Interestingly, they disclosed
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Fig. 12 The structures of probes 14–17, and their responsive mechanisms
for Fe2+.
the elevated level of Fe2+ in the lysosomes and ER in the
HT-1080 cells under ferroptosis for the first time. Nonetheless,
their work suggested that the Fe2+ level in the mitochondria
was unchanged under the same circumstance. The authors
suggested that the alterations of endogenous homeostasis of
labile iron instead of iron influx was responsible for the
intracellular Fe2+ elevation. Moreover, no obvious Fe2+ fluctua-
tions in the ferroptosis-insusceptible MCF-7 cells under ferrop-
tosis was examined. Taken together, their work provided two
robust sensors to demonstrate that Fe2+ localized in lysosomes
and ER involved in the ferroptosis pathway.
Also based on deoxygenation reaction, two fluorescent sen-
sors (18–19) for detecting Fe2+ were designed and synthesized
by Wang’s and our group, respectively.138,139 The turn-on/
colorimetric probe 18 was fabricated by connecting 4-(dimethy-
lamino) cinnamaldehyde to natural camphor through conden-
sation reaction, which was further oxidized to form the N-oxide
moiety (Fig. 13). The probe itself emitted weak red emission
owing to the existence of PET process. Upon addition of Fe2+,
about 27-fold fluorescence enhancement with a peak at 603 nm
was observed, with rapid response time (o40 s), large Stokes
shift (B208 nm) and high sensitivity (LOD = 8.3 nM). This was
attributed to the cleavage of the N-oxide bond from the
dimethylamine group, which promoted the ICT processes and
re-opened the NIR fluorescence. Unfortunately, the detection of
Fe2+ was significantly affected by polarity. They successfully
employed probe 18 for tracking Fe2+ on test paper. Further-
more, probe 18 was used to trace Fe2+ in HeLa cells and
zebrafish under ferroptosis induced by BSO. Importantly, the
results demonstrated that Bpy could effectively cause the
reduction of Fe2+ in HeLa cells, whereas Fer-1 had little effect
Fig. 13 The structure of probe 18, and the responsive mechanism
for Fe2+.
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Fig. 14 (A) Proposed response mechanism and properties of Probe 19 (FeP). (B) DHA regulates iron homeostasis and neuronal ferroptosis in KA-induced
epileptic mouse brain via the LCN2/FPN/GPx-4 pathway. (C) Representative EEG maps of KA-induced epileptic mice without (red) or with (green) DHA
regulation. Reprinted with permission from ref. 139. Copyright (2022) Elsevier B.V.

on the process, which was similar to Chang’s previous findings

(probe 14).
For probe 19, our group selected DCI as the skeleton because
it had great potential to cross BBB and to construct TP probes
(Fig. 14). The proposed sensor was initially almost non-
fluorescent due to the existence of the N-oxide moiety resulted
in the prohibition of the push-pull electron effect. However,
addition of Fe2+ to the solution of probe 19 generated remark-
able NIR fluorescence at 640 nm under excitation at 456 nm
within 300 s, which was attributed to the leaving of the N-oxide
part and thus recovered the red signals. Meanwhile, the maxi-
mum absorption shifted from 386 nm to 460 nm. By combing
HCA with the probe 19, we screened various natural products
and found the dihydroartemisinin (DHA) can alleviate the
damage of hippocampal neurons under KA-induced stress
and maintain their functions through inhibiting the ferroptosis
progression. The probe was able to detect the alterations of Fe2+
in vitro and in vivo. Importantly, we successfully applied this
probe to image Fe2+ in both KA-induced epileptic mouse model
and pentylenetetrazole (PTZ) kindling epileptic mouse model,
which suggested the overproduction of Fe2+ in epileptic brain,
whereas DHA could cause the reduction of Fe2+. Specifically,
in vivo 3D TP imaging of the epileptic mice brain verified the
potential of DHA in the prevention and treatment of epilepsy
(Fig. 15). Moreover, EEG (electroencephalography)-EMG (electro-
myography) signals indicated that DHA treatment caused the
inhibition of epileptic spikes. Besides, WB experiments con-
firmed that DHA post-treatment downregulated the expression
of lipocalin-2 (LCN2) in the epileptic brains. Thus, these results Fig. 15 (A) and (B) In vivo TPM imaging in live epileptic brains. Reprinted
suggested that DHA could be used as a potent anti-epileptic with permission from ref. 139. Copyright (2022) Elsevier B.V.

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Fig. 16 The structure of probe 20, and the responsive mechanisms for Fe2+.
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agent. Taken together, our work proposed a robust TP fluores-
cent probe for the detection of Fe2+ in the epileptic mouse brain,
provided a promising anti-epileptic compound, and explored the
links between epilepsy and ferroptosis.
On the basis of a two-step single electron transfer (SET)
mechanism, Lou’s group rationally designed and constructed a
novel fluorescent probe 20 for detecting Fe2+ in ferroptosis
(Fig. 16).140 In this probe, coumarin 343 was used as skeleton,
and the 3-nitrophenylazanyl ester simultaneously served as Fe2+
reactive site and quenching moiety. Based on the PET mecha-
nism, probe 20 emitted faint fluorescence emission due to the
quenching effect of 3-nitrophenylazanyl ester. However, about
10.9-fold fluorescence enhancement was observed in the
presence of Fe2+. The strong fluorescence was due to the N–O
bond cleavage in the N-aryl-O-acyl hydroxyl amine by Fe2+. They
used this probe to trace the Fe2+ in HT-1080 cells under erastin-
induced ferroptosis. Their observations indicated that erastin
up-regulated Fe2+ level, whereas Fer-1 could inhibit the process.
Furthermore, they found that the Fe2+ level was higher in RAW
264.7-M1 macrophages than that of both RAW 264.7-M0 macro-
phages and RAW 264.7-M2 macrophages.
Importantly, cell imaging and flow cytometry experiments
revealed that Fe2+ increased in the RAW 264.7-M0 cells upon
erastin-induced ferroptosis, while adverse results were
observed in the RAW 264.7-M1 cells. They speculated that the
main cause of the difference actions was attribute to the latter
cell state in higher Fe2+ levels inclined to protect themselves
through releasing Fe2+ under stress. Very interestingly, co-
localization fluorescence experiments suggested that probe 20
was mainly localized in the mitochondria, and that Fer-1
incubation might have some effects on its subcellular organelle
localization. They explained that the distribution of the probe
was questionable, but the Fe2+ localized in mitochondria
seemed to participate in ferroptosis.
5.2. Small-molecule fluorescent probes for monitoring
Fe3+under ferroptosis
Non-heme iron in food is mainly Fe3+, which needs to be reduced
to the soluble Fe2+ for absorption.3 Fe2+ from intestinal absorption
or erythrocyte degradation can be oxidized into Fe3+ by cerulo-
plasmin (CP) or hephaestin (HEPH).23 Fe3+ binds to transferrin
(TF), which further binds to transferrin receptor 1 (TFR1) and
endocytosed into endosomes.141 In the cell Fe3+ can be reduced to
Fe2+ by six-transmembrane epithelial antigen of the prostate 3
(STEAP3) and stored in either the unstable LIP or ferritin.5 Fe2+
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Fig. 17 (A) The structure of probe 21, and the responsive mechanisms for
Fe3+. (B) Ratiometric imaging (Fred/Fgreen) of probe 21-stained HeLa cells
under ferroptosis. Reprinted with permission from ref. 143. Copyright
(2020) American Chemical Society.
can also be oxidized to Fe3+ by ferroportin (FPN).142 During LPO,
O2  reacts with Fe3+ to produce Fe2+, which is called the Haber–
Weiss reaction. The circulation of internal iron intricately main-
tains iron homeostasis in the living system.5 In all, Fe3+ is closely
linked to ferroptosis, but few studies focus on its role in ferroptosis.
Recently, He’s group prepared a reversible ratiometric fluor-
escent probe 21 for detecting Fe3+ in ferroptosis (Fig. 17).143
In probe 21, dansylamide, spirolactam rhodamine B and
N2-hydroxyethyldiethylenetriamine (HEDTA) were selected as
the donor core, acceptor unit and specific binding site for Fe3+,
respectively. In addition, the latter also served as a linker to
bridge the first two parts to construct a FRET skeleton. Owing
to the inhibition of FRET process, the dual-excitation/dual-
emission probe itself presented the optical characteristics of
dansylamide featured by remarkable fluorescence emission
with a peak at 483 nm under the excitation wavelength of
405 nm. However, upon addition of Fe3+, the green emission
gradually decreased when excited at 405 nm, accompanied by an
inconspicuous increase of fluorescence at 576 nm under the
excitation at 543 nm. Of note, these spectral changes were attrib-
uted to the formation of ring-open form rhodamine B triggered by
Fe3+ and resulted in the ‘‘on’’ state of FRET process. Co-localization
experiments indicated that the sulfonamide moiety enabled this
probe to mainly localize in ER. Furthermore, they applied probe 21
to track exogenous Fe3+ levels in HeLa cells. Importantly, they
verified the elevation of intracellular endogenous labile Fe3+ under
ferroptosis with dual-channels for the first time. Very interestingly,
they further found that both DFO and Fer-1 could effectively
prevent the production of Fe3+, whereas the latter showed stronger
effects than the former. They suggested that it was possibly because
Fe3+ involved in the downstream of ferroptosis linked to peroxida-
tion of PUFAs. Additionally, this probe was able to monitor the
exogenous Fe3+ in kidney tissue slices.
6. Small-molecule fluorescent probes
for monitoring pathophysiological
microenvironments and other
biomolecules under ferroptosis
Pathophysiological microenvironments (viscosity, polarity,
pH, etc.) is closely correlated with the regulation of various
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biological processes such as enzyme-based catalysis, signal

transduction, proliferation, etc.60 Of note, various diseases in
the early state always show anomalous pathophysiological
microenvironments, and thus it is helpful for the early diag-
nosis through monitoring these factors.35 However, the abnor-
mal microenvironments in ferroptosis have not been yet fully
explored.

6.1. Small-molecule fluorescent probes for monitoring pH

under ferroptosis
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Autophagy, which means ‘‘self-eating’’ in Greek, is a lysosome-

dependent degradation pathway that recycles misfolded pro-
teins and dysfunctional organelles, in addition to offers nutri-
ents during starvation.44,144,145 In this process, the fusion of
lysosomes and autophagosomes produces autolysosomes,
accompanied by the fluctuations of microenvironments.146,147
Increasing evidences demonstrated that acidic pH conditions
can stimulate the autophagy process,148 whereas the raise of
lysosomal pH is able to efficiently block autophagy through
inhibition of hydrolase.149 On the other hand, autophagy can
induce ferroptosis, which is named autophagy-dependent
ferroptosis.150 Therefore, pH can serve as a key factor for
tracking ferroptosis.
Mitophagy is a specialized autophagy program that removes
dysfunctional or superfluous mitochondria in a lysosome-
dependent manner.145,151 Very recently, based on the design
Fig. 18 (A) The structures of probe 22, and the responsive mechanisms
strategy of covalent anchoring of a lysosomal probe to the for pH. (B) and (C) Dependence of red fluorescence on ferroptosis induced
mitochondrial inner membrane (CALM), a versatile pH- by erastin or RSL3, respectively. Reprinted with permission from ref. 152.
sensitive fluorescent probe 22 for visualization of mitophagy Copyright (2021) American Chemical Society.
in ferroptosis was tactfully designed and constructed by Han’s
group (Fig. 18).152 The coumarin moiety with blue signal was
and well colocalized with LAMP2-GFP, which directly suggested
taken to light up and localize in mitochondria. The classic TPP
that the pH decreased and the mitophagy occurred during
unit allowed the probe to target mitochondria. In addition, the
ferroptosis. Finally, they applied this probe for tracking mito-
X-rhodamine-lactam part was selected as the reporter for pH
phagy in K-ras mutant A549 cells under ferroptosis via CALM,
and able to illuminate the lysosomes after the mitochondria
which again suggested the correlation between mitophagy and
transferred into lysosomes, with red emission. More impor-
ferroptosis (Fig. 18). In summary, their work showed that
tantly, the dibenzocyclooctyne (DBCO) segment enabled the
mitophagy was involved in the process of ferroptosis.
probe to bind to the azido-containing phosphatidylcholine on
the mitochondrial membrane through bioorthogonal ligation,
which thus ‘‘fixed’’ the probe to mitochondrial lipids. Notably, 6.2. Small-molecule fluorescent probes for monitoring
this design strategies overcome the major bottleneck where viscosity or polarity under ferroptosis
conventional staining fluorescent sensors or dyes inclined to As changes in viscosity and polarity is associated with the
‘‘escape’’ the abnormal mitochondria. The probe displayed structures and functions of organelle (e.g. ER and LDs) or
weak red emission under neutral condition, but emitted strong cytoplasmic membrane, it was hypothesized that tracking their
red signals in acidic condition. Meanwhile, relatively stable changes can allow us to understand their functions in ferrop-
blue emission was observed with the varied pH. They demon- tosis process.
strated that HeLa cells and MCF-7 cells incubated with Azcho- LDs are the major organelles for storing neutral lipids.153
line could reduce the dissipation of probe 22 in mitochondria Recent findings showed that accelerated accumulation of lipid
under the decreased mitochondrial membrane potential (DCm) storage through the formation of LDs mitigates ferroptosis,
caused by carbonyl cyanide m-chlorophenylhydrazone (CCCP). whereas increased reduction of LDs propels ferroptosis.2 Thus,
Moreover, their work indicated that this probe was able to it is necessary to track the LDs alteration for studying ferrop-
detect mitophagy during cell starvation. Moreover, this probe tosis. In 2021, Lin et al. prepared a NIR fluorescent probe 23 to
was utilized for sensing pH in HT1080 cells expressing LAMP2- sense the fluctuations of viscosity in the LDs during ferroptosis
GFP specific for lysosomes in the process of ferroptosis. Impor- (Fig. 19(A)).154 This probe was fabricated by linking 4-(dimethyl-
tantly, the red emission in the erastin or RSL3 treated groups amino) cinnamaldehyde to benzothiazole-2-acetonitrile. The
obviously enhanced compared with that of the control groups, sensing mechanism in response to viscosity was based on its

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Fig. 19 (A) The structures of probe 23, and the responsive mechanism for
viscosity. Representative fluorescence images and relative fluorescence
intensities of HeLa cells (B) and (C), 4T1 cells (D) and (E), A545 cells (F) and
(G), and HepG2 cells (H) and (I) pretreated with probe 23 and then with
RSL3 for various times. Reprinted with permission from ref. 154. Copyright
(2021) American Chemical Society. Fig. 20 (A) The structures of probes 24–27. (B) Fluorescence images of
HepG2 cells under ferroptosis. Reprinted with permission from ref. 155.
Copyright (2022) American Chemical Society.

molecular aggregation and the twisted intramolecular charge

transfer (TICT) caused by the inhibition of the rotation of the three cytoplasm membrane in different living cell physiological
single bonds. They found that the different cell lines including processes through dual channels (Fig. 21).156 The optical char-
HeLa cells, 4T1 cells, A545 cells, or HepG2 cells incubated with acteristics of the probe were tested in ethyl acetate–ethanol and
erastin or RSL3 generated stronger NIR signals than that of the glycerin–ethanol mixtures, respectively. In the former system,
control groups, which suggested that the LDs viscosity increased the yellow emission with a band at 537 nm when excited at
during ferroptosis (Fig. 19(B)–(I)). Furthermore, they also demon- 373 nm gradually developed with the increase of ethanol
strated that Fer-1 would block the ferroptosis process. volume, which ascribed to the formation of aggregates. Moreover,
Very recently, by combining ICT and motion-induced changes the red emission with a peak at 650 nm upon exaction at the
in emission (MICE) mechanisms, Tang’s group developed a range 500 nm significantly enhanced in the latter system with rising
of viscosity-and polarity-sensitive fluorescent probes (24–27) for proportions of glycerin, which resulted from the inhibition of
monitoring LDs under ferroptosis (Fig. 20).155 These probes
featured the merocyanine scaffold bearing diethylamino or meth-
oxy moiety directly linked to the aurone group modified with or
without hydroxyl group, which constructed a classic D–p–A plat-
form for polarity detection. In the meantime, the aurone and
diethylamino parts were also expected to serve as molecular rotors
which enabled the probes to track viscosity. Probe 24 exhibited
the largest NIR emission and absorption was selected the best
suitable candidate among the four dyes. Co-localization imaging
experiments verified that the probe mainly distributed in the LDs.
Facilitated with this probe, the accumulation and viscosity
increase of LDs in HepG2 cells under autophagy induced by
rotenone, nystatin, and starvation was examined. Furthermore,
they uncovered that the viscosity increased in HepG2 cells during
the erastin-induced ferroptosis and the Fer-1 could prevent the
process. However, the roles played by polarity were not clearly
explored in this work. Finally, the probe was used to distinguish
normal cells from cancer cells. Together, both Lin’ and Tang’s
studies demonstrated that LDs participated in the process of
Fig. 21 (A) The structure of probe 28. (B) Schematic illustration of the
ferroptosis and the level of LDs viscosity increased in this
HeLa cell morphology and membrane lipid order change during ferrop-
specific RCD. tosis. (C) 3D confocal fluorescence images of HeLa cells under ferroptosis
Tong’s group reported an aggregation-induced emission at various times. Reprinted with permission from ref. 156. Copyright (2021)
(AIE)-based fluorescent probe 28 for the visualization of American Chemical Society.

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Fig. 22 (A) The structures of probes 29–32, and the represented respon-
sive mechanism for NAD (P) H and viscosity, respectively. (B) Monitoring of
viscosity and NAD (P) H change in HT-1080 cells under ferroptosis induced
by erastin and RSL3, respectively. Reprinted with permission from ref. 159.
Copyright (2020) Elsevier B.V.
TICT process. They also verified the dual-color mode in varied
compositions of giant unilamellar vesicle (GUVs). The 3D images
indicated that the red signals were definitely localized on the
outer membrane. The probe was applied to image the cell
membranes in apoptosis, ferroptosis, and mitosis for more than
15 h. Noticeable yellow signals within 11 h were obtained in HeLa
cells under ferroptosis induced by erastin, while strong yellow
emission was found at the corresponding time in the process of
apoptosis (Fig. 21(C)). Therefore, their work revealed the different
changes of membrane viscosity in the three cell processes.
Nicotinamide adenine dinucleotide phosphate (NADPH),
an essential intracellular reducing agents, plays complex roles
in ferroptosis.157 NADPH mainly synthesizes from the pentose
phosphate pathway (PPP),72 while the phosphorylation of NAD
catalyzed by NAD kinase (NADK) can also produce NADPH.3
NADPH participates in the reduction of oxidized glutathione
(GSSG) to GSH, which contributes to controlling the redox
homeostasis.158 Besides, NADPH is needed for eliminating
LOOH.17 However, NADPH can also act as an electron donor
for NADPH oxidases (NOXs), which promotes ferroptosis by
catalyzing electron transfer from NADPH to O2 to generate
O2 .10 Thus, NADPH shows dichotomous effects in ferropto-
sis. Zhang et al. recently prepared a series of versatile fluor-
escent sensors (29–32) for simultaneously sensing viscosity and
NAD (P) H under ferroptosis through green and red channels,
separately (Fig. 22).159 These probes were fabricated by con-
necting 2-(2-Methyl-4H-chromen-4-ylidene) malononitrile to
the cationic 3- or 6-quinolaldehyde derivatives. Probe 32 was
selected because only this probe displayed absorption changes
Chem. Soc. Rev.
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Fig. 23 (A) The structure of probe 33, and the responsive mechanism for
CEs, polarity and viscosity. (B) HCA for monitoring the alterations of
viscosity, polarity, and CEs induced by various drugs in living HepG2 cells.
(C) and (D) Quantification analysis of blue and red channels separately in
(B). Reprinted with permission from ref. 160. Copyright (2022) American
Chemical Society.
when incubated with NADH. In detail, probe 32 showed weak
red emission at 670 nm when excited at 595 nm in the absence
of NADH, whereas the red signals distinctly developed upon
addition of NADH. With increasing viscosity, the green emis-
sion band centered at 550 nm obviously increased under
excitation at 400 nm. They employed this probe to simulta-
neously image the fluctuations of NAD (P) H and mitochondrial
viscosity in living cells. Very interesting, they observed that
NADH- or glucose-treated HT-1080 and PANC-1 cells produced
both stronger green and red signals than that of the control
groups, while the green signals of the cells incubated with
pyruvic acid was almost unchanged. Importantly, they used
probe 32 for trapping these two targets in the ferroptosis
process induced by RSL3 and erastin, respectively. The results
demonstrated that the two ferroptosis-inducers simultaneously
upregulated viscosity and NAD (P) H, and erastin displayed
stronger effects that of RSL3. They concluded that mito-
chondrial viscosity was increased during ferroptosis, which
might offer another evidence for the role of mitochondria in
the ferroptosis.
Based on an OR logic gate, our group recently reported a
multifunctional fluorescent probe 33 for simultaneous detec-
tion of viscosity, polarity and carboxylesterases (CEs)
(Fig. 23(A)).160 The probe was initially non-fluorescent when
excited at 443 nm. However, a 139.3-fold increase in fluores-
cence emission at 593 nm was obtained in the presence of CEs.
Meanwhile, remarkable far-red signals were observed in the
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Fig. 24 The structure of probe 34, and the responsive mechanism for
viscosity.
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high viscosity or low polarity system. We used the probe to

monitor the three analytes in the living cells. Our results
suggested that the viscosity level increased or the polarity level
decreased in the early apoptosis state, whereas opposite
changes were observed in the late apoptosis state. Furthermore,
this probe was able to distinguish normal cells from cancer
cells, as well as had good potential to identify living liver cell
lines. Importantly, using HTS, we screened a number of classic
antitumor agents and explored their effects on the analytes of
interest (Fig. 23(B)–(D)). Our work showed that sorafenib-
induced ferroptosis caused an increase in the microviscosity
and down-regulation of CEs at the same time. Moreover, we
found that aristolochic acid (AA) could induce the overproduc-
tion of CEs in HepG2 cells, which suggested that AA may induce
liver cancer. In addition, the down-regulation of CEs and the
elevated level of viscosity or the decreased level of polarity was
examined in the lipopolysaccharide (LPS)-induced inflamma-
tion model. Finally, this smart probe could be used as a robust
imaging agent in vivo.
Mao’s group proposed a neutral iridium complex, probe 34,
for quantitative imaging of ER viscosity for the first time
(Fig. 24).161 This probe, [Ir (4-(2-pyridinyl) benzaldehyde) 2(acetyl-
acetone)], was capable of real-time monitoring the dynamic
changes of ER viscosity in MCF-7 cells via two photon lifetime
imaging (TPPLIM). Their observations indicated that the lifetime
of probe 34 increased from 758 ns to 841 ns when the viscosity
values changed from 122 cP to 156 cP, which indicated that the
levels of ER viscosity in MCF-7 cells elevated during ferroptosis
induced by erastin. Thus, their work provided a novel tool for
quantitatively revealing the dynamic changes of ER viscosity in
living cells under ferroptosis.
Inspired by the structures of Nile Red and Hoechst 33342,
Liu and co-workers rationally designed and synthesized a
ratiometric fluorescent probe 35 to monitor the polarity of
LDs in ferroptotic cells, with LDs/nucleus dual-targeting ability
(Fig. 25(A)).162 The coumarin moiety, a potential polarity-
dependent dye, was selected as the donor (D), while the cationic
quinolinium part served both as the acceptor (A) and the
binding site for nucleic acids. The classic ICT systems are
usually polarity sensitive. In addition to promoting the probe
to chelate with nucleic acids, the 1-(pyridin-4-yl) piperazine
moiety adopted from the Hoechst 33342 could further confer
the platform with ratiometric fluorescence emission. Upon
excitation at 405 nm, the blue emission at 470 nm was
remarkably decreased with increasing the volume of water from
Fig. 25 (A) The structure and design strategy of probe 35. (B) Application
of probe 35 to monitoring the polarity changes of LDs and nucleus in living
cells under different conditions by FLIM. Reprinted with permission from
ref. 162. Copyright (2021) Wiley-VCH Verlag GmbH & Co. KGaA.
0% to 20% in the 1,4-dioxane and water mixtures, accompanied
by a strong red emission at 670 nm significantly developed. At
the same time, the fluorescence lifetime of probe 35 attenuated
from 1.49 ns to 0.89 ns. Additionally, treatment of the probe
with ds26 DNA only resulted in noticeable red fluorescence
enhancement. This probe was able to synchronously image the
LDs and nucleus via 3D dual-color imaging. The ratiometric
fluorescence imaging experiments revealed that the LDs polar-
ity was enhanced in HT-1080 cells under ferroptosis induced by
erastin and no typical apoptotic morphology was obtained.
In addition, they found that the polarity of LDs increased from
the nuclear edge to the cell edge. Of note, long-time imaging of
the LDs polarity with the probe revealed that LDs gradually
diffused into the cytoplasm in HT-1080 cells during the RSL3-
induced ferroptosis. At the same time, the polarity of LDs and
cytoplasm gradually showed no noticeable difference. Fluores-
cence lifetime imaging (FLIM) using probe 35 further revealed
that there was no obviously change of the fluorescence lifetime
in nucleus under ferroptosis and apoptotic model, respectively
(Fig. 25(B)). Meanwhile, the lifetime values in LDs in the
erastin-treated HT1080 cells changed remarkably. In summary,
this work offered a smart imaging tool as well a new strategy for
understanding the alterations of LDs in ferroptosis.

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7. Small-molecule fluorescent probes
for monitoring other bioactive
substances under ferroptosis
Iron-containing heme oxygenase-1 (HO-1), a critical enzyme
in heme metabolism, can decompose heme into Fe2+ and
biliverdin.73 On the one hand, elevated HO-1 can induce
ferroptosis through increasing the LIP upon metabolizing
heme.163–165 In particular, ferroptosis induced by excessive
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HO-1 activity is called non-canonical ferroptosis.165 On the
other hand, the Nrf2-mediated anti-ferroptosis activity depends
on the induction of quinone oxidoreductase 1 (NQO1), HO-1,
and ferritin heavy chain (FTH1).166 Thus, HO-1 has dual role in
ferroptosis. In order to reveal the functions of HO-1 in ferrop-
tosis, Zhang et al. proposed a series of aggregation-induced
enhanced emission (AIEE)-based fluorescent probes for the
detection of HO-1 activities in the presence of ROS under
ferroptosis in vitro and in vivo (Fig. 26).167 Probe 36 with the
highest quantum yield and outstanding AIEE optical character-
istics was selected. The average hydrodynamic diameter of
probe 36 was measured to be B52  3 nm by dynamic light
scattering (DLS). Besides, the TEM imaging showed the value
was B50 nm. They demonstrated that the heme oxygenase-
catalyzed reaction could not be triggered by ROS alone when
incubated probe 36 with various ROS separately. However,
there was distinct fluorescence emission enhancement with
hypsochromic shift when probe 36 was treated with ferroptotic
cell lysate. Next, the probe was used to track the HO-1 in living
HeLa cells under ferroptosis caused by erastin. The results
uncovered the elevated level of HO-1 activities during ferropto-
sis and GSH could prevent the increase through scavenging
H2O2. Notably, their work demonstrated that cisplatin (CDDP)
and paclitaxel (PTX) had little influence on the elevation of
HO-1 activities. This probe was further utilized for monitoring
ferroptosis in the tumor-bearing nude mice. As shown in
Fig. 26, the mice treated with erastin presented stronger green
signals than that of the control groups, while the latter groups
Fig. 26 (A) The structure of probe 36, and the responsive mechanism for
HO-1. (B) Optical imaging of tumor-xenografted nude mice. (C) Confocal
imaging of tumor sections. Reprinted with permission from ref. 167.
Copyright (2018) American Chemical Society.
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showed stronger red signals than that of the former groups. The
tumor slices imaging also verified the observations (Fig. 26(C)).
The results suggested that the probe was capable of sensing
ferroptosis in vivo through monitoring the HO-1. Altogether, this
work not only provided a robust tool for the detection of endo-
genous HO-1 activity in vitro and in vivo, but also demonstrated
that HO-1 activity levels were positively correlated with ferroptosis.
Labile heme (LH) is a complex of Fe2+ and protoporphyrin
IX, which plays important roles in all of the heme-dependent
functions in living systems.168 LH is possibly correlated with
ferroptosis, although the relationship between them hasn’t
been clearly studied. Based on the biomimetic reaction of
cytochrome P450, Hirayama and co-workers designed and
prepared a class of fluorescent probes (37–40) for the visualiza-
tion of LH in living cells upon the induction of ferroptosis
(Fig. 27(A)).169 Compared with probes 39 and 40, probe 37 showed
remarkable fluorescence enhancement in response to LH, and
thus the later was selected as the candidate probe. Probe 37
comprised of a rhodol platform bearing an electron-withdrawing
4,4-difluoropiperidine N-oxide moiety, which served as the LH
reactive site. The introduction of the electron-withdrawing part
Fig. 27 (A) The structures of probes 37–40, and the responsive mecha-
nism for LH. (B) Fluorescence imaging of LH and Fe2+ mobilization during
ferroptosis. Reprinted with permission from ref. 168. Copyright (2022)
American Chemical Society.
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Table 3 A summary and comparison of representative probes

Target
No. Detection system Sensing mode Chromophore analytes lex/em (nm) LOD Time Application Ref.
Selective detection of ROS
1 DMSO : PBS = 1 : 19 ICT/turn-on Quinoline HClO 390/581 (OP), 104 nM o5 s Cells, brain 88
Chem Soc Rev

800/-(TP) tissues and mice

2 — PET/turn-on Phenothiazine and HClO 520/627 7.74 nM o5 s Cells and zebrafish 89
benzopyrylium
3 MeCN : PBS = 1 : 99 —/turn-on DCI H2O2 396/566 0.77 mM o30 min Cells 96
4 DMF : PBS = 3 : 7 ICT/turn-on Naphthalimide H2O2 450/550 1.5  107 M B120 s Cells and tumor 97
tissues slice
5 MeCN : PBS = 1 : 5 ESIPT/turn-on HBT H2O2 380/542 109 nM o45 min Cells 98
6 —/MeOH-glycerol —/turn-on, —/turn-on Indolium and anisole OH and 590/652, 400/520 8.6 nM/— B30 min/— Cells 102
Viscosity
7 PBS (containing DMF) —/turn-on, —/turn-on Coumarin OH and Cys 580/650, 400/453 84 nM/0.335 mM o30 min/B2 h Cells 103
Selective detection of RSS
8 DMSO : PBS = 1 : 19 —/turn-on DCI and anthracene H2Sn 440/640 (OP), 0.98 mM o2 h Cells 115
880/-(TP)
9 DMSO : PBS : glycerol AIE/turn-on Quinolinemalonitrile H2S and 450/670, 450/646 1.3 nM/— o40 min/— Cells and mice 119

This journal is © The Royal Society of Chemistry 2022

= 1 : 9 : 40 and thiophene viscosity
11 MeCN : PBS = 1 : 9 ICT/turn-on DCI Cys 408/568 0.1 mM o30 min Cells and zebrafish 127
12 DMSO : PBS = 1 : 99 —/ratiometric, Coumarin derivative GSH 405/487, 488/562 — — Cells 128
reversible
13 DMSO : PBS = 1 : 9 ICT/turn-on Coumarin derivative Thiol 475/578 0.49 mM Cys: 10 s, GSH: Cells 129
and chromene 10 s and
Hcy: 10 s
Selective detection of iron
14 HEPES FRET/ratiometric 5-aminomethyl fluorescein Fe2+ 448/556, 408/515 — o90 min Cells 136
and cyanine 3
15 HEPES — Rhodol Fe2+ 510/535 — o3000 s Cells 137
16 HEPES — Tetramethyl Fe2+ 550/575 — o3000 s Cells 137
hydroxymethylrhodamine
17 HEPES — Silicon-incorporated Fe2+ 630/660 — o3000 s Cells 137
rhodamine
18 EtOH : PBS = 1 : 4 ICT, PET/turn-on Dimethylamine and Fe2+ 425/603 8.3 nM o40 s Cells and 138
camphor zebrafish
2+
19 DMSO : PBS = 1 : 99 — DCI Fe 640/880 14.2 nM o300 s Cells and mice 139
20 Tris–HCl buffer ICT, PET/turn-on Coumarin 343 Fe2+ 432/488 80 nM o30 min Cells 140
(containing DMSO)
21 DMSO : H2O = 1 : 99 FRET/ratiometric, Spirolactam rhodamine Fe3+ 405/483, 405/576 0.13 mM o5 min Cells and rat 143
reversible B and dansylamide kidney slices
Selective detection of microenvironmental parameters
22 MeOH : PBS = 3 : 7 —/turn-on Coumarin and X-rhodamine pH 430/470,595/615 — — Cells 152
23 MeOH/glycerol TICT/turn-on N,N-Dimethylamino Viscosity 620/723 — — Cells 154
and benzothiazole-2-acetonitrile
24 PBS/glycerol, PBS/THF ICT, MICE/turn-on Merocyanine and aurone Viscosity and 650/745, — — Cells 155
polarity 650/740–720
28 EA/EtOH, glycerin/EtOH AIE/ratiometric Tetraphenylethylene derivatives Viscosity 373/537, 500/650 — — Cells 156
32 DMSO : PBS = ICT/turn-on Methylquinolinium NADPH and 595/670, 400/550 — 20 min/— Cells 159
1 : 1, PBS/glycerol and malononitrile motif viscosity
33 DMSO : PBS = 1 : 99, TICT, ICT/turn-on Quinoline and CEs, polarity 443/593, 3.04  o40 min Cells and mice 160
H2O/1,4-dioxane, H2O/glycerol N,N-dimethylamino and viscosity 561/752–792 561/786 106 U mL1
34 MeOH/glycerol TICT — Viscosity 405/530, 810/— — — Cells 161
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Ref.
162

167

168
promoted the deoxygenation reactions between LH and probe 37,
and thus endowed the probe with high sensitivity and selectivity

tissues and mice

for LH detection. The probe showed negligible fluorescence
emission in the absence of LH. However, 230-fold fluorescence

Cells, tumor
Application

enhancement was obtained within 10 min when the probe was

treated with hemin. Of note, the introduction of Fe2+ to the
Cells

Cells
solution of probe 37 resulted in weak fluorescence response,
which attested the high selectively of probe 37 in response to
LH. The acetylated prodrug of probe 37 (probe 38) was able to
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detect the intracellular LH levels. Furthermore, the nitric oxide

460 min

o10 min

(NO)-mediated LH accumulation from endogenous hemoproteins

Time

was verified in living HeLa cells. More importantly, using the

—

probe 38, they demonstrated that knockdown of FLVCR1a

induced the overexpression of LH when SH-SY5Y cells were
incubated with 5-aminolevulinic acid (ALA) and ferric ammonium
citrate (FAC). Besides, they also confirmed that ATP binding
LOD

cassette (ABC) subfamily G member 2 (ABCG2) was partially

—
—

responsible for exporting heme. Moreover, probe 38 was applied

to examine the roles of G-quadruplexes as a source of LH. The
405/470, 405/670

results disclosed that HT1080 cells incubated with PhenDC3

—/595, —/550

produced stronger signals than that of the control groups, which

lex/em (nm)

signified that PhenDC3 could induce G-quadruplex to liberate LH.

490/535

Finally, using probes 38 and a reported fluorescent probe

(SiRhoNox-1), they observed the synchronous increased LH and
Fe2+ in HT1080 cells under ferroptosis caused by erastin
Polarity and
nucleic acid

(Fig. 27(B)). Thus, this work provided a valuable tool for tracking
analytes

LH to elucidate the molecular mechanisms of ferroptosis.

Target

HO-1

LH

8. Conclusions and outlook

As an iron-dependent form of RCD, ferroptosis has gained
cationic quinolinium

increasing attention over the past decade. Understanding the

functions and specific molecular mechanisms of ferroptosis is
Quinoxalinone
Coumarin and
Chromophore

greatly conducive to etiological research and to provide an

alternative strategy for diagnosis and treatment of diseases.
Rhodol

Small-molecule fluorescent probes with desirable character-

istics, such as real-time visualization, noninvasiveness and
ultrahigh spatial resolution, have been used to unravel the
(LE) state/ratiometric

underlying mechanisms of ferroptosis. In this review, we sys-

ICT, locally excited

AIEE/ratiometric

tematically summarized recent advances in small-molecule

Sensing mode

fluorescent probes in ferroptosis research (Table 3). The fluc-

—/turn-on

tuations of the ferroptosis-related biomarkers were also sum-

marized to help to understand the signalling pathways of
ferroptosis (Table 4). The reported work has answered many
unresolved questions in the ferroptosis field. For example, the
detection of LDs viscosity and polarity helps us to understand
DMSO : HEPES = 1 : 499

the alterations of LDs in the process of ferroptosis. Further-

more, Han and colleagues (probe 22) revealed that mitophagy
was related to ferroptosis with direct imaging evidence, which
No. Detection system
H2O/1,4-dioxane,
(continued)

tris–HCl buffer

implicated the involvement of mitochondria in ferroptosis.

DMSO/H2O

Thus, small-molecule fluorescent probes can be used as

promising tools for studying ferroptosis.
Despite the significant recent progress in this emerging
Table 3

field, there are many remaining challenges. Some forward-

35

36

37

looking suggestions for the development of small-molecule

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Table 4 The dynamic changes of the analytes under ferroptosis been used for studying ferroptosis. However, there is still an urgent
demand to employ this kind of probes for trapping the dynamically
Classification Target Changes
changes of multianalytes to identify ferroptosis process.
ROS HClO Up-regulation (4) Organ-targeting probes are able to efficiently detect the
H2O2 Up-regulation
OH Up-regulation ferroptosis-induced diseases in specific organs. Thus, it is of
RSS H2Sn Down-regulation great significance to develop fluorescent sensors with organ
H2S Down-regulation specificity.
Cys Down-regulation
GSH Down-regulation Applications: (1) most of the existing fluorescent probes only
Iron Fe2+ Up-regulation focused on revealing the variations of bioanalytes or micro-
(in ER and lysosomes)
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environment-related parameters during ferroptosis rather than

Fe3+ Up-regulation
Pathophysiological pH Down-regulation dissecting the relationship between ferroptosis and specific
microenvironments Viscosity Up-regulation diseases. We believed that there is a great demand to determine
Polarity Up-regulation the bioanalytes sensing in ferroptosis-related disease models.
Other analytes NAD(P)H Up-regulation
LDs Up-regulation For instance, the neurodegenerative disorders, such as AD and
CEs Down-regulation Parkinson’s disease (PD), have been historically considered as
HO-1 Up-regulation one of the hotspots in medical research.182 (2) In combination
LH Up-regulation
with HCA, small-molecule fluorescent probes can be used for
screening anti-ferroptosis drugs. (3) It should be noted that the
fluorescent probes for studying ferroptosis were listed as exact molecular events responsible for the execution of
following. ferroptosis-like cell death are still unknown.25 Thus, it is not
Design strategies: (1) the selectivity of these probes should convincing to identify whether ferroptosis is involved in the
be optimized through connecting with more specific reaction process of interest via the conventional protocol, that is, treat-
sites. For example, the benzene boronic acid pinacol ester unit ing the cells or animals with an inducer to cause the assumed
has been widely used for designing fluorescent probes for the ferroptosis and then inhibiting the process with an inhibitor.
detection of H2O2.170,171 However, this moiety could also com- Towards this end, more experiments are required to identify
monly serve as a reporter for peroxynitrite (ONOO).38,50,82,172,173 the process. For example, the levels of LOOH need to be
Furthermore, Pu research team showed that a probe bearing the examined. (4) More biomolecules or microenvironmental para-
boronic ester moiety showed remarkable fluorescence enhance- meters, such as reactive nitrogen species (RNS), hypoxia, DCm
ment in response to H2O2 and ONOO, respectively.174 Therefore, and even intracellular temperature should be checked to
the specificity of the recognition units should be carefully taken explore their connections with ferroptosis.
into consideration to ensure selectivity for fluorescent probes. Overall, this review provided a summary of recent develop-
(2) On the one hand, most of the proposed chemosensors ment of small-molecule fluorescent probes for studying ferrop-
for ferroptosis are located in the visible light spectral range tosis. We hoped that this review would offer the biologists,
between 400 and 650 nm. On the other hand, some of them are pathologists and pharmacologists with a smart method and
TP probes. It should be noted that these probes with short new insights for the ferroptosis research, and we anticipated
wavelengths suffer from shallow tissue penetration depth, that the applications of small-molecule fluorescent probes in
strong autofluorescence and low SBR.52,175–177 Of note, some this area would greatly promote our understanding of ferrop-
of the commercial fluorescent probes for ferroptosis studies tosis in the near future.
(e.g. DCFH-DA and C11-BODIPY) have small Stokes shift, which
greatly hamper their bioapplication in bioimaging. Furthermore,
the TP probes are still incapable of imaging in vivo by TP Abbreviations
excitation wavelength.178 Importantly, the fluorescence emissions
of these probes are mainly determined by the fluorophores.179,180 AA Aristolochic acid
Therefore, it is necessary to design the chemosensors with NIR-I ABC ATP binding cassette
skeleton, and in particular, NIR-II fluorophores which shows the ABCG2 ATP-binding cassette subfamily G member 2
irreplaceable superiority over the former in vivo imaging.36,51 ACD Accidental cell death
(3) It is noteworthy that there is still no specific biomarkers AD Alzheimer’s disease
of ferroptosis.29 Other studies have shown that there is cross- AIE Aggregation-induced emission
talk among ferroptosis, autophagy, and other types of RCD as AIEE Aggregation-induced enhanced emission
we stated above. In addition, there are multiple cellular meta- AM Acetoxymethyl
bolic pathways and a cohort of modulators involved in ferrop- ART Artesunate
tosis process. Thus, it is difficult if not impossible to monitor ATP5G3 ATP synthase F0 complex subunit C3
and identify ferroptosis through detecting a single substance or AZ Acetazolamide
Az
event. We believed that MFP could be used as smart tools for choline Azido-choline
tracking the complex physiological processes.181 Fortunately, BBB Buthioninesulphoximine
several versatile fluorescent probes (e.g. probes 6 and 7) have CA Carbonic anhydrase

This journal is © The Royal Society of Chemistry 2022 Chem. Soc. Rev.
 View Article Online

Tutorial Review Chem Soc Rev

CALM Covalent anchoring of a lysosomal probe to the MCAO Middle cerebral artery occlusion
mitochondrial inner membrane MICE Motion-induced changes in emission
CBS Cystathionine b-synthase MFP Multifunctional fluorescent probes
CCCP Carbonyl cyanide m-chlorophenylhydrazone MPO Myeloperoxidase
CDDP Cisplatin MRI Magnetic resonance imaging
CEs Carboxylesterases NADK NAD kinase
ClO Hypochlorite NADPH Nicotinamide adenine dinucleotide phosphate
COS Carbonyl sulfide NIR Near-infrared
CP Ceruloplasmin NIR-II Second near-infrared
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CSE Cystathionine g-lyase NMDAR NMDA-receptors

Cys Cysteine NOX NADPH oxidase
Cy3 Cyanine 3 NQO1 Quinone oxidoreductase 1
CVDs Cardiovascular diseases Nrf2 Nuclear factor erythroid2-related factor2
DBCO Dibenzocyclooctyne OGD/R Oxygen glucose deprivation/re-oxygenation
DHA Dihydroartemisinin OH Hydroxyl radical
DHODH Dihydroorotate dehydrogenase ONOO Peroxynitrite
DLS Dynamic light scattering O 2  Superoxide anion radical
EEG Electroencephalography PD Parkinson’s disease
EMG Electromyography PET Photo-induced electron transfer
ER Endoplasmic reticulum POR Cytochrome P450 Oxidoreductase
ESIPT Excited-state intramolecular proton transfer PTX Paclitaxel
Fer-1 Ferrostatin-1 RCD Regulated cell death
FI Fluorescence imaging RNS Reactive nitrogen species
FLIM Fluorescence lifetime imaging ROS Reactive oxygen species
FRET Fluorescence resonance energy transfer RPL8 Ribosomal protein L8
FTH1 Ferritin heavy chain RSS Reactive sulfur species
GPX4 Glutathione peroxidase 4 PUFAs Polyunsaturated fatty acids
GSH Glutathione SBR Signal-to-background ratios
GSSG Oxidized glutathione SET Single electron transfer
GUVs Giant unilamellar vesicle SPAAC Stain-promoted azide-alkyne cyclization
HBT 2-(2-Hydroxyphenyl) benzothiazole SPECT Single-photon emission computed tomography
HCA High-content assays STEAP3 Prostate 3
HCC Hepatocellular carcinoma TEM Transmission electron microscopy
HClO Hypochlorous acid TF Transferrin
HEDTA N2-Hydroxyethyldiethylenetriamine TFR1 Transferrin receptor 1
HEPH Hephaestin TICT Twisted intramolecular charge transfer
HO-1 Hemeoxygenase-1 TP Two-photon
H2O2 Hydrogen peroxide TPM Two-photon microscopy
HRAS HRas proto-oncogene TPP Triphenylphosphine
H2Sn Hydrogen polysulfide TPPLIM Two photon lifetime imaging
H2S Hydrogen sulfide TRFI Time-resolved fluorescence imaging
HTS High-throughput screening TTC35 Tetratricopeptide repeat domain 35
ICT Intramolecular charge transfer WB Western blotting
IF Immunofluorescence 3D Three-dimensional
KA Kainic acid 5-AMF 5-aminomethyl fluorescein
L Lipid radical d2 maximum two-photon absorption cross-section
LCN2 Lipocalin 2
LDs Lipid droplets
LH Labile heme Conflicts of interest
LIP Labile iron pool
There are no conflicts to declare.
LOD The limit of detection
L-OH Lipid hydroxy
LOOH Lipid hydroperoxides Acknowledgements
LOX Lipoxygenases
LPO Lipid peroxidation The authors gratefully acknowledge the financial support from
DCm Mitochondrial membrane potential National Natural Science Foundation of China (Project no.

Chem. Soc. Rev. This journal is © The Royal Society of Chemistry 2022

Chem Soc Rev
81903623 and 22007086), the China Postdoctoral Science Foun-
dation (No. 2019M652586 and 2020M670083ZX), Henan
science and technology key project (No. 202102310148), Henan
Medical Science and Technology Program (No. 2018020601 and
SBGJ202003051).
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