English

• 1. Introduction

  Ischemia-reperfusion occurs when blood flow to organs is initially obstructed and subsequently restored. This phenomenon, often resulting from arterial occlusion from by an embolus, creates an imbalance between energy supply and demand, leading to tissue hypoxia and damage [1]. Unfortunately, the restoration of blood flow and oxygenation can exacerbate tissue damage, triggering a significant inflammatory response known as "reperfusion injury" [2]. Ischemia-reperfusion injury (IRI) contributes to various pathological conditions, including abnormal levels of reactive oxygen species (ROS) and intracellular calcium ions, inflammatory responses, energy metabolism disorders, and mitochondrial dysfunction [3]. Consequently, it can lead to challenging-to-treat diseases such as acute myocardial infarction (AMI) [4], acute kidney injury (AKI) [5], cerebral vascular accident (CVA) [6], and hepatic insufficiency (HI) [7], all of which pose significant risks to human health and life.

  In recent years, new detection techniques have emerged to identify biomarker fluctuations associated with IRI. Traditional diagnostic methods, including enzyme-linked immunosorbent assay (ELISA) [8] and immunofluorescence (IF) [9], have been extensively developed with the combination of various biomarkers. However, these methods often rely on labor-intensive, expensive, and time-consuming operations, hindering the detection of real-time biomarkers. Moreover, nanoprobes and metabolomics based on small-molecule metabolites are limited in clinical diagnostics due to their reduced sensitivity, resulting in missed opportunities for timely therapy, as IRI are frequently inadequately diagnosed [10]. Therefore, there is an urgent need to develop efficient and accurate techniques for the early detection of IRI. This advancement could significantly improve cure rates, reduce patient suffering, and provide crucial time for developing appropriate treatments for both the injuries and their complications.

  Fluorescence imaging has gained significant attention as a rapidly developing detection technique due to its outstanding characteristics, including high selectivity, ease of use, and fast visualization [11]. However, traditional fluorescent probes, such as nanoprobes [12] and quantum dots [13], suffer from limitations like high toxicity to cells and tissues and low signal intensity in diagnosis, restricting their applications in bioimaging. In contrast, small-molecule fluorescent probes (SMFPs) offer practical benefits for both in vivo experiments and in vitro diagnosis due to their excellent biocompatibility, low toxicity, and sensitive signal output. Various fluorogenic recognition groups have been identified for real-time visualization of specific biomarkers in IRI models from cells to mice, such as ROS [14], amino acids [15], reactive nitrogen species (RNS) [16], ATP [17], and biothiols [18].

  At present, a multitude of SMFPs have been developed and evaluated for various single-organ IRI, enhancing our understanding of the injury process and offering potential candidates for clinical application. The absence of a comprehensive review on this topic could hinder further progress of fluorescent probes in bioimaging fields. This review (Fig. 1) addresses this gap by presenting an overview of typical single-organ IRI, highlighting their features and pathogenesis. This review introduces the response mechanisms and design strategies of SMFPs towards to specific fluctuating biomarkers in IRI, while also evaluating their performance in vivo. Additionally, it outlines future research directions and challenges based on the current understanding of the field. Beyond providing a reference for clinical insights into IRI, this review contributes to the advancement of our understanding of fluorescent probe design. We hope it will offer a comprehensive perspective that inspires further research into the use of SMFPs in disease diagnosis.

  Figure 1

  

  Figure 1.  Overview of characteristic features involved in IRI as well as fluorescence imaging and diagnosis (created with BioRender.com).

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  2. IRI

  2.1 General features of IRI

  Ischemia occurs when blood flow to the affected organ or tissue is interrupted, disrupting the supply of oxygen and nutrients required for normal metabolism. Lactic acid is produced through the breakdown of glycogen through increased anaerobic metabolism, leading to a decrease in cellular pH. The Na⁺/H⁺ exchanger (NHE) expels excess hydrogen ions to counteract this pH imbalance, causing a significant influx of sodium ions. The disruption of ion concentrations across the mitochondrial membrane reduces membrane potential and impairs ATP production [19]. Subsequently, cellular calcium overload is induced by ATP depletion through inhibition of active calcium efflux and restriction of calcium reuptake via the endoplasmic reticulum (ER) [20]. Furthermore, the shortage of ATP results in the dysfunction of ATP-dependent ionic pumps, such as the Na⁺/K⁺ and Ca²⁺ pumps, ultimately leading to the imbalance of the transmembrane ionic gradients. Potassium ions escape from the cell into the interstitial space to maintain ionic imbalance [21]. Calcium ions are discharged from the mitochondria into the extracellular regions, activating mitochondrial calcium-dependent cytosolic proteases, such as calpain, which evolves the cellular enzyme xanthine dehydrogenase into xanthine oxidase [22].

  During the reperfusion stage, the restoration of blood flow brings essential nutrients like glucose and free fatty acids, which are crucial for ATP production, and rapidly increases oxygen delivery, helping to normalize extracellular pH levels. This rapid pH normalization reverses the inhibition of mitochondrial permeability transition caused by low pH, potentially minimizing further ischemic damage to cells [23]. However, additional tissue injury occurs during this stage. Immediate normalization of extracellular pH triggers a significant influx of Na⁺ and robust Na⁺/H⁺ exchange due to a steep H⁺ gradient across the membrane. This unnatural gradient activates the reversed operation of the surface Na⁺/Ca²⁺ exchanger, leading to abnormal intracellular Ca²⁺ levels [24]. Furthermore, reoxygenation triggers ectopic xanthine not only oxidase via Ca²⁺-sensitive proteases, rapidly restoring aerobic metabolism but also degrading uric acid and thereby causing an excess production of ROS, particularly superoxide. Superoxide undergoes conversion into hydrogen peroxide (H₂O₂) and hydroxyl radicals (^•OH) [25]. The primary effect of hydroxyl radical formation is the peroxidation of cell membrane lipids, resulting in the synthesis and extensive release of proinflammatory eicosanoids. Excessive ROS production, including unstable hydroxyl radicals, can damage cellular structures and proteins, including those in the mitochondrial membrane [26]. Oxygen-derived ROS also intensifies the inflammatory response during reperfusion by generating oxidant-dependent proinflammatory substances, increasing the expression of cytokines/chemokines and production of adhesion molecules, and heightening cell susceptibility to death [27]. Moreover, excessive ROS in IRI can elevate dynamin-related protein 1 (DRP1) levels on the mitochondrial membrane surface, leading to increased mitochondrial breakdown, degradation of mitochondrial DNA (mtDNA), mitophagy, and ultimately cell death [28]. Understanding the pathological features of IRI (Fig. 2) in organs is crucial for developing treatments to reduce excess injury and improve outcomes. Further exploration of IRI pathology can deepen our understanding of the condition and guide the development of targeted therapies.

  Figure 2

  

  Figure 2.  Molecular mechanism of IRI. Two stages are involved in the IRI process, characterized by multiple cell pathways (created with BioRender.com).

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  2.2 Mechanisms underlying IRI

  Based on what has been discussed above, the total injury comprises the damage induced in both the ischemia and reperfusion stages. More importantly, the fluctuation of biomarkers varies abnormally in the reperfusion stage, continuing to cause cell death after the onset of reperfusion [29]. Therefore, understanding the mechanisms involved (Fig. 3) is crucial for developing bioimaging detection methods to assess the extent of injury caused by IRI and potentially prolong the therapeutic window before irreversible damage occurs in humans.

  Figure 3

  

  Figure 3.  Main mechanisms underlying IRI (created with BioRender.com).

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  2.2.1   Calcium overload

  In the ischemia stage, the accumulation of acidic products from anaerobic metabolism contributes to the decrease in intracellular pH. NHE in the cell membrane pumps H⁺ out to normalize pH levels and transports Na⁺ to maintain ion balance. Subsequently, the Na⁺/Ca²⁺ exchanger facilitates the exchange of surplus Na⁺ ions for Ca²⁺ ions. NHE function is significantly enhanced upon reperfusion stage due to rapid normalization of intracellular pH, leading to increased cytosolic Ca²⁺ levels. In addition to the continuous accumulation of cytosolic Ca²⁺ in IRI, mechanisms to restore Ca²⁺ to normal levels are severely impaired by IRI, especially in sarcoplasmic reticulum Ca²⁺-ATPase (SERCA). Due to ATP depletion and lack of ATP production, the Ca²⁺ storage ability of the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) is weakened, which triggers enhanced activation of ryanodine receptors (RyR) to release more Ca²⁺. Moreover, the reuptake ratio of Ca²⁺ into ER/SR through SERCA is lowered, contributing to cytosolic Ca²⁺ overload [30,31].

  Various pathological mechanisms that cause cell death can be activated by cytosolic Ca²⁺ overload. With the recovery of blood flow upon reperfusion, SERCA consumes more ATP due to its neighboring spatial position, which accelerates the cytoplasm-SR calcium cycle into a high-load state, resulting in calcium oscillations and ultimately affecting the entire cell. Furthermore, the activation of calpains due to Ca²⁺ overload not only hydrolyzes cytoskeletal proteins and ankyrin, causing the breakdown of cell structure but also degrades signal proteins such as protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinases (CaMKs), disrupting normal cell activities. Furthermore, as an inhibitor of calpains by reversibly binding, endogenous calpastatin is degraded in IRI, resulting in continuous damage to cells from Ca²⁺ overload, thereby contributing to cell death. Additionally, elevated intracellular Ca²⁺ levels produce complexes that attach to intracellular proteins, which facilitate the generation of cytokines, such as interleukin-1 beta and tumor necrosis factor-α. These cytokines transmit inflammatory signals to immune cells, stimulating the production of additional cytokines and chemokines, which exacerbates IRI [32,33].

  2.2.2   Oxidative/nitrosative stress

  The subsequent infusion of oxygenated blood into ischemic tissue, which is essential for the recovery of aerobic ATP production, contributes to the production of ROS. The release of ROS during reperfusion can oxidatively disrupt various cellular biomolecules, leading to cell dysfunction due to their high reactivity [34]. The cellular and systemic response to IRI is also influenced by RNS. These reactive species not only have adverse impacts on cellular macromolecules and disrupt cell signaling mechanisms by altering the redox state of cells or directly affecting specific signaling and effector systems but also interact with oxygen to produce highly reactive and potentially harmful nitric oxide (ONOO⁻) and other reactive nitrogen species (RNOS) [35,36].

  The adverse consequences of RNOS in IRI encompass the impairment or alteration of macromolecules, the initiation of endothelial and parenchymal cell death within the affected tissue, the stimulation of pro-inflammatory mediator production and release by various cells, the induction of adhesion molecules that facilitate adhesive interactions between leukocytes/lymphocytes-endothelial cells, and a reduction in the availability of protective nitric oxide (NO). During the initial stages of IRI, the predominant ROS generated is the superoxide anion radical (O₂^•−), which arises from the univalent reduction of molecular oxygen. Various cytosolic and membrane enzymes generate superoxide through the mitochondria's electron transport chain (ETC). Then, O₂^•− ion can convert rapidly and spontaneously into its conjugate acid, the hydroperoxyl radical (HOO^•), a more potent oxidant. This conversion process is accelerated, particularly in low pH conditions, as in ischemic tissue. While H₂O₂ exhibits lower reactivity than O₂^•−, it can quickly permeate cell membranes, serving as a secondary messenger and regulator of cellular signaling. Through the Fenton reaction, H₂O₂ can produce free radicals that are highly reactive, such as hydroxyl (^•OH). Additionally, it can react with hemoglobin and myoglobin, resulting in the formation of detrimental ferryl derivatives. Ultimately, O₂^•− can combine with NO to create peroxynitrite anion (ONOO⁻), which can subsequently undergo protonation to yield ONOOH, a powerful oxidizing agent [37–39].

  2.2.3   Mitochondrial dysfunction

  The burst of ROS in mitochondria causes the accumulation of oxidative stress, ultimately disrupting the balance between mitochondrial fission and fusion. Abnormal mitochondrial fission and fusion activities accelerate the extrinsic apoptotic cell death program under IRI conditions, leading to reduced concentrations of ATP and elevated release of mitochondrial ROS. During the ischemia stage, ATP synthase lacks the capacity to convert ADP to ATP because of the absence of electron flow in the anaerobic environment. Instead, the hydrolysis of ATP is promoted, leading to a substantial reduction in ATP levels. Enzymes on the outer mitochondrial membrane, such as monoamine oxidases (MAO-A and MAO-B), produce ROS by breaking neurotransmitters and dietary amines during the reperfusion phase, causing the progression of mitochondria dysfunction. Furthermore, activating the mitochondrial permeability transition pore (MPT) in the inner mitochondrial membrane is essential in initiating the cell death program during IRI. The MPT pore remains inactive during ischemia due to its sensitivity to low pH. In the occurrence of reperfusion, significant rises in mitochondrial Ca²⁺ levels and a burst of ROS activate the opening of the MPT pore. The pore becomes large, allowing H⁺ ions to move back into the matrix, thereby blocking ATP synthesis. As a result, water enters the mitochondria due to an osmotic gradient, leading to swelling and potential rupture of the mitochondria [40–43].

  2.2.4   Inflammation

  Inflammation plays a crucial role in protecting the body against harmful pathogens. When an infection occurs, immune cells such as macrophages and neutrophils are activated by specific signals to eliminate the invading organisms. These cells produce additional cytokines and chemokines that trigger adaptive immune responses and activate lymphocytes. Although IRI also induces inflammation, it is characterized as sterile inflammation because microorganisms do not generally cause it. In sterile inflammation caused by IRI, the release of various damage-associated molecular patterns (DAMPs), including versican, tenascin C, mtDNA, calreticulin and so on, from the injured regions stimulates the immune system. Most DAMPs function as signal molecules for pattern recognition receptors (PRRs), mainly reacting with Toll-like receptors (TLRs). The interaction between DAMPs and PRRs initially activates immune cell neutrophils, increasing their accumulation. Moreover, proinflammatory neutrophil genes are overexpressed, generating quantities of pro-inflammatory signals, such as cytokines and chemokines. These fluctuations of inflammatory signals begin to stimulate perivascular cells, such as macrophages and mast cells, followed by secreting additional inflammatory mediators. Leukocytes start to establish adhesive connections with the endothelium of postcapillary venules. Activated white blood cells migrate into the tissues and cause dysfunction in small blood vessels by releasing oxidizing agents and enzymes that break down molecules. Leukocytes contribute to the failure of postischemic nutritive perfusion, the dysfunction of endothelium-dependent vascular regulation in arterioles, and the dysfunction of parenchymal cells. Leukocyte/endothelial cell adhesive interactions are one of the initial indicators of tissue dysfunction caused by IRI, leading to dysfunction in arterioles, capillaries, and postcapillary venules in the microcirculation [44–46].

  3. Design strategies of SMFPs for IRI

  SMFPs are designed to meet these crucial requirements for effective disease detection and evaluation, including noticeable emission wavelength changes before and after the recognition of biomarkers to maximize optical resolution and good biocompatibility for the intended human applications. Properties such as cell permeability, low cytotoxicity, and adequate water solubility should also be considered. Furthermore, recognition groups on the fluorescent probes are modified to be reactive with specific biomarkers to achieve high sensitivity and selectivity. In the occurrence of IRI, due to the variance of organs, recognition groups are tailored to selectively interact with biomarkers relevant to achieve high sensitivity and specificity. Hepatic IRI is characterized by oxidative stress, inflammation, and cellular damage, often indicated by biomarkers such as ROS, glutathione (GSH) depletion, and elevated levels of enzymes like alanine aminotransferase (ALT). SMFPs designed for hepatic IRI incorporate recognition groups that specifically react with these biomarkers. For example, probes with boronate or aryl sulfide moieties are tailored to detect ROS, enabling fluorescence activation in the liver's oxidative stress-rich environment. Besides, these probes are designed to respond to changes in viscosity or pH in injured regions, yielding real-time imaging that help assess liver function and injury severity. Cerebral IRI involves oxidative stress, disrupted calcium homeostasis, and inflammation, with biomarkers including NO, ONOO⁻, and abnormal Ca²⁺ levels. SMFPs for cerebral IRI are developed to selectively detect these biomarkers within the brain's delicate environment. To ensure effective delivery across the blood-brain barrier (BBB), cerebral IRI probes are modified with lipophilic groups or attached to brain-targeting peptides. These structural modifications enable the probes to accumulate in the brain while maintaining low cytotoxicity to protect vulnerable neuronal tissues. Myocardial IRI is marked by oxidative stress, hypoxia, and inflammation, with key biomarkers including hypoxia-inducible factor-1 (HIF-1) and ROS. SMFPs targeting myocardial IRI are equipped with recognition groups for ROS, such as hydroxyl radicals, to highlight oxidative damage in cardiomyocytes. To specifically target injured myocardial tissues, SMFPs are often designed with mitochondria-localizing moieties like triphenylphosphonium (TPP) cations, which direct the probes to regions of mitochondrial dysfunction—a hallmark of myocardial IRI. These probes are engineered to exhibit high fluorescence intensity in viscous or hypoxic environments, enabling real-time visualization of myocardial injury and recovery. In practice, SMFPs apply various identification mechanisms (Figs. S1 and S2 in Supporting information), including twisted intramolecular charge transfer (TICT), intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), and restriction of intramolecular rotation (RIR) photoinduced electron transfer (PET), and excimer/exciplex formation. Detailed information can be seen in Supporting information.

  4. SMFPs for various potential biomarkers of HIRI

  4.1 Fluorescent probes for viscosity

  In the case of the HIRI process, oxidative stress disorder is frequently observed in injured liver tissues, which could lead to organelle dysfunction in livers and extensive liver damage. As mitochondria play a vital role in energy production, mitochondrial dysfunction causes abnormal metabolism with increased mitochondrial viscosity [47]. Lysosomal dysfunction is closely associated with the failure of macromolecule decomposition. In particular, the accumulation of macromolecules results in a significant increase in lysosomal viscosity level [48]. Therefore, viscosity can be considered a crucial biomarker for the real-time identification of HIRI tissues in the HIRI process. Dyes with donor-π-acceptor structures based on TICT mechanisms are considered to detect viscosity. The free intramolecular rotation of single bonds dissipates energy via non-radiative pathways in low-viscosity solvents. As viscosity increases, high viscosity enhances the rigidity of the planar structure, which strongly restricts the free intramolecular rotation of single bonds and impairs the formation of the TICT state, resulting in the release of energy in fluorescent form [49]. The vital properties are introduced in Table S1 (Supporting information).

  The TICT-based SMFP, X-V (Fig. 4), demonstrates a remarkably sensitive reaction to viscosity (21-fold) and a notable Stokes shift reaching up to 125 nm in cell models [50]. The positively charged indole group enabled the specific targeting labeling of mitochondria [51]. Another newly developed NIR-Ⅱ fluorescent probe, DPXBI (Fig. S3 in Supporting information), operates via an AIE-active mechanism. Improved by increasing the intermolecular distance and reducing intermolecular π-π stacking through the substitution from the benzoic acid segment to the diphenylxanthene segment in the electron acceptor part, it features a larger Stokes shift of 177 nm in both cell and mouse models [52]. Moreover, DPXBI introduces sulfonate groups into the benzindoles to achieve enhanced water solubility and biocompatibility [53]. Unlike TICT-type probes, the NIR-Ⅱ fluorescent probe based on the RIR mechanism, NP-V (Fig. S4 in Supporting information), possesses a sensitive response specific to lysosomal viscosity. Owing to its linkage between cyclohexene and benzindole through flexible conjugated bonds, NP-V exhibits susceptibility to viscosity fluctuations and showed a time-dependent enhancement of red fluorescence signal in both cell and mouse models [54].

  Figure 4

  

  Figure 4.  (a) Viscosity sensing mechanism of X-V. (b) FL spectra. (c) Real-time fluorescence imaging in IRI cell models. (d) Mean FL intensities of the control group. (e) Mean FL intensities of the HIRI group. Reproduced with permission [50]. Copyright 2023, Royal Society of Chemistry.

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  4.2 Fluorescent probes for mtDNA

  mtDNA plays a vital role in regulating cellular signals. Because of its unique structure, mtDNA is particularly susceptible to oxidative stress. In the HIRI process, excess ROS production damages mitochondria, releasing mtDNA into extracellular space. As mtDNA acts as a DAMP, the release activates the inflammation response, leading to severe liver injuries [55].

  The design idea for mtDNA detection is to fabricate suitable probe dimensions with mitochondrial targeting groups, which can accumulate in mitochondria and be inserted into the DNA groove. Due to DNA's spatial limitations, the intramolecular rotation is restricted, decreasing the nonradiative transfer of the excited state and consequently causing illumination. Following the approach above, mtDNA-BP (Fig. 5) is synthesized and demonstrates a strong fluorescence intensity at 580 nm upon DNA addition. Subsequently, as the IR injury in cell models advanced, fluorescence gradually decreased, indicating that the gradual deterioration of IR worsened the harm to mtDNA, ultimately leading to its destruction [55].

  Figure 5

  

  Figure 5.  (a) mt-DNA sensing mechanism of mtDNA-BP. (b) FL spectra. (c) Real-time fluorescence imaging in IRI cell models. (d) FL intensities of (c). Reproduced with permission [55]. Copyright 2021, American Chemical Society.

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  4.3 Fluorescent probes for mitochondrial H₂S (mtH₂S)

  The production of mtH₂S in the event of the HIRI process enhances the mitochondrial electron transfer process, which modulates neural signal transmission and maintains normal mitochondrial functions. Additionally, mtH₂S enhances energy production by interacting with ROS, providing protection effects on livers [56].

  A SMFP, Mito-GW (Fig. 6), features a d-π-A structure based on the TICT mechanism and displays a highly sensitive response to mtH₂S in HIRI cell and mice models. Advantageously, the Introduction of an aryl azide moiety coupled with a pyridinium cation moiety realizes the targeting mtH₂S monitor. As a robust electron-donating moiety, naphthylamine fluorophore is incorporated into the probe's design to improve its performance in fluorescence emission at a wavelength of 602 nm with a significant Stokes shift of 178 nm. By means of this fluorescent probe, the fluctuations of mtH₂S levels in both in vitro and in vivo HIRI models are effectively monitored [57].

  Figure 6

  

  Figure 6.  (a) mt-H₂S sensing mechanism of Mito-GW. (b) FL spectra. (c) Real-time fluorescence imaging in IRI cell models. (d) FL intensities for (c). Reproduced with permission [57]. Copyright 2024, Elsevier.

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  4.4 Fluorescent probes for reactive oxygen/nitrosative species

  The outburst of ROS in the injured liver tissue is considered a vital pathophysiological mechanism of the HIRI process. It breaks the balance of redox reactions and produces severe oxidative stress that targets biological molecules, such as proteins, lipids, and DNA, eventually causing cell death and tissue damage. The primary generated ROS, known as superoxide anion radicals (O₂^•−), contributes to subsequent ROS generation. H₂O₂ is widely recognized as the most stable and abundant species in ROS. The detection of fluctuations in these two typical ROS biomarkers reflects the degree of liver damage [40,58].

  Boronated groups are considered H₂O₂ moiety because they easily react with H₂O₂ [59]. Following this classic recognition group, a NIR fluorescent probe, Cy-ArB (Fig. 7), is proposed to detect fluctuations in H₂O₂ in vivo during HIRI. Heptamethine cyanine was selected as an NIR fluorophore due to its practical utility in mitochondrial tracing. The fluorescent probe exhibits a decrease in the fluorescence signal at 806 nm and an increase in the fluorescent signal at 758 nm in the emission spectrum with an extensive rise of fluorescence signal ratio in both cell and mouse models as the reperfusion process progressed longer [60]. Later, a NIR-Ⅱ fluorescent probe BX-BOH (Fig. S5 in Supporting information) is synthesized with the addition of a quarternized pyridyl group as an electron-pulling moiety and a xanthene derivative as an electron-pushing moiety, which shows an enhancement fluorescence intensity at 938 nm [61]. Distinct from the choice of boronated groups as a recognition group, a NIR-Ⅱ fluorescent probe, BTPE-NO₂ (Fig. S6 in Supporting information), featuring an aggregation-induced emission (AIE) mechanism, is developed. The introduction of two nitrophenyloxoacetamide groups on both ends of the benzothiadiazole core provides both reaction sites for H₂O₂ and improvement of fluorescence intensity around 950–1200 nm due to strong electron-withdrawn ability. Notably, the probe enables high selectivity towards H₂O₂ in RAW264.7 cells and livers, revealing the potential application of benzothiadiazole-core molecules as NIR-Ⅱ fluorescent imaging agents in diseases [62].

  Figure 7

  

  Figure 7.  (a) H₂O₂ sensing mechanism of Cy-ArB. (b) FL spectra. (c) Fl spectra towards H₂O₂. (d) Real-time fluorescence imaging in IRI cell models. (e) FL intensities in the control group. (f) FL intensities in the HIRI group. (g) In vivo imaging of endogenous H₂O₂. (h, i) Mean ratios of (g). Reproduced with permission [60]. Copyright 2019, Royal Society of Chemistry.

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  Han and his coworkers developed a ratiometric near-infrared (NIR) fluorescent probe, Mito-Cy-Tfs (Fig. 8), to assess alterations in mitochondrial O₂^•− levels in vitro and in vivo during HIR. Three main components are included in the probe: the NIR fluorophore heptamethine cyanine, the response unit tri-fluoromethanesulfonamide, and the lipophilic TPP cation as the mitochondrial targeting moiety. Such a design strategy enables the probe to rapidly respond to O₂^•− within 3 min and successfully monitor the fluctuation of O₂^•− in RH-35 cells and mice livers in the event of HIRI [63]. In contrast to the recognition groups designed in Mito-Cy-Tfs, Tang et al. proposed a two-photon ratio fluorescent probe, CCA (Fig. S7 in Supporting information), that incorporates caffeic acid as a recognition group to detect the fluctuations of O₂^•− and an l-cysteine group to target Golgi apparatus where contains multiple reaction regions for cysteine. With a strong blue fluorescence enhancement, this probe successfully monitors the increased level of O₂^•− in the Golgi apparatus in the HIRI group. Moreover, researchers further applied CCA to evaluate the association among tumor necrosis factor-α, O₂^•− and caspase-2, proposing an underlying mechanism in which fluctuation of Golgi O₂^•− mediates the signal transduction pathway in the IR process [64]. Based on the same recognition group, another two-photon fluorescence ratio probe, CST (Fig. S8 in Supporting information), combined with mitochondrial targeting groups is developed to detect mitochondrial O₂^•− in real-time accurately [65]. They also developed a fluorescent probe DPC (Fig. S9 in Supporting information) with a rational ER-targeting design [66]. The emission wavelength of these probes demonstrates a similar range of around 430–530 nm with slight variations resulting from the choice of different organelle-targeting functional groups, affecting each probe's electron attraction ability [64–66]. To achieve a dramatic signal-to-noise ratio (130-fold) and rapid response (within 10 s) for the detection of ONOO⁻, Rhod-CN-B (Fig. S10 in Supporting information) is designed by incorporating a strong electron withdrawn group (−CH=(CN)₂) at the 2′ position of the rhodol scaffold. The probe's performance in both cell models and mouse models proves that the probe can be a powerful tool for revealing the impact of endogenous ONOO⁻ on apoptosis during the HIRI process [67].

  Figure 8

  

  Figure 8.  (a) O₂^•− sensing mechanism of Mito-Cy-Tfs. (b) FL spectra. (c) FL spectra. (d) Ratio images of cell models (d). (e) FL intensities of groups a and b. (g) In vivo imaging of mice. (h, i) FL intensities of the control group and HIRI group. Reproduced with permission [63]. Copyright 2017, Elsevier.

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  A novel dual fluorescent probe CyCA (Fig. 9) is developed to monitor real-time fluctuations in mitochondrial O₂^•−/ONOO⁻ levels directly. The design strategy for the dual-selective recognition of O₂^•−/ONOO⁻ was established by employing the caffeic acid group for targeting O₂^•− and the Cy5 group as the recognition group for ONOO⁻. These two recognition groups are connected by a piperazinyl linker. In addition, the Cy5 moiety possessed mitochondria-targeting properties due to its positive charge, which attracts negative charges in the mitochondrial membrane. Furthermore, TP microscopy allows for the achievement of separate blue and red dual-channel fluorescence signals by employing the excitation wavelength of 800 nm. The assistance of this probe enables the identification of O₂^•−-ONOO⁻-arginase 1-mediated IRI signaling pathway. Moreover, three reaction sites in arginase 1 where tyrosine nitration occurs are also investigated, which explains a deactivation mechanism in HIRI [68]. Based on the same O₂^•− recognition group, another newly dual fluorescent probe UDP (Fig. S11 in Supporting information) displayed simultaneous imaging of O₂^•− and ATP in separate blue and red fluorescence channels without any interference between each of them by replacing Cy5 moiety with rhodamine lactam skeleton. The hepatocyte imaging results demonstrate that UDP could monitor the concurrent dynamic fluctuations of endogenous O₂^•− and ATP. Furthermore, the utilization of UDP in HIRI mouse livers investigates the intracellular O₂^•−-SDH-Mito NADH-Mito ATP-intracellular ATP cascade mediated signaling pathways in HIRI, establishing relationships between various biomarkers and active molecules in the occurrence of HIRI [69].

  Figure 9

  

  Figure 9.  (a) O₂^•− and ONOO⁻ sensing mechanism of CyCA. (b) One-photon FL spectra of O₂^•−. (c) One-photon FL spectra of ONOO⁻. (d) In vivo 3D images of normal and IR mice liver for blue and red fluorescence imaging. (e) FL intensities of (a). (f) FL intensities of (b). Reproduced with permission [68]. Copyright 2019, Elsevier.

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  5. SMFPs for various potential biomarkers of CIRI

  5.1 Fluorescent probes for biothiol

  As vital members in RSS, sulfane sulfur, which include persulfates (R-S-SH), hydrogen polysulfides (H₂S_n, n ≥ 1), and protein-bound elemental sulfur (S₈), are highly reactive due to the presence of sulfur atoms in the thiosulfoxide form. At the onset of ischemia, the production of sulfane sulfur is based on reactions between endogenous H₂S and ROS. Mitochondria are closely related to the redox reactions to supply energy, contributing to the generation of sulfate sulfur. Therefore, the dynamic level of mitochondrial sulfane sulfur could indicate the pathological status of the tissues [70–73].

  A novel fluorescent probe, Mito-SH (Fig. 10), is developed for the real-time detection of sulfane sulfur concentrations in vitro and in vivo. The probe mainly consists of three parts: (1) The thiophenol group functions as a recognition group to selectively react with RSS. (2) The aza-BODIPY group provides an enhanced NIR fluorescence signal. (3) The lipophilic TPP cation facilitates the accumulation of the probe in mitochondria. When the probe was incubated with various cell models and mouse models under normal conditions, minimal fluorescent signals were displayed. On the contrary, Mito-SH exhibited a significant fluorescent enhancement at 723 nm in the CIRI group, showing the burst of Na₂S₄ was induced. Moreover, the protective effects of sulfane sulfur in response to oxidative stress via reacting with ROS could be investigated by the further application of Mito-SH to detect dynamic fluctuations of sulfane sulfur [74]. Yang et al. introduces a novel NIR fluorescent probe, NIR-HMPC, designed for the specific and sensitive detection of thiol fluxes in physiological and pathological conditions in CIRI. The probe operates through a thiol-triggered chromene "click" reaction, enabling self-immolative release of a fluorescent signal. Key features include high selectivity, rapid response, and excellent biocompatibility, which demonstrates the probe's application for in vitro imaging in living cells and in vivo tracking in mouse models, effectively visualizing thiol fluctuations during CIRI [75].

  Figure 10

  

  Figure 10.  (a) Sulfane sulfur sensing mechanism of Mito-SH. (b) FL spectra. (c) Confocal fluorescence images of cell models. Group A (cultured with DMEM/RPMI 1640/MEM containing 25 mmol/L glucose for 3 h). Group B (cultured with glucose-free DMEM under 0.1% O₂ for 3 h). Group C (pretreated with 2DG for 3 h and then cultured under 0.1% O₂). (d) Normalized FL intensities of IR mice. (e) The analysis of hippocampal slices in the IR mice models. Reproduced with permission [74]. Copyright 2018, The Royal Society of Chemistry.

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  GSH is a small molecule biothiol contributing to various pathological processes such as enzyme reactions, amino acid transport, and DNA synthesis. In addition to other vital functions of GSH, it serves as a dynamic biomolecule to keep redox homeostasis based on the self-regulate mechanism during the occurrence of the cerebral ischemia-reperfusion process. The fluctuation level of GSH reflects the CIRI-damaged brain region. Cysteine is the precursor in GSH production and the end product in the homocysteine (Hcy) breakdown, which plays a crucial role in protein synthesis, metabolism, detoxification, and other physiological functions. Studying the variation of GSH and Cys is vital for comprehending the physiological and pathological processes involved in the IRI process [76–78].

  Two NIR fluorescent probes, BCy-SeSe and BCy-SS (Fig. 11), are investigated for accurately visualizing mitochondrial GSH in living cells and in vivo in the occurrence of CIRI. The probes comprised the recently developed NIR fluorophore BCy-Keto and GSH targeting groups such as diselenide or disulfide. BCy-Keto displays strong fluorescence at a longer wavelength, specifically 728 nm, due to the extension of π-electron conjugated structure. Furthermore, positive charges inside the molecule effectively facilitate the accumulation of the probe in mitochondria due to the attraction effect from negative charges in the mitochondrial membrane. The GSH levels in the left and right cerebral hemispheres were found to be different from those in the sham operation, which indicates that mitochondrial GSH levels have a close relationship to cell apoptosis and ROS levels [79]. Later, they proposed the NIR fluorescence probe BCy-AC (Fig. S12 in Supporting information) based on their previous work, which involved a distinct Enol-Keto tautomerization process to enhance the sensitivity in identifying Cys in CIRI [80]. Then, Yang and his colleagues introduced a novel NIR SMFP, DCI-Ac-Py (Fig. S13 in Supporting information), to investigate the biothiol-mediated nuclear factor kappa-B (NF-κB) pathway in CIRI within H22 and PC12 living cells and mouse levels. Calculations of the BBB permeability coefficients demonstrate that the probe can efficiently penetrate the BBB. Due to the employment of pyridinate for recognition and isoflurone derivative for fluorescence, when it reacts with biothiols, an in-situ cascade reaction is triggered, resulting in a remarkably sensitive turn-on signal with minimal interference. The fluorophore obtained by the reaction between DCI-Ac-Py and biothiol, exhibits a noticeable emission at 713 nm, featuring a large Stokes' shift of 172 nm [81].

  Figure 11

  

  Figure 11.  (a) GSH sensing mechanism of BCY-SeSe and BCY-SS. (b) FL spectra. (c) Confocal fluorescence imaging in cell models. (d) The histograms show FCA for ΔΨ_m of different cell lines by JC-1. (e) Imaging in IRI mice models. (f) FL intensities of adult mice. Reproduced with permission [79]. Copyright 2019, American Chemical Society.

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  5.2 Fluorescent probes for malondialdehyde

  Lysosomal is particularly sensitive to CIRI-induced oxidative stress. Lysosomal malondialdehyde (MDA), a typical product from the peroxidation reaction on lysosomal lipids, is toxic and highly reactive, easily breaking the integrity structures of lysosomal proteins. It can serve as a suitable biomarker for detecting CIRI-induced extensive damage [82,83].

  A powerful fluorescence imaging probe, Lyso-MCBH (Fig. 12), designed for visualizing lysosomal MDA in vivo, is proposed by Su et al., which could accumulate in the lysosome attributed to the introduction of the morpholine group. In the presence of lysosome MDA, the quenched effect from the MDA recognition hydrazide group is terminated through its reaction with MDA, resulting in the formation of the coumarin fluorophore and fluorescence emission at 520 nm. Notably, the fluorescence intensity of the IR group significantly diminished after pretreatment with an MDA scavenger, which proves the probe's sensitivity and selectivity. Furthermore, by application of this probe, excessive lysosomal MDA was found to affect the effectiveness of vitamin B₁₂ in adjusting fluctuations of Hcy level in CIRI as it acts as a barrier to prevent the transportation of vitamin B₁₂ from the lysosome to the cytoplasm [84].

  Figure 12

  

  Figure 12.  (a) MDA sensing mechanism of Lyso-MCBH. (b) FL spectra. (c) Two-photon fluorescence imaging cell models. (d) FL intensities in (c). (e) Two-photon 3D fluorescence imaging in mice models. (f) FL intensities in (e). Reproduced with permission [84]. Copyright 2023, American Chemical Society.

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  5.3 Fluorescent probes for ROS

  Since the brain consumes a significant proportion of oxygen supply in the body to maintain energy production, it is susceptible to increased oxidation levels. As a vital ROS with a strong connection to oxidative stress, H₂O₂ easily accumulates due to redox imbalance, leading to a tremendous burden on the brain tissue and ultimately increasing apoptosis rates [35–37].

  A novel fluorescent probe, H₂O₂-CL-510 (Fig. 13) is developed for the first sensitive detection of CIRI-induced H₂O₂ in a living rat. The introduction of phenoxy-dioxetane as a skeleton that can specifically react with H₂O₂. Upon the addition of H₂O₂, salicylaldehyde functions as a recognition group and oxides into a catechol, following the subsequent ether cleavage to deprotect the phenoxy-dioxetane. The phenoxy-dioxetane ultimately undergoes chemiexcitation, resulting in the release of chemiluminescence. The imaging results from CIRI live rats induced by the middle cerebral artery occlusion (MCAO) method demonstrate that CIRI effectively triggered an outburst of H₂O₂ in the rat brain, which could be visualized through the probe [85]. Another novel two-photon H₂O₂-sensitive probe, PCAB (Fig. S14 in Supporting information), is evaluated in the OGD-treated SH-SY5Y cells and CIRI mouse models. Incorporation of pyridazine into its structure benefits as the pharmacophore of the histamine H₃ receptor agonist is practicable to the specific needs of brain-directed requirements. Moreover, modification of N² position substituent enhances the ability of the scaffold to emit fluorescence and selectively react to H₂O₂ [86].

  Figure 13

  

  Figure 13.  (a) H₂O₂ sensing mechanism of H₂O_2—CL-510. (b) FL spectra. (c) In vivo chemiluminescent imaging in mice models. (d) Quantifications of photon fluxes released in mice models. (e) Quantifications of FL intensities. Reproduced with permission [85]. Copyright 2020, Wiley-VCH.

  DownLoad: Full-Size Img PowerPoint

  Later, a novel dual mitochondria-targeting fluorescent probe following a molecular rotor strategy, Mito-IQS (Fig. 14), for separate visualization of the viscosity and H₂O₂ is investigated. The quinoline cation possesses the capacity to both locate mitochondria and exhibit lipophilicity. Additionally, trimethylene glycol monomethyl ether, which has excellent amphiphilicity, further enhances the lipophilicity of the probe, as sufficient lipophilicity is an essential theoretical basis for the passive diffusion of molecules through the BBB. By incorporating distinct recognition groups upon mitochondrial viscosity and H₂O₂, a discerning reaction can be detected through various color channels (viscosity: red channel, λ_em = 670 nm; H₂O₂: yellow channel, λ_em = 570 nm) during CIRI-treated SH-SY5Y cells and mouse models [87].

  Figure 14

  

  Figure 14.  (a) H₂O₂ and viscosity sensing mechanism of Mito-IQS. (b) FL spectra with viscosity. (c) FL spectra with H₂O₂. (d) Confocal fluorescence images in cell models. (e, f) Red and yellow FL intensities of (b). Reproduced with permission [87]. Copyright 2024, American Chemical Society.

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  6. Conclusion and prospects

  This review introduces the fundamental concept, typical features, and mechanisms of single-organ IRI. In addition, a comprehensive analysis and summary of recent advancements in SMFPs that specifically target fluctuations in various biomarkers during IRI are provided, which covers the basic structures and applicable mechanisms of probes in vitro and in vivo models. It can be discovered that significant progress has been made in developing fluorescent probes for biological imaging in recent years. Using probes for real-time visualization of distinctive biomarkers provides vital pathophysiological information about IRI. This imaging technique is non-invasive, highly sensitive, and provides real-time results, significantly improving over traditional clinical methods [88]. With the assistance of these novel fluorescent probes to detect fluctuations in viscosity, ROS, biothiol, and so on, profound insights into pathological mechanisms of IRI, such as overexpressed molecules, redox imbalance, and enhanced activation or inactivation of enzymes are revealed, which provides valuable guidance for drugs design strategy and treatments for IRI in clinical applications. Although significant progress has been achieved, fluorescent probes possess inherent limitations: (1) Fabrication of fluorescent probes with outstanding properties, including high sensitivity and selectivity, fast response, and specific targeting damage regions, remains a challenge as the treatment of IRI requires the early and precise diagnosis of damaged tissues [89]. (2) Most activable fluorescent probes discussed above follow a turn-on characteristic under confocal microscopy, which lacks ratiometric fluorescent probes to achieve a higher quantitative detection. Besides, most fluorescent probes are designed with ICT or TICT response mechanisms. There is a noticeable absence of PET-type or FRET-type fluorescent probes with low background signals [90]. (3) Many fluorescent probes above only confirm valid optical results in cell models, making it challenging to ensure their effectiveness in clinical applications [91].

  Promising strategies for SMFPs design. Appropriate hydrophilic functional groups should be modified into the structure to improve the properties of fluorescent probes. For example, introducing hydroxyl or sulfonic acid groups enhances the water solubility of the molecule, making it more biocompatible. The choice of fluorophores with distinctive electron attraction ability improves the fluorescence emission. Incorporating two or more recognition groups with contrasting or similar reactivity into the structure is vital for dual detection. The development of dual-responsive fluorescent probes relies on exploring fluorophores with multiple binding sites and recognition groups with specificity and stability. Moreover, most fluorescent probes undergo an irreversible reaction with the target biomarkers, significantly restricting the acquisition of valuable information towards IRI. Reversible probes demonstrate the ability to detect cyclic structures to provide additional details to illustrate the progression of IRI.

  Design SMFPs with innovative designs to improve fluorescent signals in IRI. The majority of fluorogenic derivatives yield fluorescence signals that are located within the visible range of 400–650 nm. These molecules cannot penetrate deeply, which restricts their implementation in bioimaging within complex biological environments [92]. Investigating fluorescence imaging in the NIR and NIR-Ⅱ regions can enhance the capabilities of fluorescent probes to provide more precise fluorescence signals. The fluorescence intensity could be improved by combining other mechanisms and detection methods, such as constructing ratiometric probes based on FRET or RIR via two-photo imaging. Other advancements in microscope methods such as super-resolution imaging (suitable for observation of microstructures in cells), persistent luminescence imaging (ideal for long time monitoring), photoacoustic imaging (provide high-resolution figures for deep tissue), ultrasound imaging (low cost and simple operation) and X-ray imaging (provide clear image for specific tissue regions) should be considered and investigated to maintain higher standard imaging results. Design SMFPs for fluctuations of various biomarkers to investigate the pathological mechanisms in IRI. Most biomarkers in IRI are ROS-related or biothiol. Other characteristic biomarkers, such as oxidative stress biomarkers (MDA), cell apoptosis biomarkers (mtDNA), and inflammation biomarkers (CRP), deserve to be investigated. Besides, most fluorescent probes target HIRI and CIRI, neglecting other standard IRIs. It is indisputable that the design of appropriate probes is challenging due to the characteristics of other organ IRI. Hence, it is crucial to consider appropriate target groups and modify physical characteristics such as lipid solubility, molecular rigidity, molecular size, and hydrogen bonding capability based on various organs' specific environments and metabolic processes. In addition to the fundamental research discussed, transitioning from cell models to mouse models presents a critical next step in the development and application of SMFPs. This shift is essential for gaining deeper insights into the in vivo behavior of these probes, which is crucial for their potential diagnostic applications in human patients. Such experiments can elucidate the pharmacokinetics, biodistribution, and metabolic stability of the probes, providing valuable data to inform their design and optimization.

  Furthermore, a focused future direction for these probes should emphasize their role in advancing our understanding of the pathogenesis of IRI and related diseases. By utilizing these probes to monitor real-time changes in relevant biomarkers within animal models, researchers can better delineate the mechanisms underlying tissue damage and recovery during IRI. This knowledge can facilitate the identification of new therapeutic targets and the development of more effective interventions.

  Moreover, clinical applications of these probes should be a primary consideration in future research efforts. As we refine their design for increased specificity and sensitivity, it is vital to explore how these probes can be integrated into clinical diagnostics and patient management strategies. This may involve collaborations with clinical researchers to validate the probes' efficacy in detecting IRI and related conditions in diverse patient populations.

  In summary, while the extension of experiments from cell models to mouse models is a necessary step, the future direction of SMFPs should prioritize their contributions to understanding disease mechanisms and their potential clinical applications. By focusing on these areas, we can enhance the translational impact of these probes and improve patient outcomes in the context of IRI and other related diseases.

  This review concisely overviews single-organ IRI, emphasizing their distinctive features, mechanisms, and associated biomarkers. Then, we introduce the structure, response mechanism, and in vivo experimentation of SMFPs to detect dynamic changes in biomarkers in IRI. Moreover, it presents further prospective directions for research and obstacles with current knowledge considered. We anticipate that this review will serve as a guide for inspiring a comprehensive investigation of SMFPs for potential clinical applications in the future.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Qingyuan Guo: Writing – review & editing, Writing – original draft, Visualization. Aojie Liu: Writing – original draft, Visualization. Yinghui Huang: Investigation, Conceptualization. Jiayu Ding: Investigation. Junjie Ding: Investigation. Limin Wang: Supervision, Investigation. Yang Ding: Visualization, Supervision. Bo Peng: Visualization, Supervision. Lin Li: Validation, Investigation. Bin Fang: Writing – review & editing, Writing – original draft, Supervision, Investigation, Conceptualization. Shan Jiang: Writing – review & editing, Supervision, Investigation. Hua Bai: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

  Acknowledgments

  This work was financially supported by the National Natural Science Foundation of China (Nos. 62288102 and 62475216), Natural Science Foundation of Shaanxi Province (No. 2022JM-130), Key Research and Development Program of Shaanxi (No. 2024GH-ZDXM-37), Innovation Capability Support Program of Shaanxi (No. 2023-CX-PT-23), Natural Science Basic Research Program of Shaanxi (No. 2024JC-YBQN-0919), Fujian Provincial Natural Science Foundation of China (No. 2024J01060), National High Level Hospital Clinical Research Funding (No. 2023-NHLHCRF-YSPY-01), the Postdoctoral Fellowship Program of CPSF (No. GZC20240889), Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University (No. CX2023098), and Fundamental Research Funds for the Central Universities.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.110943.

  
  
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  Figure 1  Overview of characteristic features involved in IRI as well as fluorescence imaging and diagnosis (created with BioRender.com).

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  Figure 2  Molecular mechanism of IRI. Two stages are involved in the IRI process, characterized by multiple cell pathways (created with BioRender.com).

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  Figure 3  Main mechanisms underlying IRI (created with BioRender.com).

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  Figure 4  (a) Viscosity sensing mechanism of X-V. (b) FL spectra. (c) Real-time fluorescence imaging in IRI cell models. (d) Mean FL intensities of the control group. (e) Mean FL intensities of the HIRI group. Reproduced with permission [50]. Copyright 2023, Royal Society of Chemistry.

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  Figure 5  (a) mt-DNA sensing mechanism of mtDNA-BP. (b) FL spectra. (c) Real-time fluorescence imaging in IRI cell models. (d) FL intensities of (c). Reproduced with permission [55]. Copyright 2021, American Chemical Society.

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  Figure 6  (a) mt-H₂S sensing mechanism of Mito-GW. (b) FL spectra. (c) Real-time fluorescence imaging in IRI cell models. (d) FL intensities for (c). Reproduced with permission [57]. Copyright 2024, Elsevier.

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  Figure 7  (a) H₂O₂ sensing mechanism of Cy-ArB. (b) FL spectra. (c) Fl spectra towards H₂O₂. (d) Real-time fluorescence imaging in IRI cell models. (e) FL intensities in the control group. (f) FL intensities in the HIRI group. (g) In vivo imaging of endogenous H₂O₂. (h, i) Mean ratios of (g). Reproduced with permission [60]. Copyright 2019, Royal Society of Chemistry.

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  Figure 8  (a) O₂^•− sensing mechanism of Mito-Cy-Tfs. (b) FL spectra. (c) FL spectra. (d) Ratio images of cell models (d). (e) FL intensities of groups a and b. (g) In vivo imaging of mice. (h, i) FL intensities of the control group and HIRI group. Reproduced with permission [63]. Copyright 2017, Elsevier.

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  Figure 9  (a) O₂^•− and ONOO⁻ sensing mechanism of CyCA. (b) One-photon FL spectra of O₂^•−. (c) One-photon FL spectra of ONOO⁻. (d) In vivo 3D images of normal and IR mice liver for blue and red fluorescence imaging. (e) FL intensities of (a). (f) FL intensities of (b). Reproduced with permission [68]. Copyright 2019, Elsevier.

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  Figure 10  (a) Sulfane sulfur sensing mechanism of Mito-SH. (b) FL spectra. (c) Confocal fluorescence images of cell models. Group A (cultured with DMEM/RPMI 1640/MEM containing 25 mmol/L glucose for 3 h). Group B (cultured with glucose-free DMEM under 0.1% O₂ for 3 h). Group C (pretreated with 2DG for 3 h and then cultured under 0.1% O₂). (d) Normalized FL intensities of IR mice. (e) The analysis of hippocampal slices in the IR mice models. Reproduced with permission [74]. Copyright 2018, The Royal Society of Chemistry.

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  Figure 11  (a) GSH sensing mechanism of BCY-SeSe and BCY-SS. (b) FL spectra. (c) Confocal fluorescence imaging in cell models. (d) The histograms show FCA for ΔΨ_m of different cell lines by JC-1. (e) Imaging in IRI mice models. (f) FL intensities of adult mice. Reproduced with permission [79]. Copyright 2019, American Chemical Society.

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  Figure 12  (a) MDA sensing mechanism of Lyso-MCBH. (b) FL spectra. (c) Two-photon fluorescence imaging cell models. (d) FL intensities in (c). (e) Two-photon 3D fluorescence imaging in mice models. (f) FL intensities in (e). Reproduced with permission [84]. Copyright 2023, American Chemical Society.

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  Figure 13  (a) H₂O₂ sensing mechanism of H₂O_2—CL-510. (b) FL spectra. (c) In vivo chemiluminescent imaging in mice models. (d) Quantifications of photon fluxes released in mice models. (e) Quantifications of FL intensities. Reproduced with permission [85]. Copyright 2020, Wiley-VCH.

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  Figure 14  (a) H₂O₂ and viscosity sensing mechanism of Mito-IQS. (b) FL spectra with viscosity. (c) FL spectra with H₂O₂. (d) Confocal fluorescence images in cell models. (e, f) Red and yellow FL intensities of (b). Reproduced with permission [87]. Copyright 2024, American Chemical Society.

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