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Construction and Theranostic Applications of Dual-Locked Fluorescent Probes

Yu Gao , Yinuo Liu , Dugang Chen

Progress in Chemistry ›› 2026, Vol. 38 ›› Issue (9) : 1636 -1649.

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Progress in Chemistry ›› 2026, Vol. 38 ›› Issue (9) : 1636-1649 DOI: 10.7536/PC20260210
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Construction and Theranostic Applications of Dual-Locked Fluorescent Probes
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Abstract

As an emerging molecular imaging technology， fluorescent probes have gained widespread attention in biomedical research due to their advantages of non-invasiveness， high sensitivity， and high spatiotemporal resolution. However， conventional fluorescent probes activated by a single analyte often suffer from false-positive signals due to the insufficient specificity of the target analyte in pathological environments， presenting significant limitations for disease diagnosis and treatment. In recent years， dual-locked fluorescent probes based on“AND”logic gate design have gradually become a research hotspot. These probes can simultaneously respond to two distinct biomarkers in pathological environments， becoming activated only when both are present. This design significantly reduces false-positive interference and markedly improves imaging specificity and signal-to-noise ratio， providing a powerful tool for high-fidelity analysis of complex pathological environments， early and precise disease diagnosis， and personalized treatment. This article systematically reviews the design strategies and recent research progress（over the past three years）of dual-locked fluorescent probes. It focuses on categorizing and discussing them based on the types of activating biomolecules， covering various systems including small biomolecules activation， macromolecules activation， and combined small-molecule and macromolecule activation. Additionally， it summarizes the typical applications of these probes in biomolecular detection and disease theranostics， analyzes the key limitations in current development， discusses the main directions requiring breakthroughs in future research， and concludes with a perspective on their clinical translation potential and development trends.

Contents

1 Introduction

2 Design strategies of dual-locked fluorescent probes

2.1 Intramolecular charge transfer

2.2 Photoinduced electron transfer

2.3 Fluorescence resonance energy transfer

3 Dual-locked fluorescent probes responsive to different biomolecules

3.1 Dual-locked fluorescent probes activated by small biomolecules

3.2 Dual-locked fluorescent probes activated by macromolecules

3.3 Dual-locked fluorescent probes activated sequentially by a small molecule and a macromolecule

4 Theranostic applications of dual-locked fluorescent probes

4.1 Liver diseases

4.2 Neurodegenerative diseases

4.3 Cardiovascular diseases

4.4 Tumors

5 Conclusion and outlook

Graphical abstract

Keywords

fluorescent probes / sensing / detection / diagnosis / therapy

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Yu Gao, Yinuo Liu, Dugang Chen. Construction and Theranostic Applications of Dual-Locked Fluorescent Probes. Progress in Chemistry, 2026, 38(9): 1636-1649 DOI:10.7536/PC20260210

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1 Introduction

Abnormal physiological states or pathological processes are often accompanied by aberrant expression and abnormal interactions of various active substances within the internal environment of the organism. Molecular imaging techniques enable the monitoring of levels of different biological factors or disease-related active molecules, thereby effectively evaluating physiological and pathological activities in living bodies[1]. Among existing molecular imaging technologies, fluorescence imaging has become a highly promising tool in both fundamental research and clinical applications due to its high spatiotemporal resolution, non-invasiveness, and real-time detection capabilities[2-3]. Fluorescent probes serve as the core functional units of fluorescence imaging technology. Based on differences in fluorophore materials, they can be classified into inorganic fluorescent probes and organic fluorescent probes[4]. Inorganic fluorescent probes typically utilize inorganic materials such as single-walled carbon nanotubes, quantum dots, and rare-earth-doped nanoparticles as fluorescent units, and have been extensively studied over the past few decades[5-7]. However, such probes still pose significant concerns regarding biocompatibility and biosafety, which limits their application in the biomedical field[8]. In contrast, organic fluorescent probes generally exhibit superior biocompatibility and offer greater flexibility in molecular structure design and functional regulation, thus demonstrating more significant application potential[9]. Utilizing organic fluorescent probe molecules, researchers have achieved visualization of various disease biomarkers, including reactive oxygen species (ROS), reactive sulfur species (RSS), enzymes, and gas molecules, providing crucial support for precise detection of biomolecules, elucidation of pathological signaling pathways, and early diagnosis and targeted therapy of diseases[10-13].
Organic fluorescent probes can be classified into two types: always-on and turn-on.[14-15]Always-on probes maintain their fluorescence state before and after target recognition. Such probes are typically modified with active targeting peptides or loaded into nanocarriers, allowing them to accumulate actively or passively at disease sites, forming a concentration gradient with normal tissues to achieve detection of diseased tissues.[16-18]However, this strategy still cannot avoid the non-specific distribution of probes in healthy tissues, leading to high background signals and low signal-to-background ratios (SBR), which significantly affects detection accuracy.[19]To overcome these limitations, researchers have developed turn-on fluorescent probes based on biomarkers for major diseases such as tumors and cardiovascular/cerebrovascular disorders.[20-22]These probes are typically prepared by covalently linking a responsive group with a fluorophore. Their fluorescence signal is activated only upon interaction with specific analytes, thereby significantly reducing background signals, improving the signal-to-noise ratio, and achieving high-fidelity imaging of the target components.[23]However, most current turn-on probes are single-lock fluorescent probes. The activation of such probes requires input from only one biomarker; therefore, they can only reflect changes in a single analyte within biological systems and cannot provide sufficient information regarding complex changes in biological components in vivo. Although some researchers have attempted to combine multiple single-response probes to achieve simultaneous detection of various biomarkers, differences in the physical properties and diffusion characteristics of different probe molecules lead to uneven distribution within the body, thereby affecting imaging reliability.[24]Furthermore, biomarkers may also be expressed at certain levels in non-target cells or healthy tissues. In such cases, traditional single-lock fluorescent probes may be non-specifically activated, generating false-positive signals that seriously compromise the accuracy of imaging results.[25]To overcome these limitations, researchers exploited the extremely low probability of co-expression of two disease biomarkers in healthy tissues to develop dual-lock fluorescent probes.[26]Such probes typically possess two independent responsive sites, and their fluorescence signal is activated only upon concurrent activation by two biomarkers. This dual-locking mechanism can significantly enhance the specificity of probe molecules and reduce off-target signals.[27]Moreover, the two responsive sites of dual-lock probes enable simultaneous detection of two biomarkers, helping to elucidate interactions between analytes in complex physiological or pathological environments, thus providing a powerful tool for research on disease-related signaling pathways. Beyond achieving accurate detection of disease biomarkers and precise diagnosis of diseases using dual-lock probes, this technology has also demonstrated significant potential in the field of personalized therapy. Currently, researchers have developed doubly activatable chemotherapy prodrugs, photosensitizers, and photothermal agents based on the dual-lock strategy. These smart agents are activated only in specific pathological environments, thereby significantly reducing toxic side effects on healthy tissues and enhancing therapeutic safety.[28-30].
In recent years, dual-lock fluorescent probes have become a research hotspot in the field of precision medicine due to their multiple advantages, including elucidating the mechanisms of interactions between biomolecules during disease pathology, reducing off-target interference from single-target imaging, enhancing imaging accuracy, and enabling targeted therapy for diseased tissues. Currently, existing reviews mostly focus on discussions from perspectives such as similar biomarkers or response sequences, while targeted analysis of design strategies and response mechanisms for dual-lock probes remains insufficient.[31-33]. Therefore, this article first systematically outlines the basic design concepts of dual-lock fluorescent probes starting from their response mechanisms. It then focuses on elaborating the types, performance, and design strategies of dual-lock fluorescent probes activated by different biomolecules over the past three years, followed by showcasing their diagnostic and therapeutic applications in various disease fields. Finally, the limitations, potential breakthrough directions, and future development prospects of current dual-lock fluorescent probe development are discussed and prospected.

2 Design strategy of dual-lock fluorescent probes

Fluorescent probes typically consist of two parts: a fluorophore and a recognition group. Among these, fluorophores possess easily modifiable molecular structures and tunable fluorescent properties. Common fluorophores include Coumarin, Naphthalimide, Fluorescein, Rhodamine, Methylene blue, Resorufin, BODIPY, and Cyanine dyes (Figure 1A). Through rational modification of these fluorophores, their emission wavelengths can cover the visible to near-infrared (NIR) range. The NIR region includes two bands: Near-Infrared I (NIR-I, 700~900 nm) and Near-Infrared II (NIR-II, 1000~1700 nm), which can meet the needs of different application scenarios.[34-42]The recognition group exhibits high specificity towards the target substance. Upon reaction with the marker, it induces changes in the conjugated structure or push-pull electronic properties of the probe molecule, thereby modulating the fluorescent signal and enhancing the probe's anti-interference capability and detection accuracy in complex physiological environments.[43-44]Therefore, the clever modification of the recognition group onto the fluorophore is a critical step determining the probe's function, directly affecting its sensitivity, selectivity, and response characteristics. To achieve the rational construction of more types of dual-lock probes, it is essential to systematically understand the compatibility between different response mechanisms and fluorophores. This section will overview the response mechanisms of common fluorophores and analyze the structure-function compatibility between different recognition strategies and commonly used fluorophores, aiming to provide references for designing high-performance probes targeting complex biological systems.

2.1 Intramolecular charge transfer

The Intramolecular Charge Transfer (ICT) effect refers to the process where electrons within a probe molecule migrate from an electron donor (D) to an electron acceptor (A) upon photoexcitation; such probes typically possess a characteristic push-pull (D-A) electronic structure.[45]. When designing fluorescent probes based on the ICT mechanism, recognition groups are often modified at the donor or acceptor sites. By regulating the electron push/pull capabilities of the donor/acceptor, this induces a blue shift in the probe's emission spectrum or fluorescence quenching (Figure 1B)[46]. This strategy is widely applied in classic fluorophores such as resorufin, methylene blue, and hemicyanine. Through rational design, the construction of dual-lock probes can be achieved relying solely on this mechanism. For example, as shown in Figure 1C, the Pβgal-MAO-Aprobe can achieve sequential response to two different biomarkers simply by connecting recognition groups responsive to each biomarker sequentially to its electron-donating group site.[47].

2.2 Photoinduced electron transfer

Photoinduced electron transfer (PeT) refers to the process of electron transfer occurring within a molecule under photoexcitation. Depending on the direction of electron transfer, it can be distinguished into acceptor-excited PeT (a-PeT) and donor-excited PeT (d-PeT).[48]In the PeT mechanism, the highest occupied molecular orbital (HOMO) or the lowest unoccupied molecular orbital (LUMO) of the recognition group interferes with the excited-state electron transition from HOMO to LUMO within the fluorophore, or inhibits the radiative relaxation of excited-state electrons from LUMO to the ground state, thereby causing fluorescence quenching.[49]This mechanism has been widely applied in the design of responsive probes based on fluorescent scaffolds such as BODIPY, naphthalimide, and cyanine. In such probes, the recognition group typically possesses electron push-pull capabilities. Upon interaction with the target analyte, the redox potential of the recognition group changes, affecting the electron transfer process between the fluorophore and the recognition group. This leads to the blocking of the PeT effect and the restoration of the fluorescence signal, thereby achieving specific detection of the target biomarker (Figure 1B)[50]When designing dual-lock probes, constructing dual-lock fluorescent probes can be achieved by relying solely on the PeT effect or by synergizing it with the ICT effect, as shown in Figure 1Cfor probes ACy-H-NTR and QN-Naph.[51-52].

2.3 Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is a non-radiative energy transfer process based on dipole-dipole interactions. Its occurrence requires good overlap between the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor, and the distance between the energy donor and the energy acceptor must be 10–100 Å (1 Å = 0.1 nm).[53]. Based on the FRET principle, connecting two spectrally matched fluorophores can construct ratiometric fluorescent probes. Such probes possess a self-calibration mechanism that effectively eliminates signal fluctuations caused by differences in probe concentration.[54]. Furthermore, combining fluorophores with compatible quenchers can be used to construct turn-on fluorescent probes, further expanding design strategies for fluorescent probes. When constructing dual-lock fluorescent probes using FRET, if optimization of spectral matching between the quencher and the fluorophore is not involved, the key to the design lies in the linker structure connecting the quencher and the fluorophore. If only a single linker is used, the quencher typically needs to be coupled with a probe having single responsiveness to achieve the construction of a dual-lock probe, as shown by the probe BBQ650-SS-BTZHC-E (Figure 1C)[55]. If two linkers are used to connect the quencher and the fluorophore, the realization of their dual-response characteristics will rely entirely on regulating the distance between them (i.e., controlling the effective distance for FRET occurrence), as shown by the probe PBM (Figure 1C)[56].

3 Dual-lock fluorescent probes activated by different biomarkers

Normal physiological processes rely on the precise regulation of various bioactive components, whose concentration levels maintain a dynamic equilibrium within the organism. Dual-lock fluorescent probes can specifically respond to two different biomarkers; this unique advantage significantly enhances their capability for dynamically monitoring the interactive behaviors of active molecules during complex physiological processes, thereby promoting an in-depth analysis of intracellular signal coordination mechanisms. This section elaborates on dual-lock fluorescent probes activated by different biomarkers, starting from their design strategies and response mechanisms.

3.1 Dual-lock fluorescent probes activated by small biomolecules

3.1.1 ROS and RNS

ROS and reactive nitrogen species (RNS) are two important oxidants in the human body, which can be produced through various endogenous or exogenous processes. Common ROS and RNS include superoxide anion (O2·-), hydroxyl radical (OH·), singlet oxygen (1O2), hydrogen peroxide (H2O2), hypochlorous acid (HClO), peroxynitrite (ONOO-), etc.[57-59]. Under normal physiological conditions, the level of oxidants in the body forms a dynamic balance with reducing substances RSS. Oxidative stress occurs when the levels of ROS and RNS abnormally increase or when RSS is excessively consumed.[60-62]. At this time, excessive oxidants cause damage to biomolecules such as deoxyribose, nucleic acids, and proteins in the body, disrupt signal transduction pathways, and ultimately induce various diseases.[63-64]. Therefore, using fluorescence imaging to visually monitor the distribution and changes of ROS and RNS in the body is of great significance for deeply understanding the redox changes of diseases and for early disease diagnosis. Liu et al.[65]designed and synthesized a dual-lock probe HA-P3 based on the 4-hydroxy-1,8-naphthalimide fluorescence scaffold for the detection of HClO and ONOO- (Figure 2). Diethyl dithiocarbamate and 1,4-dihydroxybenzene were grafted sequentially onto the hydroxyl sites of the probe's naphthalene ring, serving as recognition groups for HClO and ONOO-, respectively. In the initial state, an a-PeT effect occurs between the two electron-rich recognition groups and the naphthalimide, inhibiting the ICT effect of the fluorophore and keeping the fluorescence in a quenched state. When HClO and ONOO- are present sequentially, the recognition groups are specifically oxidized and removed, thereby unmasking the hydroxyl groups on the naphthalene ring, relieving the a-PeT inhibition, and forming a strong ICT between the hydroxyl and imide structures, ultimately observing significant fluorescence recovery at 550 nm. The detection limits of HA-P3 for HClO and ONOO- are 56 nmol/L and 63 nmol/L, respectively, and the probe exhibits a good linear response within the concentration ranges of 0~4 μmol/L for HClO and 0~15 μmol/L for ONOO-. Using this probe, differentiation between normal liver cells and liver cancer cells can be achieved, and changes in HClO and ONOO- levels can be monitored in drug-induced liver injury models in cells, zebrafish, and mice.

3.1.2 ROS and RSS

The occurrence of oxidative stress is not only related to the excessive generation of ROS but also closely associated with abnormal RSS metabolism[66]. Therefore, accurately assessing the redox state requires evaluation from both ROS and RSS perspectives. As a common RSS in the body, sulfur dioxide (SO2) usually exists in plasma in the ionic forms of sulfite (SO32-) and bisulfite (HSO3-), playing a key role in maintaining cellular homeostasis and regulating redox state[67-69]. Fang et al.[70]designed and synthesized a dual-lock fluorescent probe BN-DUAL using benzopyran-chromone as the fluorescent scaffold for the simultaneous detection of hydrogen peroxide (H2O2) and SO2 (Figure 3). In the initial state, due to a strong intramolecular ICT effect, the probe exhibited pink fluorescence emission. When SO2is present, SO2undergoes a nucleophilic reaction with BN-DUAL, disrupting the conjugation between malononitrile and benzopyran, leading to the blockade of the ICT process and subsequent fluorescence quenching. The generated intermediate BN-SO2further reacts with H2O2, undergoing structural rearrangement to form the product BN; at this point, the ICT effect is restored along with the recovery of conjugation. Since the original strong electron-withdrawing group malononitrile is replaced by an aldehyde group with weaker electron-withdrawing ability, the fluorescence emission of BN exhibits a significant blue shift (>100 nm) compared to BN-DUAL, presenting yellow fluorescence, which effectively avoids spectral crosstalk. BN-DUAL possesses a strict sequential response mechanism; fluorescence is activated only when SO2and H2O2are added sequentially, and the detection limits for SO2and H2O2are 0.36 μmol/L and 0.21 μmol/L, respectively. In cell experiments, the probe successfully monitored the dynamic changes of exogenous and endogenous SO32-and H2O2. In further in vivo experiments, visual differentiation of SO32-and H2O2can also be achieved through changes in pink and yellow dual-channel signals.

3.2 Dual-lock fluorescent probes activated by biomacromolecules

3.2.1 Leucine aminopeptidase and nitroreductase

Leucine aminopeptidase (LAP) is an important proteolytic enzyme that specifically cleaves N-terminal leucine residues in substrates, and its dysregulated expression is closely associated with cancer cell proliferation.[71]Furthermore, the rapid proliferation of tumor cells often causes the local oxygen consumption rate to exceed blood supply capacity, thereby creating a tumor hypoxic environment that induces the overexpression of the hypoxia-specific enzyme nitroreductase (NTR).[72]Based on this pathological feature, Chen et al.[73]designed and prepared a NIR-I dual-lock fluorescent probe, Si-NTR-LAP, using silicon-based rhodamine as the fluorophore, which is activated by both NTR and LAP (Figure 4). The researchers sequentially connected the LAP recognition group (L-leucine) and the NTR recognition group (4-nitrophenyl chloroformate) to the aniline site of the fluorophore via amide condensation reactions. Acylation of the amino group weakens its electron-donating ability, reducing the ICT effect; additionally, the 4-nitrophenyl ester interacts with the fluorophore through a d-PeT mechanism, jointly achieving fluorescence quenching. This probe can be sequentially activated only when both NTR and LAP are concurrently overexpressed in tumor cells, thereby restoring the NIR fluorescence signal. In two-dimensional imaging studies of cancer cells co-cultured with normal cells, Si-NTR-LAP effectively distinguished tumor cells from normal cells, with a tumor-to-normal cell fluorescence intensity ratio (T/N) of 3.1-fold, significantly higher than that of the single-lock probes Si-LAP (1.2-fold) and Si-NTR (1.6-fold). In both subcutaneous and orthotopic liver cancer mouse models, the probe achieved high-contrast imaging of tumor tissues, accurately delineating the boundaries between tumor and normal tissues, with imaging results highly consistent with pathological analysis.

3.2.2 Gamma-glutamyl transferase and caspase-1

Pyroptosis is a form of programmed cell death that has demonstrated unique potential in the field of tumor therapy. By inducing pyroptosis in tumor cells via specific drugs, it is possible not only to directly kill tumor cells but also to significantly alter the tumor microenvironment and activate systemic anti-tumor immune responses, offering hope to overcome challenges such as apoptosis resistance and immunosuppression existing in traditional treatments.[74-76]. However, the regulatory mechanisms of pyroptosis are not yet fully elucidated, and there is a lack of means for real-time and precise assessment of dynamic changes within tumors, which limits the in-depth understanding of pyroptosis therapeutic effects and efficacy monitoring. To address these challenges, Wang et al.[77]selected the key pyroptosis protease Caspase-1 and γ-glutamyltransferase (GGT), which is overexpressed on tumor cell membranes, as biomarkers. Using a hemicyanine dye with a typical D-A structure as the fluorescent skeleton, they designed and constructed a dual-locked tandem activation-type NIR-I fluorescent probe, DTAP (Figure 5). At the phenolic hydroxyl site of the fluorophore, this probe sequentially connects a Caspase-1 responsive peptide (N-acetyl-Tyr-Val-Ala-Asp-OH) and a GGT responsive unit (N-Glu-γ-OH). This structure blocks the ICT process, achieving efficient fluorescence quenching. To further improve the biocompatibility of the probe and reduce systemic toxicity, researchers introduced a polyethylene glycol chain (PEG2k) at the indole salt site via click chemistry. This modification significantly optimized the pharmacokinetic properties of the probe, shortening its serum half-life to 1.6 min and achieving approximately 61% renal clearance within 24 h, demonstrating favorable metabolic safety characteristics. In in vivo applications, the probe restores its fluorescence signal only after being sequentially cleaved by Caspase-1 and GGT within the tumor microenvironment. This dual-enzyme sequential activation mechanism enables DTAP to specifically identify pyroptosis processes within tumors, effectively avoiding false-positive signals generated by pyroptosis activity in normal tissues.

3.2.3 Caspase-1 and NS3 protease

In addition to tumors, viruses are also one of the major threats facing global public health[78]. Viral infections typically trigger host antiviral immune responses to clear pathogens; however, some viruses can evade immune surveillance and hijack immune cells, converting them into reservoirs for their own replication and spread, thereby weakening host defense mechanisms[79]. As key immune cells with phagocytic functions, macrophages participate in pathogen clearance but also face the risk of viral infection, and in certain cases, may even assist in viral persistence and dissemination[80-81]. Currently common detection methods (such as flow cytometry and immunostaining) are mostly invasive and difficult to achieve real-time dynamic monitoring in vivo; therefore, there is an urgent need to develop non-invasive imaging methods to elucidate the dynamic process of macrophage polarization after viral infection and its exact role in viral dissemination[82]. To address this challenge, Yu et al.[83]developed two activatable NIR-I fluorescent probes, ZIP and ZIMP, for broad-spectrum detection of Zika virus (ZIKV) and specific recognition of ZIKV-infected macrophages, respectively (Figure 6). In terms of molecular design, ZIP is constructed using hemicyanine (CyNH2) as the fluorophore, linked with a peptide segment specifically recognized by the ZIKV NS3 protease, which is a key enzyme for viral protein processing and replication. ZIMP is based on the ZIP structure, further incorporating a Caspase-1-specific peptide substrate highly expressed in macrophages, forming a dual-enzyme sequential activation mechanism, thereby achieving highly specific imaging of infected macrophages. This probe can not only be used for in vivo NIR fluorescence imaging but also serves as a novel tool for flow cytometry, enabling specific identification and sorting of M1-type macrophages infected with ZIKV in tissues such as lymph nodes. Leveraging this imaging system, researchers revealed important pathological processes of ZIKV infection: viral infection first induces macrophage polarization toward the M1 phenotype in lymph nodes, and subsequently, these infected M1 macrophages may act as carriers, mediating viral migration and spread to brain tissue. This study provides an important dynamic visualization tool for understanding the interaction between viruses and immune cells.

3.3 Dual-lock fluorescent probe co-activated by small and macromolecules

3.3.1 ROS and enzymes

ROS plays an indispensable role in maintaining human life activities, extensively participating in various cellular signaling pathways and pathological processes. In tumor cells, the Warburg effect (i.e., aerobic glycolysis) leads to mitochondrial metabolic disorder and dysfunction, subsequently causing excessive accumulation of ROS[84]. As a highly aggressive malignant tumor, melanoma exhibits significantly upregulated expression levels of tyrosinase (Tyr), which is involved in melanin synthesis within tumor cells and is closely related to disease progression[85-86]. Against this pathological background, Guo et al.[87]designed and synthesized a dual-locked fluorescent probe DR using resorufin as the fluorophore for detecting H2O2and Tyr in melanoma cells. The probe was constructed by sequentially modifying the 7-position hydroxyl group of resorufin with benzyl bromide pinacol boronate and 3-hydroxybenzyl alcohol. The probe first reacts with H2O2, triggering the cleavage of the boronate ester bond, followed by the removal of the benzyl group via a 1,6-rearrangement-elimination reaction to expose the active site for Tyr. Tyr further catalyzes the 4-position hydroxylation of the 3-hydroxyphenyl moiety, initiating another 1,6-rearrangement-elimination, ultimately releasing the phenolic hydroxyl group that was blocking the fluorophore. After dissociation of the phenolic hydroxyl group, the intramolecular charge transfer effect of the fluorophore is restored, thereby turning on fluorescence and achieving approximately a 56-fold fluorescence enhancement in solution. Further studies demonstrated that DR can accurately distinguish between normal cells and melanoma cells, and has been successfully applied to in vivo melanoma imaging, enabling dynamic monitoring of tumor growth and early lung metastasis processes.
Unlike the design strategy mentioned above that connects two response modules sequentially, Lü et al.[88]proposed a more sophisticated integrated design concept, fusing the H2O2and Tyr recognition units into a single reaction module, thereby simplifying the synthesis steps. In this design, the phenylboronic acid unit, commonly used for responding to ROS, was adjusted from conventional para-substitution to meta-substitution and modified onto a hemicyanine fluorescent skeleton to construct the dual-lock probe m-BA-Hcy (Figure 7).
This probe successfully achieved cascade activation of H2O2 and Tyr. Meta-phenylboronic acid is oxidized by H2O2 to generate a meta-phenolic hydroxyl intermediate. Unlike ortho- or para-substituted groups that readily undergo spontaneous elimination, this meta-phenolic hydroxyl structure is relatively stable and can be further recognized and catalyzed by Tyr. Subsequently, it releases ortho-hydroxybenzoquinone via spontaneous tautomerization, thereby activating the hemicyanine fluorophore. m-BA-Hcy exhibits high sensitivity towards both H2O2 and Tyr, with detection limits of 120 nmol/L and 0.73 U/mL (1 U = 1 μmol/min), respectively. m-BA-Hcy can respond to endogenous H2O2 and Tyr in zebrafish, and achieves differentiation between melanoma and liver cancer at both cellular and mouse levels.

3.3.2 RSS and enzymes

As an important class of RSS in the body, GSH plays a key role in maintaining cellular redox homeostasis. During tumor initiation and progression, to cope with persistent oxidative stress and support rapid proliferation, tumor cells typically significantly upregulate GSH levels.[89]. Therefore, high intracellular expression of GSH has become a universal and hallmark metabolic feature in the tumor microenvironment (TME). Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor; studies have shown that LAP is upregulated in HCC tissues and is often used as a biomarker for HCC.[90]. To achieve detection of the two tumor markers GSH and LAP, Xia et al.[91], based on a hemicyanine skeleton, adopted a "stepwise optimization" strategy to design and synthesize PCLT, a dual-locked NIR-I probe with sequential response to GSH and LAP. The turning on and off of the probe's fluorescence is achieved by regulating ICT. First, the LAP recognition group (L-leucine amide) is connected to the phenolic hydroxyl group via a self-immolative linker (4-aminobenzyl alcohol), caging the electron-donating phenol group. Subsequently, the GSH-responsive group (2,4-dinitrobenzenesulfonate) is grafted onto the amino group of the L-leucine amide via an amide bond, further blocking the LAP recognition site to form a sequential recognition unit. Finally, using an azide-alkyne cycloaddition reaction, an azide-modified PEG chain (N3-TEG-OH) is connected to the probe, significantly improving its water solubility. In in vivo imaging, PCLT demonstrated strong specificity for HCC tissues, effectively avoiding interference from overexpressed LAP in alcoholic liver injury (ALI), showing its potential application in precise HCC detection. Cathepsin B (Cat B) is another overexpressed enzymatic biomarker in HCC cells, highly correlated with tumor progression, angiogenesis, and metastasis.[92]. Xu et al.[93], based on the nitrile-aminothiol (NAT) click chemistry reaction (where nitriles react with 1,2-aminothiols to form thiazolines), designed and prepared CyNAp-SS-FK, a NIR-I dual-locked fluorescent probe specifically responsive to GSH and Cat B (Figure 8). The researchers replaced the phenolic hydroxyl group on the oxacycle with a cyano group, a strong electron-withdrawing group. Utilizing the strong electron-withdrawing ability of the cyano group, the ICT process of the fluorophore was blocked, quenching its fluorescence emission. Unlike the dual-lock design strategy achieved by modifying electron-donating groups (phenol or aniline) and electron-withdrawing groups (indole benzene ring) on hemicyanine, the two responsive groups of this probe were modified on the PEG chain at the N-position of the indole salt. The GSH-responsive S-S and the Cat B-cleavable peptide (AcFK) were grafted onto the thiol and amino groups of the aminothiol, respectively, to block the orthogonal reaction between the amino and thiol groups with the cyano group. When both Cat B and GSH are present, the two capping groups in the probe CyNAP-SS-FK are removed, exposing the amino and thiol heads. These undergo intramolecular cyclization via the NAT click reaction, converting the cyano group into a thiazole structure with weaker electron-withdrawing ability, thereby relieving the ICT restriction and restoring NIR fluorescence emission. Simultaneously, the intramolecularly cyclized probe can self-assemble to form nano-aggregates. These aggregates can significantly prolong the retention time of the probe at the tumor site, thus overcoming the limitation of traditional small-molecule probes being rapidly cleared from the lesion area. In a mouse orthotopic HCC model, CyNAp-SS-FK achieved detection of micro-tumor lesions with a diameter of only 2 mm, indicating its excellent detection sensitivity for Cat B and GSH.

4 Therapeutic and diagnostic applications of dual-lock fluorescent probes

4.1 Liver disease

Liver diseases such as nonalcoholic fatty liver disease (NAFLD), drug-induced liver injury, hepatitis, and cirrhosis have become a major global health challenge due to their high incidence.[94-96]. The clinical diagnosis of NAFLD primarily relies on liver biopsy, an invasive method whose subjective histological assessment may lead to misdiagnosis. The non-invasiveness and high sensitivity of fluorescence imaging can effectively overcome these limitations. A typical pathological feature of NAFLD is the excessive accumulation of lipids within hepatocytes, a process often accompanied by increased cellular viscosity. Furthermore, hepatocytes in NAFLD patients exhibit overexpression of heme oxygenase-1, an enzyme associated with the production of carbon monoxide (CO); consequently, CO concentrations are correspondingly elevated in NAFLD lesions. Han et al.[97]innovatively designated CO as another biomarker for NAFLD and designed and synthesized a NIR-I dual-lock fluorescent probe (HCO-9) co-activated by CO and viscosity (Figure 9A). The lactam aromatic hydrazone group and the rotatable carbon-carbon double bond (C=C) were designed as recognition sites for CO and viscosity, respectively. Fluorescence signal activation occurs only when CO undergoes electrophilic addition with the amide bond (C=N) and the intramolecular rotation of C=C is inhibited by viscosity. In cellular experiments, HCO-9 responded to endogenous or exogenous CO and high-viscosity environments. At the living organism level, upon in situ injection of HCO-9 into the livers of NAFLD mouse models, significantly enhanced fluorescence signals appeared in the liver regions of NAFLD mice, whereas normal mice exhibited only weak fluorescence. In ex vivo organ imaging, fluorescence signals were primarily concentrated in liver tissue, indicating that this probe enables visualization of CO content and viscosity in the livers of NAFLD mice (Figure 9B).

4.2 Neurodegenerative diseases

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases[98]. Due to the diversity and insidious nature of AD inducing factors, patients usually present obvious symptoms only at advanced stages. Traditional clinical detection methods suffer from high costs and insufficient accuracy, whereas optical imaging technology based on fluorescent probes holds promise for advancing AD treatment thanks to its high sensitivity and selectivity. Compared with normal brains, the level of butyrylcholinesterase (BChE), a neurotransmitter hydrolase, in the brains of AD patients increases by 900%[99]. Meanwhile, studies have shown upregulation of myeloperoxidase (MPO) and 3-chlorotyrosine levels associated with hypochlorous acid in the brains of neurological disorders, indicating that oxidative stress and abnormal enzyme activity also play important roles in the progression of AD[100]. In response to this pathological feature, Guo et al.[101]designed and synthesized a two-photon dual-lock fluorescent probe (S1) responsive to HClO and BChE using 4-methylumbelliferone as the fluorophore (Figure 10A). In cellular experiments, S1 successfully achieved dynamic monitoring of changes in HClO and BChE activity and could effectively distinguish between normal cells and AD model cells. More importantly, the probe exhibits good blood-brain barrier penetration capability; after tail vein injection, it achieved two-photon imaging at a depth of 117 μm within the mouse brain. Its signal intensity was significantly higher in AD model brains than in the normal group, partially overlapping with Aβ plaque distribution, revealing its potential for in vivo visualization of the spatial correlation between HClO/BChE and AD pathology (Figure 10B).

4.3 Cardiovascular disease

Cardiovascular diseases (CVDs) primarily affect heart and brain function, exhibiting high mortality and disability rates, and represent one of the most severe threats to human health. Among these, atherosclerosis (AS) is a major factor leading to CVDs.[102]. The main component of atherosclerotic plaques in the arterial wall is foam cells, which are formed by the transformation of macrophages or vascular smooth muscle cells through the phagocytosis of oxidized low-density lipoprotein (ox-LDLs).[103]. Excessive phagocytosis of ox-LDLs leads to abnormal accumulation of lipid droplets (LDs); therefore, LDs are often used as biomarkers for identifying AS.[104]. Furthermore, overexpressed ONOO- in foam cells is also an important pathological factor in the progression of atherosclerosis; thus, ONOO- can serve as another biomarker for identifying atherosclerotic plaques.[105]. Sang et al.[106]designed a dual-lock fluorescent probe, CNN2-B, activated by an ONOO- and LD sequential activation mechanism (Figure 11A). This probe possesses unique subcellular localization migration characteristics: when unactivated, it localizes to mitochondria; upon activation, it shifts from mitochondrial targeting to lipid droplet enrichment. The dynamic changes in its fluorescence intensity and spatial location facilitate more intuitive confirmation of intracellular pathological activities. In a mouse carotid artery plaque model, after administering CNN2-B via in situ patching or local spraying, the fluorescence signal rapidly and specifically enriched at the plaque sites, clearly outlining the contour and extent of the plaques, while simultaneously reflecting lipid accumulation and oxidative stress levels within the plaques, forming a sharp contrast with healthy vessel walls (Figure 11B). These results confirm that the CNN2-B probe exhibits specificity for plaques in vivo, indicating the significant potential of dual-lock fluorescent probes based on ONOO- and LD sequential activation for the precise diagnosis of AS.

4.4 Tumor

As a highly invasive and lethal disease, the incidence and mortality rates of cancer continue to rise. Precise diagnosis and treatment of tumors are key to improving patient survival rates, but this process still faces numerous challenges.[107]. Xu et al.[93]developed a dual-lock fluorescent probe (CyNAp-SS-FK) co-activated by GSH and Cat B (Figure 12A). In LM3 human liver cancer cells, CyNAp-SS-FK can be activated by endogenous GSH and Cat B; when cells were pretreated with a GSH inhibitor, the fluorescence intensity decreased by 74%. In an orthotopic liver cancer mouse model, when the tumor had been implanted for 5 days with a diameter of only 2 mm, CyNAp-SS-FK achieved precise tumor detection with a 5-fold SBR, and the imaging window exceeded 36 h (Figure 12B). In contrast, the clinically commonly used detection methods for Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) require waiting until 15 days after tumor transplantation, when the tumor diameter reaches 6.5 mm, to detect a significant rise in indicators. These results indicate that CyNAp-SS-FK not only achieves early and sensitive detection of HCC but also provides superior imaging contrast and a longer detection window.
Drug therapy is another tumor treatment option besides surgical resection; however, the off-target effects of traditional chemotherapeutic drugs in the body lead to severe toxic side effects, and multidrug resistance in tumors also severely limits their efficacy.[108-109]. Amonafide (ANF) is a naphthalimide-based small-molecule anticancer drug with the potential to overcome multidrug resistance, but its clinical application is limited by significant side effects.[110]. To address this issue, Cheng et al.[111]developed an ANF prodrug, AcKLP, based on a dual-locking strategy to achieve efficient and low-toxicity treatment for glioblastoma (GBM). ANF can only be cascadingly activated by histone deacetylase (HDAC) and cathepsin L (CTSL), which are overexpressed in GBM cells, thereby significantly reducing toxicity to normal cells. The half-maximal inhibitory concentration (IC50) of AcKLP for U87 glioblastoma cells is 2.26 μmol/L, while the IC50 for normal human umbilical vein endothelial cells is > 100 μmol/L, demonstrating excellent tumor selectivity. In both 3D cellular tumor spheroid models and U87 xenograft mouse models, AcKLP exhibited significant antitumor activity with a tumor inhibition rate of approximately 72.4% and low systemic toxicity.
Photodynamic therapy (PDT) is an anti-tumor strategy that utilizes light to activate photosensitizers to generate cytotoxic reactive oxygen species (ROS) for killing tumor cells. Due to its advantages such as non-invasiveness, spatiotemporal controllability, and extremely low drug resistance, it has been extensively studied.[112]. However, the continuous activation state of photosensitizers can still lead to systemic side effects, such as skin sensitization, swelling, and organ damage. To overcome this limitation, Zhan et al.[113]prepared a pH/GSH dual-responsive and endoplasmic reticulum-targeting nano-photosensitizer, CySD@POD, to achieve activable Type I PDT and photothermal therapy (PTT). This probe remains in an "off" state in normal tissues, where fluorescence is quenched and the generation of ROS and heat is suppressed. The nanoparticles are specifically activated only under the acidic pH and high concentration of GSH in the tumor microenvironment, which triggers the recovery of NIR fluorescence and the efficacy of Type I PDT and PTT. Furthermore, its endoplasmic reticulum-targeting unit can precisely localize photoactivity to the endoplasmic reticulum, enhancing endoplasmic reticulum stress and protein inactivation, thereby improving the killing effect on hypoxic tumors. In vitro experiments demonstrated that CySD@POD exhibits significant phototoxicity against tumor cells while showing extremely low toxicity to normal cells. In vivo studies indicated that these nanoparticles efficiently accumulate at tumor sites, significantly inhibit tumor growth under NIR laser irradiation, and possess low systemic toxicity.

5 Conclusion and Outlook

This article systematically summarizes the progress of typical dual-lock fluorescent probes over the past three years and their applications in disease diagnosis and treatment, providing an in-depth analysis from the perspectives of molecular design strategies and response mechanisms. Dual-lock fluorescent probes possess the capability to simultaneously detect two biomarkers, offering powerful tool support for deeply analyzing biological signaling pathways, revealing disease progression mechanisms, and enabling early disease diagnosis. However, related research still faces the following major challenges: constructing a dual-lock structure on a single fluorophore requires balancing fluorescence quenching mechanisms with the covalent linkage of dual-response groups, significantly increasing the difficulty of molecular design and synthesis; the introduction of two response groups often leads to increased probe molecular weight, which may affect cellular permeability and increase potential biological toxicity; the recognition processes at dual recognition sites may interfere with each other, thereby reducing probe sensitivity and response rates; tandem probes typically have strict requirements for activation sequences, limiting their application flexibility in diverse biological environments; some small-molecule probes lack sufficient targeting specificity and are prone to non-specific uptake by the liver, affecting their in vivo distribution and efficacy; furthermore, the clinical translation of dual-lock probes remains extremely limited, with only a very few probes successfully applied to clinical sample detection. To address these challenges, future design of dual-lock probes should focus on the following directions: utilizing nanocarriers to construct composite probe systems to enhance targeting specificity, biocompatibility, and metabolic performance while reducing non-target tissue uptake; introducing high-specificity targeting ligands to minimize off-target effects; selecting biomarkers with greater disease specificity and functional groups with higher response efficiency; developing multimodal imaging fusion strategies, such as chemiluminescence imaging, phosphorescence imaging, photoacoustic imaging, magnetic resonance imaging, and ultrasound imaging, to compensate for the limitations of single-mode fluorescence imaging in biological applications; to advance the clinical translation and practical application of dual-lock fluorescent probes, rigorous characterization of their pharmacokinetic parameters and comprehensive toxicological analysis are required.
Furthermore, the application potential of dual-lock fluorescent probes in the biomedical field remains to be deeply explored. Most currently reported dual-lock probes primarily focus on cancer diagnosis and prognosis assessment, with only a few developed for fluorescence-guided surgery or therapy. Notably, the co-activation mechanism of two biomarkers by dual-lock probes can effectively avoid false positives caused by single biomarkers, significantly reduce interference from background signals in normal tissues, and improve the accuracy and real-time capability of tumor boundary identification, providing more reliable fluorescence guidance for precise resection. For various disease types such as metabolic disorders, infectious diseases, and kidney diseases, the imaging and diagnostic capabilities of dual-lock fluorescent probes remain largely unexplored. For instance, in metabolic diseases like liver cirrhosis, fatty liver, and atherosclerosis, as well as kidney diseases such as acute kidney injury and chronic kidney disease, concurrent abnormalities in biomarkers like adenosine triphosphate, reactive oxygen species, or lipid content often occur[114-115]; whereas during viral infections involving Severe Acute Respiratory Syndrome Coronavirus 2, Dengue virus, Adenovirus, etc., pathogen invasion can trigger dynamic changes in various enzyme activities and immune-related molecules within host cells[83,116]. In response to the aforementioned diseases or infection processes, traditional single-lock fluorescent probes struggle to achieve precise monitoring, and dual-lock response systems capable of simultaneously recognizing multidimensional signals are still lacking. With the deep integration of chemistry, biology, and medicine, future efforts should fully leverage the diagnostic advantages of dual-lock fluorescent probes, conducting targeted structural design and optimization based on the pathological characteristics of different diseases. This will promote exploratory research in broader disease areas, accumulate more data and possibilities for their clinical translation, and thereby provide key support for the development of precision medicine.

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Funding

National Natural Science Foundation of China(51703171)

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