Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells.

Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.

Lanthanide Inorganic Nanoparticles Enhance Semiconducting Polymer Nanoparticles Afterglow Luminescence for In Vivo Afterglow/Magnetic Resonance Imaging.

Dual/multimodal imaging strategies are increasingly recognized for their potential to provide comprehensive diagnostic insights in cancer imaging by harnessing complementary data. This study presents an innovative probe that capitalizes on the synergistic benefits of afterglow luminescence and magnetic resonance imaging (MRI), effectively eliminating autofluorescence interference and delivering a superior signal-to-noise ratio. Additionally, it facilitates deep tissue penetration and enables noninvasive imaging. Despite the advantages, only a limited number of probes have demonstrated the capability to simultaneously enhance afterglow luminescence and achieve high-resolution MRI and afterglow imaging. Herein, we introduce a cutting-edge imaging platform based on semiconducting polymer nanoparticles (PFODBT) integrated with NaYF4@NaGdF4 (Y@Gd@PFO-SPNs), which can directly amplify afterglow luminescence and generate MRI and afterglow signals in tumor tissues. The proposed mechanism involves lanthanide nanoparticles producing singlet oxygen (1O2) upon white light irradiation, which subsequently oxidizes PFODBT, thereby intensifying afterglow luminescence. This innovative platform paves the way for the development of high signal-to-background ratio imaging modalities, promising noninvasive diagnostics for cancer.

Organic Afterglow Nanoparticles in Bioapplications.

Organic afterglow nanoparticles are unique optical materials that emit light long after cessation of excitation. Due to their advantages of no need for real-time light excitation, avoiding autofluorescence, low imaging background, high signal-to-background ratio, deep tissue penetration, and high sensitivity, afterglow imaging technology has been widely used in cell tracking, biosensing, cancer diagnosis, and cancer therapy, which provides an effective technical method for the acquisition of molecular information with high sensitivity, specificity and real-time at the cellular and living level. In this review, we summarize and illustrate the recent progress of organic afterglow imaging, focusing on the mechanism of organic afterglow materials and their biological application. Furthermore, we also discuss the potential challenges and the further directions of this field.

The rational design of nanozymes for imaging-monitored cancer therapy.

Nanozymes are nanoscale materials that display enzyme-like properties, which have been improved to eliminate the limitations of natural enzymes and further broaden the use of conventional artificial enzymes. In the last decade, the research and exploration of nanozymes have attracted considerable attention in the chemical and biological fields, especially in the fields of biomedicine and tumor therapy. To date, plenty of nanozymes have been developed with the single or multiple activities of natural enzymes, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), glucose oxidase (GOx). Tumor-characteristic metabolites can be transformed into toxic substances under the catalysis of nanozymes to kill tumor cells. However, the therapeutic effects of nanozymes greatly depend on their catalytic activity, which displays a lot of differences in vitro and in vivo. Moreover, the complex tumor environment (low pH, high H2O2 and GSH concentration, hypoxia, etc.) plays an important role in affecting their catalytic activity. Besides, the uncontrollable catalysis of nanozymes may lead to the destruction of normal tissues. To solve these problems, researchers have exploited several imaging methods to monitor the reaction processes during catalysis, including optical imaging methods (fluorescence and chemiluminescence), photoacoustic imaging, and magnetic resonance imaging. In this review, we have summarized the development of tumor treatment using nanozymes in recent years, along with the current imaging tools to monitor the catalyzing activity of nanozymes. Representative examples have been elaborated on to show the current development of these imaging tools. We hope this review will provide some instructive perspectives on the development of nanozymes and promote the applications of imaging-guided tumor therapeutics.

Zinc-Carnosine Metallodrug Network as Dual Metabolism Inhibitor Overcoming Metabolic Reprogramming for Efficient Cancer Therapy.

The targeting of tumor metabolism as a novel strategy for cancer therapy has attracted tremendous attention. Herein, we develop a dual metabolism inhibitor, Zn-carnosine metallodrug network nanoparticles (Zn-Car MNs), which exhibits good Cu-depletion and Cu-responsive drug release, causing potent inhibition of both OXPHOS and glycolysis. Notably, Zn-Car MNs can decrease the activity of cytochrome c oxidase and the content of NAD+, so as to reduce ATP production in cancer cells. Thereby, energy deprivation, together with the depolarized mitochondrial membrane potential and increased oxidative stress, results in apoptosis of cancer cells. In result, Zn-Car MNs exerted more efficient metabolism-targeted therapy than the classic copper chelator, tetrathiomolybdate (TM), in both breast cancer (sensitive to copper depletion) and colon cancer (less sensitive to copper depletion) models. The efficacy and therapy of Zn-Car MNs suggest the possibility to overcome the drug resistance caused by metabolic reprogramming in tumors and has potential clinical relevance.

Hydrogen-bond-driven self-assembly of chemiluminophore affording long-lasting in vivo imaging.

Developing chemiluminescence probe with a slow kinetic profile, even a constant emission within analytical time, would improve the analytical sensitivity, but still remains challenging. This work reports a novel strategy to afford long-lasting in vivo imaging by developing a self-assembled chemiluminophore HPQCL-Cl via the introduction of the hydrogen-bond-driven self-assembled dye HPQ to Schaap's dioxetane. Compared with classical chemiluminophore HCL, self-assembled HPQCL-Cl was isolated from the physiological environment, thereby lowering its deprotonation and prolonging its half-life. Based on HPQCL-Cl, the long-lasting in vivo imaging of 9L-lacz tumor was achieved by developing a ß-gal-responsive probe. Its signals remained constant (<5% change) for about 20 min, which may provide a wide time window for the determination of ß-gal. This probe also showed high tumor-to-normal tissue ratio throughout tumor resection, highlighting its potential in image-guided clinical surgery.

A novel afterglow nanoreporter for monitoring cancer therapy.

Rationale: Immunogenic cell death (ICD)-associated immunogenicity evoked through reactive oxygen species (ROS) is an efficient way to fight against the immune-dysfunctional microenvironment, so as to provoke potent anti-tumor immunity. However, the unknown ROS dose during cancer therapies may induce adverse immune responses (e.g., insufficient ICD, toxicity toward normal tissues or immune system). Methods: Herein, we developed a pyrido pyrazine - thiophene based semiconducting polymer as novel near-infrared (NIR) organic afterglow nanoparticles for the real-time visualization of self-generated ROS, during photodynamic-mediated immunogenic cell death. Specifically, we introduced the strong "acceptor" (pyrido pyrazine) into thiophene based semiconducting polymer to redshift emission wavelength, and further modulate the "donor" to afford more afterglow reaction sites and reducing ΔEst, so as to enhance luminescence intensity. Results: The semiconducting polymer-based afterglow nanoparticles exhibit strong afterglow emission with longer-wavelength emission (> 800 nm), compared with the reported organic afterglow nanoparticles (e.g., MEHPPV, PFODBT or Chlorin, < 690 nm), which endows this afterglow nanoparticles with a greatly improvement of signal to noise ratio. Moreover, the photodynamic effect of this afterglow nanoparticles can induce immunogenic cell death of cancer cells and further cause immune responses in mice. Conclusions: The NIR afterglow signal presents a good relationship with ROS generation, immunogenic cell death and outcome of treatment. Therefore, it was able to provide a non-invasive tool for predicting the degree of ICD that occurs during ROS-mediated cancer therapy and may contribute to precise immunotherapy.

Molecular engineering of organic-based agents for in situ bioimaging and phototherapeutics.

In situ monitoring of the location and transportation of bioactive molecules is essential for deciphering diverse biological events in the field of biomedicine. In addition, obtaining the in situ information of lesions will provide a clear perspective for surgeons to perform precise resection in clinical surgery. Notably, delivering drugs or operating photodynamic therapy/photothermal therapy in situ by labeling the lesion regions of interest can improve treatment and reduce side effects in vivo. In various advanced imaging and therapy modalities, optical theranostic agents based on organic small molecules can be conveniently modified as needed and can be easily internalized into cells/lesions in a non-invasive manner, which are prerequisites for in situ bioimaging and precision treatment. In this tutorial review, we first summarize the in situ molecular immobilization strategies to retain small-molecule agents inside cells/lesions to prevent their diffusion in living organisms. Emphasis will be focused on introducing the application of these strategies for in situ imaging of biomolecules and precision treatment, particularly pertaining to why targeting therapy in situ is required.

Smart Nanozyme Platform with Activity-Correlated Ratiometric Molecular Imaging for Predicting Therapeutic Effects.

Nanozymes with intrinsic enzyme-like characteristics have attracted enormous research interest in biological application. However, there is a lack of facile approach for evaluating the catalytic activity of nanozymes in living system. Herein, we develop a novel manganese-semiconducting polymer-based nanozyme (MSPN) with oxidase-like activity for reporting the catalytic activity of itself in acid-induced cancer therapy via ratiometric near-infrared fluorescence (NIRF)-photoacoustic (PA) molecular imaging. Notably, MSPN possess oxidase-like activity in tumor microenvironment, owing to the mixed-valent MnOx nanoparticles, which can effectively kill cancer cells. Because the semiconducting polymer (PFODBT) is conjugated with oxidase-responsive molecule (ORM), the catalytic activity of nanozyme can be correlated with the ratiometric signals of NIRF (FL695 /FL825 ) and PA (PA680 /PA780 ), which may provide new ideas for predicting anticancer efficacy of nanozymes in living system.

Oxygen-embedded quinoidal acene based semiconducting chromophore nanoprobe for amplified photoacoustic imaging.

In this chapter, we summarize the advantages of photoacoustic imaging and the current methods of enhancing photoacoustic. We then provide detailed procedures for the synthesis and characterization of a photoacoustic imaging molecule, Nano(O-Nonacene)-PEG, developed in our research group. At the same time, we proved that the incorporation of Zn0.4Fe2.6O4 can enhance the photoacoustic imaging effect of Nano(O-Nonacene)-PEG. This provides a new material for photoacoustic imaging to guide tumor treatment.

Tumor-Specific Multipath Nucleic Acid Damages Strategy by Symbiosed Nanozyme@Enzyme with Synergistic Self-Cyclic Catalysis.

The high proliferation efficiency, redox imbalance, and elevated nucleic acid repair capabilities of tumor cells severely restrict the theranostic efficacy. Selectively interference chaotic tumors with devastating nucleic acid damages (NUDs) properties are expected to overcome theranostic barriers. Here, an exquisite catalytic-based strategy with comprehensive NUDs mechanisms is demonstrated. In this regard, enzyme (glucose oxidase, GOD) symbioses nanozyme Cu3+x (PO4 )2 through biomineralization (abbreviated as Cu@GOD), GOD can disorder the metabolism by consuming glucose, thereby inhibiting the nutrition supply for nucleic acid repair. GOD-catalyzed H2 O2 guarantees the self-cyclic glutathione depletion and reactive oxygen species generation caused by Cu3+x (PO4 )2 , resulted the reduced antioxidation defense and enhanced oxidation assault, ensures an indiscriminate NUDs ability. Moreover, the high photothermal effect of Cu3+x (PO4 )2 induces effective tumor inhibition. Consequently, this substantial multipath NUDs strategy, with potentials of suppressing the cytoprotective mechanisms, amplifying the cellular oxidative stress, and disrupting the redox balance to ensure substantial irreversible NUDs, completely breaks the obstacle of chaotic tumors, providing new conceptual thinking for tumor proliferation inhibition.

Monitoring Immunotherapy With Optical Molecular Imaging.

Immunotherapy is an effective way to mobilize the body's own immune system to confront tumor cells. However, the efficacy of immunotherapy is affected by tumor heterogeneity, and the low therapeutic response to immunotherapy may lead to negative outcomes, which reinforces the urgency for early benefit predictors. Evaluating the infiltration of immune cells in solid tumors and metabolism changes of tumors provide potential response targets for monitoring immune response. Non-invasive imaging identifying prognostic biomarkers can select the beneficiaries of targeted immunotherapy from non-responses. Quantitative biomarkers may eventually improve the cancer management, help customize individual treatment plans and predict the treatment outcomes. In this review, we summarize the non-invasive optical molecular imaging methods for monitoring immunotherapy. With the combination of imaging and immunotherapy, the prediction of immunotherapy response may promote the development of precision medicine.

Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors.

Aminopeptidase N (APN) is capable of cleaving N-terminal amino acids from peptides with alanine in the N-terminal position and plays a key role in the growth, migration, and metastasis of cancer. However, reliable in situ information is hard to be obtained with the current APN-responsive molecular probes because the released fluorophores are cytoplasmic soluble and thus rapidly depart from the enzymatic reaction sites and spread out all over the cytoplasm. Here, we report a de novo precipitated fluorophore, HBPQ, which is completely insoluble in water and shows strong yellow solid emission when excited with a 405 nm laser. Owing to the controllable solid fluorescence of HBPQ by the protection-deprotection of phenolic hydroxyl, we further utilized HBPQ to design an APN-responsive fluorogenic probe (HBPQ-A) for the imaging of intracellular APN. Importantly, HBPQ-A can not only perform in situ imaging of APN in different organelles (e.g., lysosomes, mitochondria, endoplasmic reticula, and so forth) but also display a stable and indiffusible fluorescent signal for reliable mapping of the distribution of APN in living cells. In addition, through real-time imaging of APN in 4T1 tumors, we found that the fluorescent signal with high fidelity generated by HBPQ-A could remain constant even after 12 h, which further confirmed its diffusion-resistant ability and long-term reliable imaging ability. We believe that the precipitated fluorophore may have great potential for long-term in situ imaging.

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

A two-photon fluorescence self-reporting black phosphorus nanoprobe for the in situ monitoring of therapy response.

The in situ and real-time supervision of reactive oxygen species (ROS) generated during photodynamic therapy (PDT) is of great significance for lessening nonspecific damage and guiding personalized therapy. However, photosensitizers frequently fail to deliver successful treatment accompanying the ROS-related imaging signals produced, impeding simple treatment outcome predictions and therapeutic schedule adjustments. Here, we report a two-photon fluorescence self-reporting strategy for the in situ and real-time monitoring of treatment response via a novel black phosphorus-based two-photon nanoprobe (TPBP). TPBP effectively generated singlet oxygen (1O2) under near-infrared laser irradiation for PDT, and 1O2 stimulated a two-photon molecule to emit fluorescence signals for feedback of 1O2 generation, which facilitated the regulation of treatment parameters to achieve precise and personalized medicine in deep tissue.

Oxygen-Embedded Pentacene Based Near-Infrared Chemiluminescent Nanoprobe for Highly Selective and Sensitive Visualization of Peroxynitrite In Vivo.

Peroxynitrite (ONOO-) is involved in neurodegenerative, inflammatory, cardiovascular disorders, cancers, and other pathological progress. However, current imaging methods for sensing ONOO- usually suffer from high background/autofluorescence for fluorescent probes and poor selectivity/short emission wavelength for chemiluminescent probes. Herein, we present a novel chemiluminescent molecule (oxygen-embedded quinoidal pentacene) responsive to ONOO- for the first time, on the basis of which we rationally construct a near-infrared nanoprobe for detecting ONOO- via chemiluminescence resonance energy transfer (CRET) mechanism. Notably, our nanoprobe exhibits good selectivity, ultrahigh sensitivity (nanomole level), low background noise, fast response, and high water solubility. Moreover, the near-infrared emission from CRET offers higher tissue penetration of the chemiluminescent signal. Finally, our nanoprobe is further successfully applied to detecting endogenous ONOO- in mice with abdominal inflammation, drug-induced hepatotoxicity, or tumor models in vivo. In summary, the self-luminescing nanoprobes can act as an alternative visualizable tool for illuminating the mechanism of ONOO- involved in the specific pathological process.

Oxygen-Embedded Quinoidal Acene Based Semiconducting Chromophore Nanoprobe for Amplified Photoacoustic Imaging and Photothermal Therapy.

Photoacoustic (PA) imaging as a noninvasive biomedical imaging technology exhibits high spatial resolution and deep tissue penetration for in vivo imaging. In order to fully explore the potential of PA imaging in biomedical applications, new contrast agents with improved PA stability and efficiency are in high demand. Herein, we present a new PA agent based on an oxygen-embedded quinoidal nonacene chromophore that is self-assembled into nanoparticles (Nano(O-Nonacene)-PEG), assisted by polyethylene glycol (PEG). Notably, the photothermal conversion efficiency of Nano(O-Nonacene)-PEG is 1.5 fold that of semiconducting polymer nanoparticles (Nano(PCPDTBT)-PEG) and 2.8 fold that of Au nanorods, owing to the low quantum yield of Nano(O-Nonacene)-PEG. Thereby, Nano(O-Nonacene)-PEG possess a greatly elevated PA signal intensity, compared to Nano(PCPDTBT)-PEG and Au nanorods, which have been widely explored for PA imaging. Due to the high resistance to photo bleaching, Nano(O-Nonacene)-PEG exhibits higher PA signal stability, which may be employed for long-term PA imaging. Moreover, when magnetic Zn0.4Fe2.6O4 nanoparticles are incorporated into Nano(O-Nonacene)-PEG, not only are magnetic resonance signals generated but also the photoacoustic efficacy is greatly enhanced. Therefore, Nano(O-Nonacene)-PEG offers distinct properties: (i) the elevated photoacoustic effect allows for high-resolution photoacoustic imaging, (ii) small size (10 nm in diameter) results in efficient tumor-targeting, and (iii) the facile application of efficient photothermal therapy in vivo. The current work offers the possibility of oxygen-embedded quinoidal acene as a promising PA probe for precision phototheranostics.

Nitric Oxide-Activated "Dual-Key-One-Lock" Nanoprobe for in Vivo Molecular Imaging and High-Specificity Cancer Therapy.

Cancer treatments are confounded by severe toxic effects toward patients. To address these issues, activatable nanoprobes have been designed for specific imaging and destruction of cancer cells under the stimulation of specific cancer-associated biomarkers. Most activatable nanoprobes were usually activated by some single-factor stimulation, but this restricts therapeutic specificity between diseased and normal tissue; therefore, multifactor activation is highly desired. To this end, we herein develop a novel dual-stimuli responsive theranostic nanoprobe for simultaneously activatable cancer imaging and photothermal therapy under the coactivation of "dual-key" stimulation of "nitric oxide (NO)/acidity", so as to further improve the therapeutic specificity. Specifically, we have integrated a weak electron acceptor (benzo[c][1,2,5]thiadiazole-5,6-diamine) into a donor-π-acceptor-π-donor type chromophore. When the weak acceptor was oxidized by NO in acidic conditions to form a stronger acceptor (5H-[1,2,3]triazolo[4,5-f]-2,1,3-benzothiadiazole), the molecule absorption was significantly increased in the near-infrared region, based on the intramolecular charge transfer (ICT) mechanism. Under the dual-key stimulation of NO/acidity within the tumor associated with inflammation, the nanoprobe can correspondingly output dual signals for ratiometric photoacoustic and photothermal imaging of cancer in vivo and do so with enhanced accuracy and specificity. Our novel nanoprobe exhibited higher photoacoustic signal enhancement under dual-factor activation at 9.8 times that of NO and 132 times that of acidity alone, respectively. Moreover, through such dual activation of NO/acidity, the nanoprobe produces more differentiation of hyperthermia between tumor and normal tissues, to afford satisfactory photothermal therapy with minimal toxic side effects. Thus, our work presents a promising strategy for significantly improving the precision and specificity of cancer imaging and therapy.

A cell membrane-anchored fluorescent probe for monitoring carbon monoxide release from living cells.

Carbon monoxide (CO) acts as an important gasotransmitter in delivering intramolecular and intermolecular signals to regulate a variety of physiological processes. This lipid-soluble gas can freely pass through the cell membrane and then diffuse to adjacent cells acting as a messenger. Although many fluorescent probes have been reported to detect intracellular CO, it is still a challenge to visualize the release behavior of endogenous CO. The main obstacle is the lack of a probe that can anchor onto the cell membrane while having the ability to image CO in real time. In this work, by grafting a polar head onto a long and linear hydrophobic Nile Red molecule, a cell membrane-anchored fluorophore ANR was developed. This design strategy of a cell membrane-anchored probe is simpler than the traditional one of using a long hydrophobic alkyl chain as a membrane-anchoring group, and endows the probe with better water solubility. ANR could rapidly bind to the cell membrane (within 1 min) and displayed a long retention time. ANR was then converted to a CO-responsive fluorescent probe (ANRP) by complexation with palladium based on a metal palladium-catalyzed reaction. ANRP exhibited a fast response to CO with a 25-fold fluorescence enhancement in vitro. The detection limit was calculated to be 0.23 µM, indicating that ANRP is sensitive enough to image endogenous CO. Notably, ANRP showed excellent cell membrane-anchoring ability. With ANRP, the release of CO from HepG2 cells under LPS- and heme-stimulated conditions was visualized and the cell self-protection effect during a drug-induced hepatotoxicity process was also studied. Moreover, ANRP was successfully applied to the detection of intracellular CO in several cell lines and tissues, and the results demonstrated that the liver is the main organ for CO production, and that cancer cells release more CO from their cells than normal cells. ANRP is the first membrane-anchored CO fluorescent probe that has the ability to reveal the relationship between CO release and diseases. It also has prospects for the studying of intercellular signaling functions of CO.

In Situ Imaging of Furin Activity with a Highly Stable Probe by Releasing of Precipitating Fluorochrome.

Furin, a kind of trans-Golgi proprotein convertases, plays important role in various physiological processes. It is overexpressed in many cancers and relates to tumor growth and migration. In situ detection and imaging of furin is of great significance for obtaining real-time information about its activity. However, the previously reported fluorescent probes for furin usually failed to realize in situ detection and long-term bioimaging, because these probes are based on water-soluble fluorophores, which tend to diffuse away from the reaction sites after converted by furin. Such a problem can be addressed by designing a probe, which releases a precipitating fluorophore upon furin conversion. Herein, we developed a probe HPQF for in situ detection of endogenous furin activity and long-term bioimaging by integrating a strictly insoluble solid-state fluorophore 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (Cl-HPQ) with a furin specific peptide substrate (RVRR) through a self-immolative linker. The HPQF probe shows high selectivity and sensitivity to furin. Upon converted by furin, HPQF releases free Cl-HPQ, which precipitates near the enzyme active site. The precipitates emit bright solid-state fluorescence for in situ imaging. HPQF could truly visualize the location of intracellular furin, which was further confirmed by colocalization and immunofluorescence experiments. Excitingly, the long-term bioimaging was also achieved benefiting from its outstanding signal-stability and antidiffusion ability. HPQF was further utilized to monitor the level change of furin under stabilizing of hypoxia-inducible factor (HIF) regulated by cobalt chloride (CoCl2) as well as visualization of furin in MDA-MB-468 cell tumor tissues.