Dual-Responsive Near-Infrared BODIPY-Based Fluorescent Probe for the Detection of F- and HClO in Organisms.

Fluoride anions (F-) play a crucial role in human physiological processes. However, excessive intake of F- would affect oxygen metabolism and promote the generation of oxygen-free radicals. Hence, it is essential to develop a precise and efficient fluorescent probe for visualizing F--induced oxidative stress. In this work, we developed the first bifunctional BODIPY-based fluorescent probe dfBDP with p-tert-butyldimethylsilanolate benzyl thioether as the sensing site for the detection of F- and HClO via two distinct reactions, the self-immolative removal and the thioether oxidation, which generate the sensing products with two nonoverlap fluorescence bands: 800-1200 and 500-750 nm, respectively. The probe dfBDP displays rapid response, high specificity, and sensitivity for the detection of F- (LOD, 316.2 nM) and HClO (LOD, 33.9 nM) in vitro. Cellular imaging reveals a correlation between F--induced oxidative stress and the upregulation of HClO. Finally, probe dfBDP was employed to detect F- and HClO in mice under the stimulation of F-. The experimental results display that the level of HClO elevates in the liver of mice.

ß-Galactosidase-Activatable Fluorescent and Photoacoustic Imaging of Tumor Senescence.

ß-Galactosidase (ß-gal) is the gold standard marker of cellular senescence, which is linked with various age-related diseases. Therefore, it is essential to develop more excellent probes that can real-time monitor ß-gal activity in cellular senescence in vivo. Fluorescent/photoacoustic (FL/PA) dual-modal imaging possesses excellent sensitivity and spatial resolution. To our knowledge, there has been no tumor-targeted FL/PA probe to image cellular senescence by monitoring the activity of ß-gal in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Gal-HCy-Biotin) for ß-gal-activatable imaging of tumor senescence. Gal-HCy without tumor-targeted biotin is used as a control probe. Gal-HCy-Biotin is superior to Gal-HCy due to the higher kinetic parameter of Gal-HCy-Biotin than Gal-HCy in vitro. Moreover, biotin could help Gal-HCy-Biotin enter and accumulate in tumor cells with higher FL/PA signal. In detail, Gal-HCy-Biotin or Gal-HCy could image senescent tumor cells with 4.6-fold or 3.5-fold FL enhancement and 4.1-fold or 3.3-fold PA enhancement. Gal-HCy-Biotin or Gal-HCy could image tumor senescence with 2.9-fold or 1.7-fold FL enhancement and 3.8-fold or 1.3-fold PA enhancement. We envision that Gal-HCy-Biotin will be applied for FL/PA imaging of tumor senescence in clinic.

Caspase-3-Responsive Fluorescent/Photoacoustic Imaging of Tumor Apoptosis.

Caspase-3 is an essential executor in apoptosis, and its activation has been regarded as a biomarker of cell apoptosis. The development of Caspase-3-responsive multimodal probes is a promising research prospect. Fluorescent/photoacoustic (FL/PA) imaging has attracted considerable attention due to the high sensitivity of FL as well as the high spatial resolution and penetration depth of PA. To our knowledge, there has been no tumor-targeted FL/PA probe to monitor the activity of Caspase-3 in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Bio-DEVD-HCy) for Caspase-3-responsive imaging of tumor apoptosis. Ac-DEVD-HCy without tumor-targeted biotin is used as a control probe. In vitro experiments indicated that Bio-DEVD-HCy is superior to Ac-DEVD-HCy because of the higher kinetic parameter of Bio-DEVD-HCy in comparison to Ac-DEVD-HCy. Cell and tumor imaging results suggested that Bio-DEVD-HCy could enter and accumulate in tumor cells with higher FL/PA signal with the help of tumor-targeted biotin. In detail, Bio-DEVD-HCy or Ac-DEVD-HCy could image apoptotic tumor cells with 4.3-fold or 3.5-fold FL enhancement and 3.4-fold or 1.5-fold PA enhancement. Bio-DEVD-HCy or Ac-DEVD-HCy could image tumor apoptosis with 2.5-fold or 1.6-fold FL enhancement and 4.1-fold or 1.9-fold PA enhancement. We envision that Bio-DEVD-HCy will be applied for FL/PA imaging of tumor apoptosis in clinical settings.

A Golgi-Targeting and Dual-Color "Turn-On" Probe for Spatially Precise Imaging of Furin.

Development of fluorescence probes for highly accurate detection of cancer-related enzyme activity is important in early cancer diagnosis. Herein, we report a Golgi-targeting and dual-color "Turn-On" probe Q-RVRR-DCM for imaging furin with high spatial precision. By integrating the principles of Förster resonance energy transfer and intramolecular charge transfer, the probe was designed to be non-fluorescent. Upon furin cleavage, Q-RVRR-DCM was converted into Q-RVRR and DCM-NH2, turning the dual fluorescence color "On" at 420 and 640 nm without spectral cross-talk. In furin-overexpressing HCT116 cells, Q-RVRR-DCM showed not only furin-specific, dual-color "Turn-On" fluorescence but also superior colocalization with a Golgi tracker than the single-color "Turn-On" probe RVRR-DCM. We envision that, with the excellent properties of Golgi-targeting and dual fluorescence color "Turn-On", our furin probe Q-RVRR-DCM could be applied for accurate early diagnosis of cancer in the near future.

Cathepsin B-Activated Fluorescent and Photoacoustic Imaging of Tumor.

Early diagnosis is crucial to the treatment of cancer. Cathepsin B (CTB) plays an important role in numerous cancers, which is a promising biomarker for early diagnosis of cancer. It is necessary to exploit new probes for visualization of CTB in vivo. Fluorescent/photoacoustic (FL/PA) imaging is a powerful tool for in vivo study which possesses both excellent sensitivity and spatial resolution. To our knowledge, there has been no FL/PA probe to image CTB in vitro or in vivo. Therefore, we developed two CTB-activated FL/PA probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly, which could successfully monitor CTB activity in vivo. Both two probes had excellent sensitivity and selectivity in vitro. Cell imaging showed that HCy-Cit-Val or HCy-Gly-Leu-Phe-Gly could image endogenous CTB in lysosome with 6.8-fold or 5.1-fold enhancement of the FL signal and 5.8-fold or 3.4-fold enhancement of the PA signal compared to their inhibitor contrast groups. Tumor imaging in vivo further confirmed the good applicability of these two probes to monitor CTB activity with high sensitivity and spatial resolution. Moreover, the property of HCy-Cit-Val is superior to HCy-Gly-Leu-Phe-Gly due to the higher catalytic efficiency of CTB toward HCy-Cit-Val than HCy-Gly-Leu-Phe-Gly. We envision that our FL/PA probe HCy-Cit-Val will be suitable for clinical early diagnosis of CTB-related cancer in the near future.

Mitochondria-Targeted Fluorescent and Photoacoustic Imaging of Hydrogen Peroxide in Inflammation.

Hydrogen peroxide (H2O2) is a prominent reactive oxygen species with relative stability, which makes it a potential diagnostic marker for pathological states. Excessive H2O2 in mitochondria leads to oxidative stress and inflammation. However, precisely monitoring the level of H2O2 at specific organelles (e.g., mitochondria) in vivo is still of urgent necessity. Therefore, we rationally designed a mitochondria-targeted near-infrared probe TPP-HCy-BOH for fluorescent/photoacoustic (FL/PA) dual-modal imaging of overproduced H2O2 in an inflamed mouse model. TPP-HCy-BOH had a low LOD (0.348 µM), which is comparable to those of recently reported probes for H2O2 detection. The high kinetic rate constant (kobs = 4.72 × 10-3 s-1) of TPP-HCy-BOH toward H2O2 is superior to recently reported H2O2 probes. Compared to control probe HCy-BOH without the mitochondrial targeting moiety, TPP-HCy-BOH successfully images exogenous or endogenous H2O2 in mitochondria with an additional 2.4-fold FL increase and 4.7-fold PA increase in HeLa cells or additional 2.1-fold FL increase and 3.3-fold PA increase in RAW 264.7 cells. In LPS-induced acute inflammation in vivo, TPP-HCy-BOH is more competent to image overproduced H2O2 with additional 1.6-fold higher sensitivity of FL in abdomen and 2.0-fold higher sensitivity of PA in liver and longer retention time of 0.5 h than HCy-BOH. We anticipate that TPP-HCy-BOH could be employed for the FL/PA dual-modal diagnosis of pathological inflammation in clinic in near future.

Multi-Stimuli-Triggered Shape Transformation of Polymeric Filaments Derived from Dynamic Covalent Block Copolymers.

Using dynamic polymers to achieve the morphology transformation of polymeric assemblies under different conditions is challenging. Herein, we reported diversiform shape transformation of multi-responsive polymer filaments, which were self-assembled by a new kind of amphiphilic block copolymer (PVEG-PVEA) possessing dynamic and reversible acylhydrazone bonds through reacting benzaldehyde-containing block copolymers poly(vinylbenzaldehyde)-b-poly(N-(4-vinylbenzyl)-N,N-diethylamine) (PVBA-PVEA) with acylhydrazine-modified oligoethylene glycol. It was found that the resulting amphiphilic and dynamic PVEG-PVEA was capable of hierarchically self-assembling into intriguing core-branched filaments in aqueous solution. Notably, the features of acylhydrazone bonds and PVEA block endow the filaments with multi-responsiveness including acid, base, and temperature, leading to the multiple morphological transformations under such stimuli. Moreover, the core-branched filaments would further transform into polymeric braided bundles driven by hydrogen-bonding interactions of amide bonds. It is noteworthy that both core-branched filaments and braided bundles made from polymers are quite rare. These diversiform polymeric assemblies and their morphological evolution were characterized by TEM, Cryo-TEM, SEM, and DLS. Finally, we used PVBA-PVEA as a platform to facilely prepare functional polymers, such as glycopolymers via the reaction of amino-containing sugars and aldehyde groups. The obtained glycopolymers self-assembled into glycofibers for the biomimicry of glycans via binding with lectins. These findings not only are conducive to understanding of the stimulated shape change process of dynamic polymeric assemblies in water but also provide a new method for the facile fabrication of smart and functional polymeric assemblies for different potential applications, such as biomimicry and targeted drug nanocarriers or delivery vehicles.

γ-Glutamyltranspeptidase-Triggered Intracellular Gadolinium Nanoparticle Formation Enhances the T2-Weighted MR Contrast of Tumor.

Magnetic resonance imaging (MRI) is advantageous in the diagnosis of deep internal cancers, but contrast agents (CAs) are always needed to improve MRI sensitivity. Gadolinium (Gd)-based agents are routinely used as T1-dominated CAs in clinic but using intracellularly formed Gd nanoparticles to enhance the T2-weighted MRI of tumor in vivo at high magnetic field has not been reported. Herein, we rationally designed a "smart" Gd-based probe Glu-Cys(StBu)-Lys(DOTA-Gd)-CBT (1), which was subjected to γ-glutamyltranspeptidase (GGT) cleavage and an intracellular CBT-Cys condensation reaction to form Gd nanoparticles (i.e., 1-NPs) to enhance the T2-weighted MR contrast of tumor in vivo at 9.4 T. Living cell experiments indicated that the 1-treated HeLa cells had an r2 value of 27.8 mM-1 s-1 and an r2/r1 ratio of 10.6. MR imaging of HeLa tumor-bearing mice indicated that the T2 MR contrast of the tumor enhanced 28.6% at 2.5 h post intravenous injection of 1. We anticipate that our probe 1 could be employed for T2-weighted MRI diagnosis of GGT-related cancers in the future when high magnetic field is available in clinic.

Cathepsin B Turning Bioluminescence "On" for Tumor Imaging.

Cathepsin B (CTSB) is a lysosomal protease, and several human cancers are reported highly expressing CTSB. Many optical methods have been developed for CTSB detection but not a bioluminescence (BL) probe. Herein, a CTSB-specific bioluminescence probe Val-Cit-AL was rationally designed for selectively sensing CTSB activity in vitro with a 67-fold "Turn-On" of BL intensity and an excellent limit of detection. Inhibitory experiments indicated that Val-Cit-AL is capable of sensing CTSB activity in living cells and tumors. We anticipate that Val-Cit-AL might be applied to diagnose CTSB-related diseases in rodent models or evaluate CTSB roles in more biological processes in the near future.

Legumain-Specific Near-Infrared Fluorescence "Turn On" for Tumor-Targeted Imaging.

Legumain is one of the cysteine proteases which can serve as an essential indicator for cancer diagnosis. Near-infrared (NIR) nanoprobes with fluorescence "Turn On" property are advantageous in cancer diagnosis. However, to the best of our knowledge, using a completely organic NIR nanoprobe to image legumain activity either in vitro or in vivo has not been reported. Herein, employing a CBT-Cys click condensation reaction, we used a rationally designed NIR probe Cys(StBu)-Ala-Ala-Asn-Lys(Cy5.5)-CBT (1) to synthesize its nanoprobes 1-NPs with self-quenched fluorescence. Cell and animal experiments indicated that our nanoprobes were able to specifically image legumain activity in living cells and tumors with a NIR fluorescence "Turn On" manner. We envision that the nanoprobes could be applied for the diagnosis of legumain-related diseases in the near future.

Smart Dual Quenching Strategy Enhances the Detection Sensitivity of Intracellular Furin.

Development of sensitive fluorescence "Turn-On" strategies for imaging enzyme activity in living cells is of disease-diagnostic importance but remains challenging. Herein, by employing a click condensation reaction and rational design of a single quenched probe Cys(StBu)-Lys(Gly-Lys(DABCYL)-Gly-Gly-Arg-Arg-Val-Arg-Gly-FITC)-CBT (1), we developed a "smart" dual quenching strategy and applied it to detect intracellular furin activity with enhanced sensitivity. At physiological conditions, 1 was subjected to reduction-controlled condensation reaction to form 1-NPs and its fluorescence intensity further dropped to 1/2.8 of its original. Upon furin cleavage in vitro, the dual quenched 1-NPs had fluorescence "Turn-On" contrast 11-fold more than that of single quenched control probe FITC-Gly-Arg-Val-Arg-Arg-Gly-Gly-Lys(DABCYL)-Gly-OH (1-P). Live cell imaging results indicated that 1 showed fluorescence "Turn-On" contrast 6.3-fold of that of 1-P for sensing intracellular furin activity. We envision that, by replacing the RVRR substrate with other enzyme-cleavable ones, our versatile "smart" dual quenching strategy could be easily adjusted for the detection (or imaging) of other intracellular enzymes' activity with enhanced sensitivity.

Alkaline Phosphatase-Triggered Simultaneous Hydrogelation and Chemiluminescence.

Chemiluminescence (CL) has a higher signal-to-noise ratio than fluorescence, but the use of CL to track an enzyme-instructed self-assembly (EISA) process has not been reported. In this work, by coincubation of the hydrogelator precursor Fmoc-Phe-Phe-Tyr(H2PO3)-OH (1P) and the CL agent AMPPD (2) with alkaline phosphatase (ALP), we employed CL to directly characterize and image the simultaneous EISA process of 1P. Hydrogelation processes of 1P with and without 2 and the CL properties of 2 with and without 1P under ALP catalysis were systematically studied. The results indicated that 2 is an ideal CL indicator for ALP-triggered hydrogelation of 1P. Using an IVIS optical imaging system, we obtained time-course CL images of 2 to track the simultaneous hydrogelation process of 1P in the same solution. We envision that our CL method could be employed to track more biological EISA events in the near future.

Intracellular Proteolytic Disassembly of Self-Quenched Near-Infrared Nanoparticles Turning Fluorescence on for Tumor-Targeted Imaging.

The design of tumor-targeting, intracellular protease-activatable near-infrared fluorescence (NIRF) nanoprobes is broadly interesting but remains challenging. In this work, we report the rational design of a NIR probe Cys(StBu)-Lys(Biotin)-Lys-Lys(Cy5.5)-CBT (1) to facilely prepare the self-quenched nanoparticles 1-NPs for tumor-targeted imaging in vitro and in vivo. The biotinylated 1-NPs could be actively uptaken by biotin receptor-overexpressing tumor cells via receptor-mediated endocytosis. Upon intracellular proteolytic cleavage, 1-NPs were disassembled to yield the small molecular probe Lys(Cy5.5)-Luciferin-Lys(Biotin)-Lys-OH (1-D-cleaved), accompanied by fluorescence "Turn-On". With this NIRF "Turn-On" property, 1-NPs were successfully applied for tumor-targeted imaging. We envision that our nanoparticles could be applied for fluorescence-guided tumor surgery in the near future.

Bioluminescence Sensing of γ-Glutamyltranspeptidase Activity In Vitro and In Vivo.

γ-Glutamyltranspeptidase (GGT) is an important tumor biomarker but using a bioluminescence (BL) probe to real time monitor its activity has not been reported. Herein, we rationally designed two GGT-cleavable BL probes Glu-AmLH2 (1) and Glu-p-aminobenzyloxycarbonyl-AmLH2 (2), and successfully applied them for sensing GGT activity with high sensitivity and excellent selectivity both in vitro and in vivo. The results indicated that, although 2 had lower background BL signal than 1, GGT had higher catalytic efficiency for 1 than 2, and 1 was superior to 2 for sensing GGT activity in living cells and tumors. We envision that our probe 1 could be widely applied for the diagnosis of important GGT-related diseases in animal models in the near future.

Alkaline Phosphatase-Instructed Self-Assembly of Gadolinium Nanofibers for Enhanced T2-Weighted Magnetic Resonance Imaging of Tumor.

Alkaline phosphatase (ALP) is an important enzyme but using ALP-instructed self-assembly of gadolinium nanofibers for enhanced T2-weighted magnetic resonance imaging (MRI) of tumor has not been reported. In this work, we rationally designed a hydrogelator Nap-FFFYp-EDA-DOTA(Gd) (1P) which, under the catalysis of ALP, was able to self-assemble into gadolinium nanofibers to form hydrogel Gel I for enhanced T2-weighted MR imaging of ALP activity in vitro and in tumor. T2 phantom MR imaging indicated that the transverse relaxivity (r2) value of Gel I was 33.9% higher than that of 1P and both of them were 1 order of magnitude higher than that of Gd-DTPA. In vivo T2-weighted MR imaging showed that, at 9.4 T, ALP-overexpressing HeLa tumors of 1P-injected mice showed obviously enhanced T2 contrast. We anticipate that, by replacing ALP with other enzymes, our approach could be applied for MR diagnosis of other diseases in the future.

Enzymatic Hydrogelation-Induced Fluorescence Turn-Off for Sensing Alkaline Phosphatase in Vitro and in Living Cells.

Alkaline phosphatase (ALP)-catalyzed hydrogelation has been extensively explored and found wide applications. Spectroscopic and electrochemical approaches are commonly employed for the detection of ALP activity. Herein, by rational design of a fluorescence probe Fmoc-K(FITC)FFYp (P1) (where FITC is fluorescein), we incorporated sol-gel transition with fluorescence "turn-off" and developed a new method for quantitative sensing ALP activity in vitro and in living cells. Under the catalysis of ALP, P1 was converted to hydrogelator Fmoc-K(FITC)FFY (1) which self-assembles into nanofibers to form Gel I. Accompanying this sol-gel transition, the fluorescence emission of P1 was turned off. Our assay was employed to detect ALP activity over the range of 0-2.8 U/mL with a limit of detection (LOD) of 0.06 U/mL. ALP-inhibitor-treated cell imaging indicated that P1 could be applied for sensing ALP activity in living cells. Our method provides a new option for real time and quantitative sensing ALP activity in vitro and even in living cells.

Pyridine-biquinoline-metal complexes for sensing pyrophosphate and hydrogen sulfide in aqueous buffer and in cells.

Herein, we report a new pyridine-biquinoline-derivative fluorophore L for effectively sensing pyrophosphate (PPi) and monohydrogen sulfide (HS(-)) in aqueous buffer and in living cells. L could selectively coordinate with metal ions (M(n+)) in Groups IB and IIB to form L-M(n+) complexes with 1:1 stoichiometry, resulting in fluorescence quenching via photoinduced electron transfer (PET) mechanism. L-Zn(2+) complex was applied to competitively coordinate with PPi to form a new "ate"-type complex, turning on the fluorescence by a 21-fold-increase. The limit of detection (LOD) of this assay for PPi detection in aqueous buffer is 0.85 µM. L-Cu(2+) complex was applied for highly selective detection of HS(-) with an excellent sensitivity by 25-fold decomplexation-induced fluorescence increase. LOD of L-Cu(2+) complex for HS(-) detection in aqueous buffer is 2.24 µM. With the in vitro data obtained, we successfully applied these two complexes for sequential imaging Zn(2+) and PPi, Cu(2+) and HS(-) in living cells, respectively. Since PPi and HS(-) occur in vascular calcification in positive correlation, our multifunctional probe L might help doctors to more precisely diagnose this disease in vivo in the future. For example, we could use radioactive tracer L-(64)Cu for qualitative and quantitative positron emission tomography/computed tomography (PET/CT) imaging of HS(-) in vivo.

Discriminative fluorescence sensing of biothiols in vitro and in living cells.

Simultaneous discriminative sensing of biothiols in vitro and in living cells has remained challenging. Herein, we report a new sulfonamide-based self-quenched fluorescent probe 1 for this purpose with high sensitivity and good selectivity. Treatment of 1 with cysteine (Cys), homocysteine (Hcy), or glutathione (GSH) yields aminoluciferin, 2-cyano-6-aminobenzothiazole homocysteine (CBTHcy), or 2-cyano-6-aminobenzothiazole (CBT), turning "on" the fluorescence at wavelengths of 522, 517, or 490 nm, respectively. Kinetic study indicated that 1 reacts with Cys faster than with Hcy or GSH. With these unique properties of 1, we applied 1 for highly sensitive sensing of Cys, Hcy, and GSH among other 19 natural amino acids (AAs) with good selectivity. Confocal fluorescence microscopic imaging of 1-treated HepG2 cells at two channels (522 ± 8 and 490 ± 8 nm), together with quantitative analysis, indicated that the "turn-on" fluorescence was induced by intracellular Cys-dominating condensation and reduction of 1 but not by intracellular GSH-dominating reduction of 1. This suggests that 1 could be applied for discriminative sensing of intracellular Cys from the abundant GSH. Further development of 1 might bring about an efficient tool for probing cellular functions that relate to biothiols.

Intracellular Formation of Hemicyanine Nanoparticle Enhances Tumor-Targeting Photoacoustic Imaging and Photothermal Therapy.

Alkaline phosphatase (ALP) is a tumor marker for early diagnosis and treatment. Tumor targeting can recognize and fight tumor cells more accurately from healthy cells. Glycyrrhetinic acid (GA) is a targeting ligand of liver tumors. Photoacoustic imaging (PAI) and photothermal therapy (PTT) are promising techniques for tumor diagnosis and treatment. The outstanding characteristics of Hemicyanine (HCy) dye make it suitable for tumor diagnosis and treatment. However, using HCy nanoparticle (HCy NP) for liver tumor-targeting PAI and PTT has not been reported. Herein, Probe-1 is developed to enhance PAI and PTT of liver tumors due to GA targeting and intracellular ALP-instructed self-assembly of HCy NP. Compared to Probe-2 without self-assembly ability, Probe-1 displays a 4.6-fold higher PAI signal or 1.7-fold lower half inhibitory concentrations in HepG2 cells. Moreover, Probe-1 shows extended retention time (10 vs 6 h) and 2.1-fold higher PAI signal than Probe-2 in HepG2 tumors. The HepG2 tumors in Group Probe-1 obviously increase 18 °C (Tmax : 55 °C) with a 3.3-fold decreased volume while that in Group Probe-2 mildly increase 9.8 °C (Tmax : 46.8 °C) with a 4.3-fold increased volume. It is envisioned that this smart self-assembly strategy can be easily adjusted for PAI and PTT of more tumors.

Caspase-1-responsive fluorescence biosensors for monitoring endogenous inflammasome activation.

The activation of inflammasome leads to secretion of inflammatory factors and cell pyroptosis that are critical in the pathogenesis of various chronic and acute inflammatory diseases. Recruitment and activation of caspase-1 is a marker of inflammasome activation. However, there is still lack of real-time and efficient methods to detect the activation of inflammasome, especially in vivo. Herein, we developed two activatable caspase-1-responsive fluorescence biosensors, WEHD-HCy and YVAD-HCy, to specifically monitor the activation of inflammasome in vivo. Our in vitro study demonstrated that WEHD-HCy and YVAD-HCy can sensitively and specifically respond to caspase-1 activation. Moreover, these biosensors can efficiency and specifically activated in the common inflammatory disease model, including inflammatory bowel disease, Salmonella infection, and acute arthritis. In particular, WEHD-HCy is more advantageous than YVAD-HCy to specifically image of caspase-1 activity both in vitro and in vivo. These caspase-1-responsive fluorescence biosensors provide an efficient, rapid, and in situ tool for monitoring inflammasome activation, and have the potential to be suitable for clinical diagnosis of various inflammatory diseases associated with inflammasome activation.