Search

The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe-biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.

Bovine pericardium (BP) is a vascular biomaterial used in cardiovascular surgery that is typically cross-linked for masking antigenicity and enhance stability. There is a need for biochemical evaluation of the tissue properties prior to implantation to ensure that quality and reliability standards are met. Here, engineered antigen removed BP (ARBP) that was cross-linked with 0.2% and 0.6% glutaraldehyde (GA), and further calcified in vitro to simulate graft calcifications upon implantation was characterized nondestructively using fluorescence lifetime imaging (FLIm) to identify regions of interest which were then assessed by Raman spectroscopy. We observed that the tissue fluorescence lifetime shortened, and that Raman bands at 856, 935, 1282, and 1682 cm-1 decreased, and at 1032 and 1627 cm-1 increased with increasing GA cross-linking. Independent classification analysis based on fluorescence lifetime and on Raman spectra discriminated between GA-ARBP and untreated ARBP with an accuracy of 91% and 66%, respectively. Pearson's correlation analysis showed a strong correlation between pyridinium cross-links measured with high-performance liquid chromatography and fluorescence lifetime measured at 380-400 nm (R = -0.76, p = 0.00094), as well as Raman bands at 856 cm-1 for hydroxy-proline (R = -0.68, p = 0.0056) and at 1032 cm-1 for hydroxy-pyridinium (R = 0.74, p = 0.0016). Calcified areas of GA cross-linked tissue showed characteristic hydroxyapatite (959 and 1038 cm-1) bands in the Raman spectrum and fluorescence lifetime shortened by 0.4 ns compared to uncalcified regions. FLIm-guided Raman imaging could rapidly identify degrees of cross-linking and detected calcified regions with high chemical specificity, an ability that can be used to monitor tissue engineering processes for applications in regenerative medicine.

Transparent surfaces within microfluidic devices are essential for accurate quantification of chemical, biological, and mechanical interactions. Here, we report how to create low-cost, rapid 3D-printed microfluidic devices that are optically free from artifacts and have transparent surfaces suitable for visualizing a variety of fluid phenomenon. The methodology described here can be used for creating high-pressure microfluidic systems (significantly higher than PDMS-glass bonding). We develop methods for annealing Poly-Lactic Acid (PLA) microfluidic devices demonstrating heat resistance typically not achievable with other plastic materials. We show DNA melting and subsequent fluorescent imaging analysis, opening the door to other high-temperature applications. The FDM techniques demonstrated here allow for fabrication of microfluidic devices for precise visualization of interfacial dynamics, whether mixing between two laminar streams or droplet tracking. In addition to these characterizations, we include a printer troubleshooting guide and printing recipes for device fabrication to facilitate FDM printing for microfluidic device development.

Aminopeptidase N (APN/CD13) is a widely expressed transmembrane ectoenzyme that is crucial for maintaining normal physiological activities. It exhibits abnormal activity closely associated with hepatic fibrosis and nonalcoholic fatty liver disease (NAFLD). Therefore, there is a high demand for noninvasive detection of aminopeptidase N (APN) in the diagnosis and research of related diseases. Here, we developed a small molecule fluorescent probe, Hcy-APN, which is a fluorescent probe with high sensitivity and selectivity for the detection of APN. Furthermore, we synthesized the fluorescent nanoprobe Hcy-APN@MSN by self-assembling Hcy-APN and mesoporous silica nanoparticles in solution using a combination of molecular probe design and nanofunctionalization strategies. The detection limit of this probe was 1.5 ng/mL. Hcy-APN@MSN exhibits more stable spectral characteristics compared to Hcy-APN and is suitable for detecting APN activity in live cells and mice. Hcy-APN@MSN was utilized for in vivo and intracellular imaging of NAFLD and hepatic fibrosis at different stages, as well as for a systematic assessment of APN levels in the liver. The results confirm an elevation in the expression levels of APN in NAFLD and hepatic fibrosis models. Furthermore, we investigated the inhibitory effect of the APN inhibitor bestatin in nonalcoholic fatty liver and hepatic fibrosis disease models, confirming its regulatory effect on APN levels in cells and in vivo in both disease models. Therefore, this study may offer diagnostic possibilities for detecting NAFLD and hepatic fibrosis.

Bioluminescence imaging (BLI) is an important noninvasive optical imaging technique that has been widely used to monitor many biological processes due to its high sensitivity, resolution, and signal-to-noise ratio. However, the BLI technique based on the firefly luciferin-luciferase system is limited by the expression of exogenous luciferase and the short half-life of firefly luciferin, which pose challenges for long-term tracking in vivo. To solve the problems, here we rationally designed an intelligent strategy for persistent BLI in tumors by combining luciferase-loaded calcium phosphate nanoparticles (Luc@CaP NPs) to provide luciferase and the probe Cys(SEt)-Lys-CBT (CKCBT) to slowly produce the luciferase substrate amino luciferin (Am-luciferin). Luc@CaP NPs constructed with CaP as a carrier could enable luciferase activity to be maintained in vivo for at least 12 h. And compared to the conventional substrate luciferin, CKCBT apparently prolonged the BL time by up to 2 h through GSH-induced intracellular self-assembly and subsequent protease degradation-induced release of Am-luciferin. We anticipate that this strategy could be applied for clinical translation in more disease diagnosis and treatment in the near future.

The pursuit of advanced mRNA detection methods has been driven by the need for sensitive, accurate approaches that are particularly suited for live-cell analysis. Herein, we proposed a cascaded and localized assembly (CLA) system, integrating branched catalytic hairpin assembly (bCHA) with a localized hybridization chain reaction (LHCR) for enhanced mRNA imaging. The CLA system employed a dual-nanosphere (NS) platform, NSABC and NS12, and the interaction between the target and NSABC initiated the bCHA process and activated a split trigger. The newly generated trigger served as the initiator for the LHCR on NS12, leading to amplified fluorescent signals. Notably, this work introduced the first integration of a splitting strategy in a bCHA-HCR cascaded system, reducing false-positive signals and enhancing specific detection. The dual-NS platform further minimized background noise and improved the reaction kinetics through spatial confinement. As a result, the system achieved a detection limit of 1.23 pM. With these advantages, the CLA system demonstrated successful application in both living cells and clinical tissues, underscoring its potential in biomolecular research and clinical diagnostics.

Aggrephagy describes lysosomal transport and degradation of protein aggregates via cellular macroautophagy, a key mechanism to prevent neurodegenerative diseases. Here, we develop a dual-probe method to visualize the aggrephagy process and resolve its viscosity heterogeneity using fluorescence lifetime imaging (FLIM). The dual-probe system consists of (1) a near-infrared lysosomal targeting FLIM probe (Lyso-P1) that is derived from a rhodamine scaffold with a tailored p K a value to accommodate an acidic lysosomal environment and (2) a green BODIPY-based FLIM probe (Agg-P2) that reports on degradation of cellular aggregates via HaloTag. Both probes exhibit acid-resistant, viscosity-dependent fluorescence intensity and lifetime (τ) responses, which are ready for intensity- and FLIM-based imaging. Photochemical, theoretical, and biochemical characterizations reveal the probes' mechanism-of-actions. In cells, we exploit Lyso-P1 and Agg-P2 to simultaneously quantify both lysosomal and protein aggegates' viscosity changes upon the aggrephagy process via FLIM. We reveal orthogonal changes in microenvironmental viscosities and morphological heterogeneity upon various cellular stresses. Overall, we provide an imaging toolset to quantitatively study aggrephay, which may benefit screening of aggrephay modulators for disease intervention.

Atherosclerosis (AS) is the leading cause of cardiovascular disease. Foam cells, with elevated lipid droplets (LDs) and HClO levels, are the main components of the atherosclerotic plaques that are characteristic of AS. Super-resolution imaging can be used to visualize the distribution of LDs in foam cells at the nanometer level, facilitating the identification of LDs and HClO. In the present study, we report the development of a ratiometric fluorescent probe, SFL-HClO , for super-resolution imaging of LDs and HClO. Super-resolution imaging with this probe revealed the precise structure of LDs at the suborganelle level. Moreover, the fluorescence behavior of SFL-HClO on the surface of LDs verified its excellent performance in detecting HClO in the foam cells. SFL-HClO can sequentially and specifically respond to LDs and HClO via "turn-on" and ratiometric signal output, respectively, thus contributing to precise imaging of foam cells. Importantly, we demonstrate that SFL-HClO can be used to report on upregulated HClO in atherosclerotic plaques in the aorta of AS mice, providing a suitable fluorescent tool for early atherosclerotic disease assessment.

The mitochondria, as one of the essential organelles in cells, are closely associated with numerous biological processes. Therefore, the realization of clear and real-time imaging for tracking mitochondria is of profound significance. Here, we present a mitochondria-targeting fluorescent probe, N(CH 2 ) 3 -PD-NEt , for the real-time fluorescence imaging of mitochondria in living cells. Using the probe, the fluorescence changes of mitochondria stimulated by different drugs were successfully observed by fluorescence imaging. In addition, the dynamic processes of mitochondria and lysosomes during apoptosis were also explored. Importantly, we observed several novel dynamic interaction patterns between mitochondria and lysosomes. Among them, the most prominent pattern involved the noncontact movements of two lysosomes, that is, one lysosome gradually approached the other lysosome over time, eventually coming into contact and merging with it while gradually combining with mitochondria to form new mitochondria. Notably, the protrusions of the mitochondria became increasingly evident during this process. Meanwhile, we successfully observed the dynamic changes of mitochondria with SIM super-resolution imaging. The study provides promising help for the in-depth study of the dynamic processes of mitochondrial physiology and pathology and the study of the interactions between organelles.

Small-molecule fluorescent probes have emerged as potential tools for cancer cell imaging-based diagnostic and therapeutic applications, but their limited selectivity and poor imaging contrast hinder their broad applications. To address these problems, we present the design and construction of a novel near-infrared (NIR) biotin-conjugated and viscosity-activatable fluorescent probe, named as QL-VB , for selective recognition and imaging of cancer cells. The designed probe exhibited a NIR emission at 680 nm, with a substantial Stokes shift of 100 nm and remarkably sensitive responses toward viscosity changes in solution. Importantly, QL-VB provided an evidently enhanced signal-to-noise ratio (SNR: 6.2) for the discrimination of cancer cells/normal cells, as compared with the control probe without biotin conjugation (SNR: 1.8). Moreover, we validated the capability of QL-VB for dynamic monitoring of stimulated viscosity changes within cancer cells and employed QL-VB for distinguishing breast cancer tissues from normal tissues in live mice with improved accuracy (SNR: 2.5) in comparison with the control probe (SNR: 1.8). All these findings indicated that the cancer-targeting and viscosity-activatable NIR fluorescent probe not only enables the mechanistic investigations of mitochondrial viscosity alterations within cancer cells but also holds the potential as a robust tool for cancer cell imaging-based applications.

The rapid and sensitive quantification of low-abundance protein markers holds immense significance in early disease diagnosis and treatment. Single-molecule fluorescence imaging exhibits very high detection sensitivity and thus has great application potential in this area. The single-molecule signal, however, is often susceptible to interference from background noise due to its inherently weak intensity. A variety of signal amplification techniques based on cascading reactions have been developed to improve the signal-to-noise ratio of single-molecule imaging. Nevertheless, the operation of these methods is typically complicated and time-consuming, which limits the clinical application. Herein, we introduce an enzyme-free, photonic-crystal-based single-molecule (PC-SM) biochip for cost-effective, time-efficient, and ultrasensitive detection of disease markers. The PC-SM biochip can enhance the signal-to-noise ratio of single molecules by nearly 3-fold compared with unamplified samples, through coupling of the single-molecule photon energy with the optical band gap of the photonic crystal. We used the PC-SM biochip to detect the low-abundance leukemia inhibitory factor in the blood of pancreatic cancer patients and healthy people and achieved a detection limit of 2.0 pg/L and an AUC of 0.9067. The method exhibits exceptional sensitivity and specificity, showing great application potential in various clinical settings.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

Although targeted therapy has revolutionized oncotherapy, engineering a versatile oncotherapy nanoplatform integrating both diagnostics and therapeutics has always been an intractable challenge to overcome the limitations of monotherapy. Herein, a theranostics platform based on DI/MP-MB has successfully realized the fluorescence detection of disease marker miR-21 and the gene/photothermal/chemo triple synergetic cancer therapy, which can trace the tumor through photothermal and fluorescence dual-mode imaging and overcome the limitations of monotherapy to improve the treatment efficiency of tumors. DI/MP-MB was prepared by magnetic mesoporous silicon nanoparticles (M-MSNs) loaded with doxorubicin (Dox) and new indocyanine green (IR820), and subsequently coating polydopamine as a "gatekeeper", followed by the surface adsorbed with molecular beacons capable of targeting miR-21 for responsive imaging. Under the action of enhanced permeability retention and external magnetic field, DI/MP-MB were targeted and selectively accumulated in the tumor. MiR-21 MB hybridized with miR-21 to form a double strand, which led to the desorption of miR-21 MB from the polydopamine surface and the fluorescence recovery to realize gene silencing and fluorescence imaging for tracking the treatment process. Meanwhile, with the response to the near-infrared irradiation and the tumor's microacid environment, the outer layer polydopamine will decompose, releasing Dox and IR820 to realize chemotherapy and photothermal therapy. Finally, the ability of DI/MP-MB to efficiently suppress tumor growth was comprehensively assessed and validated both in vitro and in vivo. Noteworthily, the excellent anticancer efficiency by the synergistic effect of gene/photothermal/chemo triple therapy of DI/MP-MB makes it an ideal nanoplatform for tumor therapy and imaging.

The occurrence and development of diseases are accompanied by abnormal activity or concentration of biomarkers in cells, tissues, and blood. However, the insufficient sensitivity and accuracy of the available fluorescence probes hinder the precise monitoring of associated indexes in biological systems, which is generally due to the high probe intrinsic fluorescence and false-negative signal caused by the reactive oxygen species (ROS)-induced probe decomposition. To resolve these problems, we have engineered a ROS-stable, meso-carboxylate boron dipyrromethene (BODIPY)-based fluorescent probe, which displays quite a low background fluorescence due to the doubly quenched intrinsic fluorescence by a combined strategy of the photoinduced electron transfer (PET) effect and "ester-to-carboxylate" conversion. The probe achieved a high S/N ratio with ultrasensitivity and good selectivity toward biothiols, endowing its fast detection capability toward the biothiol level in 200×-diluted plasma samples. Using this probe, we achieved remarkable distinguishing of liver injury plasma from normal plasma even at 80× dilution. Moreover, owing to its good stability toward ROS, the probe was successfully employed for high-fidelity imaging of the negative fluctuation of the biothiol level in nonsmall-cell lung cancer (NSCLC) during dihydroartemisinin-induced ferroptosis. This delicate design of suppressing intrinsic fluorescence reveals insights into enhancing the sensitivity and accuracy of fluorescent probes toward the detection and imaging of biomarkers in the occurrence and development of diseases.

The viscosity that ensures the controlled diffusion of biomolecules in cells is a crucial biophysical parameter. Consequently, fluorescent probes capable of reporting viscosity variations are valuable tools in bioimaging. In this field, red-shifted probes are essential, as the widely used and gold standard probe remains green-emitting molecular rotors based on BODIPY. Here, we demonstrate that pyrrolyl squaraines, red-emissive fluorophores, exhibit high sensitivity over a wide viscosity range from 30 to 4890 mPa·s. Upon alkylation of the pyrrole moieties, the probes improve their sensitivity to viscosity through an enhanced twisted intramolecular charge transfer phenomenon. We utilized this scaffold to develop a plasma membrane probe, pSQ-PM, that efficiently stains the plasma membrane in a fluorogenic manner. Using fluorescence lifetime imaging, pSQ-PM enabled efficient sensing of viscosity variations in the plasma membrane under various conditions and in different cell lines (HeLa, U2OS, and NIH/3T3). Moreover, upon incubation, pSQ-PM stained the membrane of intracellular vesicles and suggested that the lysosomal membranes displayed enhanced fluidity.

Accurate diagnosis and effective treatment of tumors remain significant clinical challenges. While fluorescence imaging is essential for tumor detection, it has limitations in terms of specificity, penetration depth, and emission wavelength. Here, we report a novel glutathione (GSH)-responsive peptide self-assembly excimer probe ( pSE ) that optimizes two-photon tumor imaging and self-assisted counteraction of the cisplatin resistance in cancer cells. The GSH-responsive self-assembly of pSE induces a monomer-excimer transition of coumarin, promoting a near-infrared redshift of fluorescence emission under two-photon excitation. This process enhances penetration depth and minimizes interference from biological autofluorescence. Moreover, the intracellular self-assembly of pSE impacts GSH homeostasis, modulates relevant signaling pathways, and significantly reduces GSTP1 expression, resulting in decreased cisplatin efflux in cisplatin-resistant cancer cells. The proposed self-assembled excimer probe not only distinguishes cancer cells from normal cells but also enhances the efficacy of cisplatin chemotherapy, offering significant potential in tumor diagnosis and overcoming cisplatin-resistant tumors.

Developing a novel strategy to improve the optical performances of fluorescent probes is a vital factor in elevating its practical application; viz., novel biocompatible fluorescent probes with excellent multifunctions exhibited unparalleled advantages in probing functions of intracellular molecules to elucidate intracellular events in living systems. Herein, we have successfully constructed a new strategy that aggregation and coordination synergistically induce (2-hydroxylphenyl-benzothiazole) HBT derivatives to form excimers with large red-shifted fluorescence and application for insight into stress-response zinc fluctuations in living systems. We have synthesized four HBT-based derivatives and deeply investigated the response mechanism by fluorescent spectral studies, demonstrating that probes 3 and 4 showcased large red shifts in emission wavelength due to J -aggregation. More interestingly, the fluorescence of probe 4 was significantly enhanced in the presence of a zinc ion, suggesting that zinc coordination synergistically induced J -aggregation. Probe 4 was successfully applied to image zinc fluctuations in different models of living systems, proving that this probe is a powerful tool to unveil the relationship between invasive stress and diseases by monitoring endogenous zinc fluctuations.

Dipeptidyl peptidase 4 (DPP4) plays a key role in glucose metabolism, which has been a close target for diabetes pathology and treatment. It is significant for the evaluation of cellular DPP4 activity in various biological systems. Fluorescence imaging technology is currently a popular method for detecting enzymes in living cells due to its advantages of high selectivity, high sensitivity, high spatiotemporal resolution, and real-time visualization. Herein, a near-infrared (NIR)-emissive probe NEDP with a large Stokes shift (153 nm) was developed for the assay of DPP4 activity. Upon addition of DPP4, NEDP can emit a significant turn-on NIR fluorescence signal (673 nm) with high sensitivity and specificity. Moreover, NEDP can successfully be used for imaging of intracellular DPP4, confirming the regulation of DPP4 expression in hyperglucose and its treatment in living cells. Most importantly, NEDP can not only monitor the changes of DPP4 in vivo but also show that DPP4 in diabetes is mainly up-regulated in the liver, and the level of DPP4 is positively correlated with the pathological damage of the liver. In addition, NEDP can identify the serum of diabetic patients from healthy people through the fluorescence response to DPP4. These results demonstrated that the designed probe NEDP provides a prospective visual tool to explore the relationship between DPP4 and diabetes and would be applied for detecting serum of diabetes in the clinic.

Drug resistance in multiple myeloma (MM) poses a significant challenge to treatment efficacy, primarily attributed to P-glycoprotein (P-gp) dysfunction. This study delves into the elusive spatial organization of P-gp, aiming to enhance our understanding of its role in MM drug resistance by exploring the intricate relationship between molecular function and spatial arrangement. Employing super-resolution imaging of P-gp with the inhibitor probe Tariquidar-TAMR labeling on MM cell membranes, the research uncovered a more pronounced clustering distribution of P-gp in drug-resistant cells (MM1R) compared to drug-sensitive counterparts (MM1S). Further exploration revealed the clustering distribution of P-gp was heightened as cellular drug resistance increased in hypoxic condition, directly emphasizing the strong correlation between P-gp cluster morphology and drug resistance. Additionally, stable P-gp cluster formation was influenced by cross-linking of membrane carbohydrates, and disrupting these glycoprotein clusters could reduce cellular drug resistance, suggesting that altering distribution patterns of P-gp can modulate drug responsiveness. Finally, dexamethasone (Dex) treatment was revealed to enhance P-gp clustering distribution, particularly in MM1S cells, indicating that change degree in P-gp distribution correlate with the modifiable space of cellular drug responsiveness. This study provides insights into the correlation between P-gp assembly and cellular drug responsiveness, deepening our understanding of functional changes in MM drug resistance and offering valuable perspectives for overcoming this challenge.

Oxidative stress, characterized by an imbalance between oxidative and antioxidant processes, results in excessive accumulation of intracellular reactive oxygen species. Among these responses, the regulation of intracellular hydroxyl radicals ( • OH) and glutathione (GSH) is vital for physiological processes. Real-time in situ monitoring these two opposing bioactive species and their redox interactions is essential for understanding physiological balance and imbalance. In this study, we developed a dual-site fluorescence chemosensor OG-3 , which can independently image both exogenous and endogenous • OH and GSH in separate channels both within cells and in vivo, eliminating issues of spatiotemporal inhomogeneous distribution and cross-interference. With its imaging capabilities of monitoring • OH-GSH redox, OG-3 elucidated two different pathways for ferroptosis induction: (i) inhibition of system x c - to block cystine uptake (extrinsic pathway) and (ii) GPX4 inactivation, leading to the loss of antioxidant defense (intrinsic pathway). Moreover, we assessed the antiferroptotic function and effects of ferroptosis inhibitors by monitoring • OH and GSH fluctuations during ferroptosis. This method provides a reliable platform for identifying potential ferroptosis inhibitors, contributing to our understanding of relevant metabolic and physiological mechanisms. It shows potential for elucidating the regulation of ferroptosis mechanisms and investigating further strategies for therapeutic applications.

Activatable probes with a higher signal-to-background ratio and accuracy are essential for monitoring liver cancer as well as intraoperative fluorescence navigation. However, the presence of only one biomarker is usually not sufficient to meet the high requirement of a signal-to-background ratio in cancer surveillance, leading to the risk of misdiagnosis. In this work, a dual-locked activation response probe, Si-NTR-LAP , for nitroreductase and leucine aminopeptidase was reported. This dual-locked probe provides better tumor recognition and a higher signal-to-noise ratio than that of single-locked probes ( Si-LAP and Si-NTR ). In both the subcutaneous tumor model and the more complex orthotopic hepatocellular carcinoma model, the probe was able to identify tumor tissue with high specificity and accurately differentiate the boundaries between tumor tissue and normal tissue. Therefore, the dual-locked probe may provide a new and practical strategy for applying to real patient tumor tissue samples.

Aging represents a significant risk factor for compromised tissue function and the development of chronic diseases in the human body. This process is intricately linked to oxidative stress, with HClO serving as a vital reactive oxygen species (ROS) within biological systems due to its strong oxidative properties. Hence, conducting a thorough examination of HClO in the context of aging is crucial for advancing the field of aging biology. In this work, we successfully developed a fluorescent probe, OPD , tailored specifically for detecting HClO in senescent cells and in vivo. Impressively, OPD exhibited a robust reaction with HClO, showcasing outstanding selectivity, sensitivity, and photostability. Notably, OPD effectively identified HClO in senescent cells for the first time, confirming that DOX- and ROS-induced senescent cells exhibited higher HClO levels compared to uninduced normal cells. Additionally, in vivo imaging of zebrafish demonstrated that d-galactose- and ROS-stimulated senescent zebrafish displayed elevated HClO levels compared to normal zebrafish. Furthermore, when applied to mouse tissues and organs, OPD revealed increased fluorescence in the organs of senescent mice compared to their nonsenescent counterparts. Our findings also illustrated the probe's potential for detecting changes in HClO content pre- and post-aging in living mice. Overall, this probe holds immense promise as a valuable tool for in vivo detection of HClO and for studying aging biology in live organisms.

Inflammatory bowel disease (IBD) is an idiopathic intestinal inflammatory disease, whose etiology is intimately related to the overproduction of hypochlorous acid (HClO). Optical monitoring of HClO in the living body favors real-time diagnosis of inflammatory diseases. However, HClO-activated near-infrared (NIR) fluorescent probes with rapid response and high inflammatory cell uptake are still lacking. Herein, we report an activatable acceptor-π-acceptor (A-π-A)-type NIR fluorescent probe ( Cy-DM ) bearing two d-mannosamine groups for the sensitive detection of HClO in early IBD and stool testing. Once reacted with HClO, nonfluorescent Cy-DM could be turned on within 2 s by generating a donor-π-acceptor (D-π-A) structure due to the enhanced intramolecular charge transfer mechanism, showing intense NIR fluorescence emission at 700 nm and a large Stokes shift of 115 nm. Moreover, it was able to sensitively and selectively image exogenous and endogenous HClO in the lysosomes of living cells with a detection limit of 0.84 μM. More importantly, because of the d-mannosamine modification, Cy-DM was efficiently taken up by inflammatory cells in the intestine after intravenous administration, allowing noninvasive visualization of endogenous HClO in a lipopolysaccharide-induced IBD mouse model with a high fluorescence contrast of 6.8/1. In addition, water-soluble Cy-DM has also been successfully applied in ex vivo optical fecal analysis, exhibiting a 3.4-fold higher fluorescence intensity in the feces excreted by IBD mice. We believe that Cy-DM is promising as an invaluable tool for rapid diagnosis of HClO-related diseases as well as stool testing.

Fluorescence sensing and imaging techniques are being widely studied for detecting carbon monoxide (CO) in living organisms due to their speed, sensitivity, and ease of use to biological systems. Most fluorescent probes used for this purpose are based on heavy metal ions like Pd, with a few using elements like Ru, Rh, Ir, Os, Tb, and Eu. However, these metals can be expensive and toxic to cells. There is a need for more affordable and biologically safe fluorescent probes for CO detection. Drawing inspiration from the robust affinity exhibited by heme iron toward CO, in this work, a rhodamine derivative called RBF was developed for imaging CO in living cells by binding to Fe(III) and could be used for CO sensing. A Fe(III)-based fluorescent probe for CO imaging in living cells offers advantages of cost effectiveness, low toxicity, and ease of use. The fluorescence detection using the RBF-Fe system showed a direct correlation with increasing levels of CORM-3 (LOD = 146 nM) or the exposure time of CO gas, displaying reduced fluorescence. A CO test paper based on RBF-Fe was created for simple on-site CO detection, where fluorescence would diminish in response to CO exposure, allowing rapid (2 min) visual identification. Imaging of CO in living cells was successfully conducted using the probe system, showing a decrease in fluorescence intensity as CORM-3 concentrations increased, indicating its effectiveness in monitoring CO levels accurately within living cells.

Ferroptosis is an iron-dependent programmed cell death that is characterized by the dysregulation of lipid reactive oxygen species (ROS) production, causing abnormal changes in hypochlorous acid (HClO) levels in lysosomes. Super-resolution imaging can observe the fine structure of the lysosome at the nanometer level; therefore, it can be used to detect lysosome HClO levels during ferroptosis at the suborganelle level. Herein, we utilize a ratiometric fluorescent probe, SRF-HClO , for super-resolution imaging of lysosome HClO. Structured-illumination microscopy (SIM) improves the accuracy of lysosome targeting and enables the probe SRF-HClO to be successfully applied to rapidly monitor the up-regulated lysosome HClO at the nanoscale during inflammation and ferroptosis. Importantly, the probe SRF-HClO can also detect HClO changes in inflammatory and ferroptosis mice and evaluate the inhibitory effect of ferroptosis on mice tumors.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho -quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.

Carboxylesterase (CE), an enzyme widely present in organisms, is involved in various physiological and pathological processes. Changes in the levels of CEs in the liver may predict the presence of type 2 diabetes mellitus (T2DM). Here, a novel dicyanoisophorone (DCI)-based proximity-labeled far-red fluorescent probe DCI2F-Ac with endoplasmic reticulum targeting was proposed for real-time monitoring and imaging of the CEs activity. DCI2F-Ac featured very low cytotoxicity and biotoxicity and was highly selective and sensitive for CEs. Compared with traditional CEs probes, DCI2F-Ac was covalently anchored directly to CEs, thus effectively reducing the loss of in situ fluorescent signals due to diffusion. Through the "on-off" fluorescence signal readout, DCI2F-Ac was able to distinguish cell lines and screen for CEs inhibitors. In terms of endoplasmic reticulum (ER) stress, it was found that thapsigargin (Tg) induced upregulation of CEs levels but not tunicamycin (Tm), which was related to the calcium homeostasis of the ER. DCI2F-Ac could efficiently detect downregulated CEs in the livers of T2DM, and the therapeutic efficacy of metformin, acarbose, and a combination of these two drugs was assessed by tracking the fluctuation of CEs levels. The results showed that combining metformin and acarbose could restore CEs levels to near-normal levels with the best antidiabetic effect. Thus, the DCI2F-Ac probe provides a great opportunity to explore the untapped potential of CEs in liver metabolic disorders and drug efficacy assessment.

The uptake of plastic particles by plants and their transport through the food chain make great risks to biota and human health. Therefore, it is important to trace plastic particles in the plant. Traditional fluorescence imaging in plants usually suffers significant autofluorescence background. Here, we report a persistent luminescence nanoplatform for autofluorescence-free imaging and quantitation of submicrometer plastic particles in plant. The nanoplatform was fabricated by doping persistent luminescence nanoparticles (PLNPs) onto polystyrene (PS) nanoparticles. Cr 3+ -doped zinc gallate PLNP was employed as the dopant for autofluorescence-free imaging due to its persistent luminescence nature. In addition, the Ga element in PLNP was used as a proxy to quantify the PS in the plant by inductively coupled plasma mass spectrometry (ICP-MS). Thus, the developed nanoplatform allows not only dual-mode autofluorescence-free imaging (persistent luminescence and laser-ablation ICP-MS) but also ICP-MS quantitation for tracking PS in plant. Application of this nanoplatform in a typical plant model Arabidopsis thaliana revealed that PS mainly distributed in the root (>99.45%) and translocated very limited (<0.55%) to the shoot. The developed nanoplatform has great potential for quantitative tracing of submicrometer plastic particles to investigate the environmental process and impact of plastic particles.

Hydrogen peroxide (H 2 O 2 ) overexpressed in mitochondria has been regarded as a key biomarker in the pathological processes of various diseases. However, there is currently a lack of suitable mitochondria-targetable near-infrared (NIR) probes for the visualization of H 2 O 2 in multiple diseases, such as PM 2.5 exposure-induced lung injury, hepatic ischemia-reperfusion injury (HIRI), nonalcoholic fatty liver (NAFL), hepatic fibrosis (HF), and malignant tumor tissues containing clinical cancer patient samples. Herein, we conceived a novel NIR fluorescent probe ( HCy-H 2 O 2 ) by introducing pentafluorobenzenesulfonyl as a H 2 O 2 sensing unit into the NIR hemicyanine platform. HCy-H 2 O 2 exhibits good sensitivity and selectivity toward H 2 O 2 , accompanied by a remarkable "turn-on" fluorescence signal at 720 nm. Meanwhile, HCy-H 2 O 2 has stable mitochondria-targetable ability and permits monitoring of the up-generated H 2 O 2 level during mitophagy. Furthermore, using HCy-H 2 O 2 , we have successfully observed an overproduced mitochondrial H 2 O 2 in ambient PM 2.5 exposure-induced lung injury, HIRI, NAFL, and HF models through NIR fluorescence imaging. Significantly, the visualization of H 2 O 2 has been achieved in both tumor-bear mice as well as surgical specimens of cancer patients, making HCy-H 2 O 2 a promising tool for cancer diagnosis and imaging-guided surgery.

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.

Epinephrine (EP) is an essential catecholamine in the human body. Currently, most EP detection methods are not suitable for in vivo detection due to material limitations. An organic small molecule fluorescent probe based on a chemical cascade reaction for the detection of EP was designed. Anionic heptamethine cyanine dye was selected as a fluorescent dye because of its NIR fluorescence emission with excellent biocompatibility. The secondary amine of EP nucleophilically attacks the carbonate of the probe with its stronger nucleophilicity and further undergoes intramolecular nucleophilic cyclization to release the fluorophore. Other substances containing only primary amines or no β-OH lack reaction competitiveness due to their weaker nucleophilicity or inability to undergo further cyclization. The fluorescence recovery of the probe was linearly related to the EP concentration of 2-75 μmol/L. The detection limit was 0.4 μmol/L. The recovery rate was 94.78-111.32%. Finally, we successfully achieved bioimaging of EP in living cells and EP analogue in nematodes.

Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1 AP , and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1 AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1 AP ). Subsequently, miRNA-21 facilitates the hybridization of activated H1 AP on tFNA-H1 AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1 AP at the vertices of tFNA-H1 AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.

Ferroptosis is a pattern of cell death caused by iron-dependent accumulation of lipid peroxides and is closely associated with the occurrence and development of multiple diseases. Acrolein (ACR), one of the final metabolites of lipid peroxidation, is a reactive carbonyl species with strong biotoxicity. Effective detection of ACR is important for understanding its role in the progression of ferroptosis and studying the specific mechanisms of ferroptosis-mediated diseases. However, visualization detection of ACR during ferroptosis has not yet been reported. In this work, the first ratiometric fluorescent probe ( HBT-SH ) based on 2-(2'-hydroxyphenyl) benzothiazole (HBT) was designed for tracing endogenous ACR with an unprecedented regiospecific ACR-induced intramolecular cyclization strategy, which employs 2-aminoethanethiol as an ACR-selective recognition receptor. The experimental results showed that HBT-SH has excellent selectivity, high sensitivity (LOD = 0.26 μM) and good biocompatibility. More importantly, the upregulation of ACR levels was observed during ferroptosis in HeLa cells and zebrafish, indicating that ACR may be a specific active molecule that plays an essential biological role during ferroptosis or may serve as a potential marker of ferroptosis, which has great significance for studying the pathological process and treatment options of ferroptosis-related diseases.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate " n " secondary targets, thereby initiating " n " nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

Visualization of the mitochondrial state is crucial for tracking cell life processes and diagnosing disease, while fluorescent probes that can accurately assess mitochondrial status are currently scarce. Herein, a fluorescent probe named " SYN " was designed and prepared, which can target mitochondria via the mitochondrial membrane potential. Upon pathology or external stimulation, SYN can be released from the mitochondria and accumulate in the nucleolus to monitor the status of mitochondria. During this process, the brightness of the nucleolus can then serve as an indicator of mitochondrial damage. SYN has demonstrated excellent photostability in live cells as well as an extremely inert fluorescence response to bioactive molecules and the physiological pH environment of live cells. Spectroscopic titration and molecular docking studies have revealed that SYN can be lit up in nucleoli due to the high viscosity of the nucleus and the strong electrostatic interaction with the phosphate backbone of RNA. This probe is expected to be an exceptional tool based on its excellent imaging properties for tracking mitochondrial state in live cells.

Ulcerative colitis is a persistent inflammatory bowel disease characterized by inflammation and ulceration in the colon and gastrointestinal tract. It was indicated that the generation of hypochlorous acid (HClO) through the enzymatic activity of myeloperoxidase is significantly linked to ulcerative colitis. In this study, by assembling two hairpins (Hpa and Hpb) onto a quadrivalent cruciform DNA nanostructure, a novel HClO-activatable fluorescent probe was developed based on DNA nanomaterials (denoted MHDNA), which is sensitive, economic, simple, and stable. In the presence of HClO, the Trigger (T) was liberated from the MHDNA probe through a hydrolysis reaction between HClO and phosphorothioate (PS), which is modified on the MHDNA probe and has proved to exhibit particular susceptibility to the HClO. The liberated T subsequently initiated the opening of Hpa and Hpb to facilitate the catalyzed hairpin assembly (CHA) reaction, resulting in the changes of fluorescence and releasing T for recycled signal amplification to achieve sensitive detection of HClO (with a limit of detection 9.83 nM). Additionally, the MHDNA-based spatial-confinement effect shortens the physical distance between Hpa and Hpb and yields a high local concentration of the two reactive hairpins, achieving more rapid reaction kinetics in comparison to conventional CHA methods. Inspirationally, the MHDNA probe was effectively utilized for imaging HClO in ulcerative colitis mice, yielding valuable diagnostic insights for ulcerative colitis.

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.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

Gold nanorods (AuNRs) have been considered highly compelling materials for early cancer diagnosis and have aroused a burgeoning fascination among the biomedical sectors. By leveraging the versatile tunable optical properties of AuNRs, herein, we have developed a novel tumor-targeted dual-modal nanoprobe (FFA) that exhibits excellent bioluminescence and photoacoustic imaging performance for early tumor diagnosis. FFA has been synthesized by anchoring the recombinant bioluminescent firefly luciferase protein (Fluc) on the folate-conjugated AuNRs via the PEG linker. TEM images and UV-vis studies confirm the nanorod morphology and successful conjugation of the biomolecules to AuNRs. The nanoprobe FFA relies on the ability of the folate module to target the folate receptor-positive tumor cells actively, and simultaneously, the Fluc module facilitates excellent bioluminescent properties in physiological conditions. The success of chemical engineering in the present study enables stronger bioluminescent signals in the folate receptor-positive cells (Skov3, Hela, and MCF-7) than in folate receptor-negative cells (A549, 293T, MCF-10A, and HepG2). Additionally, the AuNRs induced strong photoacoustic conversion performance, enhancing the resolution of tumor imaging. No apparent toxicity was detected at the cellular and mouse tissue levels, manifesting the biocompatibility nature of the nanoprobe. Prompted by the positive merits of FFA, the in vivo animal studies were performed, and a notable enhancement was observed in the bioluminescent/photoacoustic intensity of the nanoprobe in the tumor region compared to that in the folate-blocking region. Therefore, this synergistic dual-modal bioluminescent and photoacoustic imaging platform holds great potential as a tumor-targeted contrast agent for early tumor diagnosis with high-performance imaging information.

This study explores the synthesis and characterization of aggregation-induced emission enhancement (AIEE)-active gold nanoclusters (AuNCs), focusing on their near-infrared luminescence properties and potential applications in biological imaging. These AIEE-active AuNCs were synthesized via the NaBH 4 -mediated reduction of HAuCl 4 in the presence of peptides. We systematically investigated the influence of the peptide sequence on the optical features of the AuNCs, highlighting the role of glutamic acid in enhancing their quantum yield (QY). Among the synthesized peptide-stabilized AuNCs, EECEE-stabilized AuNCs exhibited the maximum QY and a pronounced AIEE effect at pH 5.0, making them suitable for the luminescence imaging of intracellular lysosomes. The AIEE characteristic of the EECEE-stabilized AuNCs was demonstrated through examinations using transmission electron microscopy, dynamic light scattering, zeta potential analysis, and single-particle imaging. The formation of the EECEE-stabilized AuNCs was confirmed by size-exclusion chromatography and mass spectrometry. Spectroscopic and electrochemical examinations uncover the formation process of EECEE-stabilized AuNCs, comprising EECEE-mediated reduction, NaBH 4 -induced nucleation, complex aggregation, and subsequent cluster growth. Furthermore, we demonstrated the utility of these AuNCs as luminescent probes for intracellular lysosomal imaging, leveraging their pH-responsive AIEE behavior. Additionally, cyclic arginylglycylaspartic acid (RGD)-modified AIEE dots, derived from cyclic RGD-linked peptide-induced aggregation of EECEE-stabilized AuNCs, were developed for single- and two-photon luminescence imaging of α v β 3 integrin receptor-positive cancer cells.

Herein, a fluorescence light-up 3D DNA walker (FLDW) was powered and accelerated by endogenous adenosine-5'-triphosphate (ATP) molecules to construct a biosensor for sensitive and rapid label-free detection and imaging of microRNA-221 (miRNA-221) in malignant tumor cells. Impressively, ATP as the driving force and accelerator for FLDW could significantly accelerate the operation rate of FLDW, reduce the likelihood of errors in signaling, and improve the sensitivity of detection and imaging. When FLDW was initiated by output DNA H1-op transformed by target miRNA-221, G-rich sequences in the S strand, anchored to AuNP, were exposed to form G-quadruplexes (G4s), and thioflavin T (ThT) embedded in the G4s emitted intense fluorescence to realize sensitive and rapid detection of target miRNA-221. Meanwhile, the specific binding of ThT to G4 with a weak background fluorescence response was utilized to enhance the signal-to-noise ratio of the label-free assay straightforwardly and cost-effectively. The proposed FLDW system could realize sensitive detection of the target miRNA-221 in the range of 1 pM to 10 nM with a detection limit of 0.19 pM by employing catalytic hairpin assembly (CHA) to improve the conversion of the target. Furthermore, by harnessing the abundant ATP present in the tumor microenvironment, FLDW achieved rapid and accurate imaging of miRNA-221 in cancer cells. This strategy provides an innovative and high-speed label-free approach for the detection and imaging of biomarkers in cancer cells and is expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Singlet oxygen ( 1 O 2 ) plays imperative roles in a variety of biotic or abiotic stresses in crops. The change of its concentration within a crop is closely related to the crop growth and development. Accordingly, there is an urgent need to develop an efficient analytical method for on-site quantitative detection of 1 O 2 in crops. Here, we judiciously constructed a novel ratiometric fluorescent probe, SX-2 , for the detection of 1 O 2 in crops. Upon treating with 1 O 2 , probe SX-2 displayed highly selective ratiometric fluorescence response, which is favorable for the quantitative detection of 1 O 2 . Concurrently, the fluorescence solution color of probe SX-2 was varied, obviously from blue to yellow, indicating that the probe is beneficial for on-site detection by the naked eye. Sensing reaction mechanism studies showed that the 2,3-diphenyl imidazole group in SX-2 could function as a new selective recognition group for 1 O 2 . Probe SX-2 was utilized for the detection of photoirradiation-induced 1 O 2 and endogenous 1 O 2 in living cells. The changes in the 1 O 2 level in zebrafish were also tracked by fluorescence imaging. In addition, the production of 1 O 2 in crop leaves under a light source of different wavelengths was studied. The results demonstrated more 1 O 2 were produced under a light source of 365 nm. Furthermore, to achieve on-site quantitative detection, a mobile fluorescence analysis device has been made. Probe SX-2 and mobile fluorescence analysis device were capable of on-site quantitative detecting of 1 O 2 in crops. The method developed herein will be convenient for the on-site quantitative measurement of 1 O 2 in distinct crops.

Allysine, a pivotal biomarker in fibrogenesis, has prompted the development of various radioactive imaging probes. However, fluorogenic probes targeting allysine remain largely unexplored. Herein, by leveraging the equilibrium between the nonfluorescent spirocyclic and the fluorescent zwitterionic forms of rhodamine-cyanine hybrid fluorophores, we systematically fine-tuned the environmental sensitivity of this equilibrium toward the development of fluorogenic probes for fibrosis. The trick lies in modulating the nucleophilicity of the ortho -carboxyl group, which is terminated with a hydrazide group for allysine conjugation. Probe B2 was developed with this strategy, which featured an N -sulfonyl amide group and exhibited superior fibrosis-to-control imaging contrast. Initially presenting as nonfluorescent spirocyclic aggregates in aqueous solutions, B2 displayed a notable fluorogenic response upon conjugation with protein allysine through its hydrazide group, inducing deaggregation and switching to the fluorescent zwitterionic form. Probe B2 outperformed the traditional Masson stain in imaging contrast, achieving an about 260-2600-fold ratio for fibrosis-to-control detection depending on fibrosis severity. Furthermore, it demonstrated efficacy in evaluating antifibrosis drugs. Our results emphasize the potential of this fluorogenic probe as an alternative to conventional fibrosis detection methods. It emerges as a valuable tool for antifibrosis drug evaluation.

Despite recent advancements in colorectal cancer (CRC) treatment, the prognosis remains unfavorable primarily due to high recurrence and liver metastasis rates. Fluorescence molecular imaging technologies, combined with specific probes, have gained prominence in facilitating real-time tumor resection guided by fluorescence. Hepatocyte growth factor (HGF) is overexpressed in CRC, but the advancement of HGF fluorescent probes has been impeded by the absence of effective HGF-targeting small-molecular ligands. Herein, we present the targeted capabilities of the novel V-1-GGGK-MPA probe labeled with a near-infrared fluorescent dye, which targets HGF in CRC. The V-1-GGGK peptide exhibits high specificity and selectivity for HGF-positive in vitro tumor cells and in vivo tumors. Biodistribution analysis of V-1-GGGK-MPA revealed tumor-specific accumulation with low background uptake, yielding signal-to-noise ratio (SNR) values of tumor-to-colorectal >6 in multiple subcutaneous CRC models 12 h postinjection. Quantitative analysis confirmed the probe's high uptake in SW480 and HT29 orthotopic and liver metastatic models, with SNR values of tumor-to-colorectal and -liver being 5.6 ± 0.4, 4.6 ± 0.5, and 2.1 ± 0.3, 2.0 ± 0.5, respectively, enabling precise tumor visualization for surgical navigation. Pathological analysis demonstrated the excellent tumor boundaries discrimination capacity of the V-1-GGGK-MPA probe at the molecular level. With its rapid tumor targeting, sustained tumor retention, and precise tumor boundary delineation, V-1-GGGK-MPA merges as a promising HGF imaging agent, enriching the toolbox of intraoperative navigational fluorescent probes for CRC.

Lipids are essential for various cellular functions, including energy storage, membrane flexibility, and signaling molecule production. Maintaining proper lipid levels is important to prevent health problems such as cancer, neurodegenerative disorders, cardiovascular diseases, obesity, and diabetes. Monitoring cellular lipid droplets (LDs) in real-time with high resolution can provide insights into LD-related pathways and diseases owing to the dynamic nature of LDs. Fluorescence-based imaging is widely used for tracking LDs in live cells and animal models. However, the current fluorophores have limitations such as poor photostability and high background staining. Herein, we developed a novel fluorogenic probe based on a push-pull interaction combined with aggregation-induced emission enhancement (AIEE) for dynamic imaging of LDs. Probe 1 exhibits favorable membrane permeability and spectroscopic characteristics, allowing specific imaging of cellular LDs and time-lapse imaging of LD accumulation. This probe can also be used to examine LDs in fruit fly tissues in various metabolic states, serving as a highly versatile and specific tool for dynamic LD imaging in cellular and tissue environments.

Development of reliable methods for the detection of potential biomarkers is of the utmost importance for an early diagnosis of critical diseases and disorders. In this study, a novel lanthanide-functionalized carbon dot-based fluorescent probe Zn-CD@Eu is reported for the ratiometric detection of dipicolinic acid (DPA) and uric acid (UA). The Zn-CD@Eu nanoprobe was obtained from a simple room-temperature reaction of zinc-doped carbon dots (Zn-CD) and the EDTA-Eu lanthanide complex. Under optimal conditions, a good linear response was obtained for DPA in two concentration ranges of 0-55 and 55-100 μM with a limit of detection of 0.53 and 2.2 μM respectively, which is significantly below the infectious dosage of anthrax (∼55 μM). Furthermore, the Zn-CD@Eu/DPA system was employed for the detection of UA with a detection limit of 0.36 μM in the linear range of 0-100 μM. The fluorescent probe was successfully implemented for determining DPA and UA in human blood serum, sweat, and natural water bodies with considerable recovery rates. In addition, the potential of the nanoprobe for ex vivo visualization of UA was demonstrated in fruit fly ( Drosophila melanogaster ) as a model organism.

Tumor microenvironment-responsive phototheranostic agents are highly sought after for their ability to improve diagnostic accuracy and treatment specificity. Here, we introduce a novel single-molecule probe, POZ-NO , which is activated by nitric oxide (NO) and weak acidity, enabling dual-mode imaging and photothermal therapy (PTT) of tumors. In acidic environments with elevated NO levels, POZ-NO exhibits a distinctive ratiometric fluorescence signal shift from the red to near-infrared, accompanied by a 700 nm photoacoustic signal. Additionally, POZ-NO demonstrated potent photothermal effects upon NO and acidity activation, achieving an impressive conversion efficiency of 74.3% under 735 nm laser irradiation. In vivo studies confirm POZ-NO 's ability to accurately image tumors through ratiometric fluorescence and photoacoustic modes while selectively treating tumors with PTT.