Iron is an essential element for life owing to its ability to participate in a diverse array of oxidation-reduction reactions. However, misregulation of iron-dependent redox cycling can also produce oxidative stress, contributing to cell growth, proliferation, and death pathways underlying aging, cancer, neurodegeneration, and metabolic diseases. Fluorescent probes that selectively monitor loosely bound Fe(II) ions, termed the labile iron pool, are potentially powerful tools for studies of this metal nutrient; however, the dynamic spatiotemporal nature and potent fluorescence quenching capacity of these bioavailable metal stores pose challenges for their detection. Here, we report a tandem activity-based sensing and labeling strategy that enables imaging of labile iron pools in live cells through enhancement in cellular retention. Iron green-1 fluoromethyl (IG1-FM) reacts selectively with Fe(II) using an endoperoxide trigger to release a quinone methide dye for subsequent attachment to proximal biological nucleophiles, providing a permanent fluorescent stain at sites of elevated labile iron. IG1-FM imaging reveals that degradation of the major iron storage protein ferritin through ferritinophagy expands the labile iron pool, while activation of nuclear factor-erythroid 2-related factor 2 (NRF2) antioxidant response elements (AREs) depletes it. We further show that lung cancer cells with heightened NRF2 activation, and thus lower basal labile iron, have reduced viability when treated with an iron chelator. By connecting labile iron pools and NRF2-ARE activity to a druggable metal-dependent vulnerability in cancer, this work provides a starting point for broader investigations into the roles of transition metal and antioxidant signaling pathways in health and disease.

Copper participates in a range of critical functions in the nervous system and human brain. Disturbances in brain copper content is strongly associated with neurological diseases. For example, changes in the level and distribution of copper are reported in neuroblastoma, Alzheimer\'s disease, and Lewy body disorders, such as Parkinson disease and dementia with Lewy bodies (DLB). There is a need for more sensitive techniques to measure intracellular copper levels to have a better understanding of the role of copper homeostasis in neuronal disorders. Here, we report a reaction-based near-infrared (NIR) ratiometric fluorescent probe CyCu1 for imaging Cu2+ in biological samples. High stability and selectivity of CyCu1 enabled the probe to be deployed as a sensor in a range of systems, including SH-SY5Y cells and neuroblastoma tumors. Furthermore, it can be used in plant cells, reporting on copper added to Arabidopsis roots. We also used CyCu1 to explore Cu2+ levels and distribution in post-mortem brain tissues from patients with DLB. We found significant decreases in Cu2+ content in the cytoplasm, neurons, and extraneuronal space in the degenerating substantia nigra in DLB compared with healthy age-matched control tissues. These findings enhance our understanding of Cu2+ dysregulation in Lewy body disorders. Our probe also shows promise as a photoacoustic imaging agent, with potential for applications in bimodal imaging.

Nitric oxide (NO) generated within the tumor microenvironment is an established driver of cancer progression and metastasis. Recent efforts have focused on leveraging this feature to target cancer through the development of diagnostic imaging agents and activatable chemotherapeutics. In this context, porphyrins represent an extraordinarily promising class of molecules, owing to their demonstrated use within both modalities. However, the remodeling of a standard porphyrin to afford a responsive chemical that can distinguish elevated NO from physiological levels has remained a significant research challenge. In this study, we employed a photoinduced electron transfer strategy to develop a panel of NO-activatable porphyrin photosensitizers (NOxPorfins) augmented with real-time fluorescence monitoring capabilities. The lead compound, NOxPorfin-1, features an o-phenylenediamine trigger that can effectively capture NO (via N2O3) to yield a triazole product that exhibits a 7.5-fold enhancement and a 70-fold turn-on response in the singlet oxygen quantum yield and fluorescence signal, respectively. Beyond demonstrating excellent in vitro responsiveness and selectivity toward NO, we showcase the potent photodynamic therapy (PDT) effect of NOxPorfin-1 in murine breast cancer and human non-small cellular lung cancer cells. Further, to highlight the in vivo efficacy, two key studies were executed. First, we utilized NOxPorfin-1 to ablate murine breast tumors in a site-selective manner without causing substantial collateral damage to healthy tissue. Second, we established a nascent human lung cancer model to demonstrate the unprecedented ability of NOxPorfin-1 to halt tumor growth and progression completely. The results of the latter study have tremendous implications for applying PDT to target metastatic lesions.

The detection of analytes with small molecular probes is crucial for the analysis and understanding of chemical, medicinal, environmental and biological situations as well as processes. Classic detection approaches rely on the concept of molecular recognition and bond formation reactions. Bond breakage reactions have been less explored in similar contexts. This concept article introduces metal-salen and metal-imine complexes as \"covalent-disassembly\"-based (DB)-probes for detecting polyoxophosphates, thiols, amino acids, HCN and changes in pH. It discusses the roles, importance and combinations of structurally functionalized molecular building blocks in the construction of DB-probes. Applications of optimized DB-probes for analyte detection in live cells and foodstuff are also discussed. Furthermore, the mechanism of the disassembly of a Fe(III)-salen probe upon pyrophosphate binding is presented. Extraordinary selectivity for this analyte was achieved by a multistep disassembly sequence including an unprecedented structural change of the metal complex (i. e. \"induced-fit\" principle). Design principles of probes for sensing applications following the \"covalent-disassembly\" approach are summarized, which will help improving current systems, but will also facilitate the development of new DB-probes for challenging analytic targets.

We report the development of a new class of protease activity sensors called DNA-barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide-DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease-mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a-based assay as a proxy for protease activity. As proof-of-concept, we show activity-based, multiplexed detection of the SARS-CoV-2-associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25-fold turn-on signals, 100-fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation-dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer-associated protease, cathepsin B, in three different patient-derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

The monitoring of metabolites in biofluids provides critical clues for disease diagnosis and evaluation. Yet, the quantitative detection of metabolites remains challenging for surface-enhanced Raman spectroscopy (SERS) due to poor reproducibility in preparation and manipulation of SERS nanoprobes. Herein, we develop an activity-based, slippery liquid-infused porous surface SERS (abSLIPSERS) sensor for facile quantification of metabolites with unmodified naked metal nanoparticles (NPs) by integrating biocatalysis-boronate oxidation cascades with SLIPS-driven self-concentration and delivering. Upon mixing the target metabolite with a specific oxidase, a H2O2-sensitive phenylboronate probe, and the naked Au NPs, H2O2 produced from the biocatalytic reaction oxidizes the phenylboronate probe to phenol, resulting in a ratiometric SERS response. Meanwhile, the SLIPS enables the complete enrichment of molecules and NPs within an evaporating liquid droplet, delivering the probes to the SERS-active sites for Raman amplification. Compared with conventional SERS biosensors, abSLIPSERS avoids multistep synthesis and biofunctionalization of nanoprobes, which significantly simplifies the detection workflow and improves the reproducibility. The abSLIPSERS sensor also shows tunable dynamic range beyond 4 orders of magnitude and allows quantifying any other metabolites with specific enzymes. We demonstrate abSLIPSERS sensing of lactate, glucose, and choline in human serum for exploring energy metabolism in lung cancer. This study opens up a new opportunity for future point-of-care testing of circulating metabolites by SERS and will help to facilitate the translation of SERS bioanalysis to clinical settings.

Copper is an essential metal nutrient for life that often relies on redox cycling between Cu(I) and Cu(II) oxidation states to fulfill its physiological roles, but alterations in cellular redox status can lead to imbalances in copper homeostasis that contribute to cancer and other metalloplasias with metal-dependent disease vulnerabilities. Copper-responsive fluorescent probes offer powerful tools to study labile copper pools, but most of these reagents target Cu(I), with limited methods for monitoring Cu(II) owing to its potent fluorescence quenching properties. Here, we report an activity-based sensing strategy for turn-on, oxidation state-specific detection of Cu(II) through metal-directed acyl imidazole chemistry. Cu(II) binding to a metal and oxidation state-specific receptor that accommodates the harder Lewis acidity of Cu(II) relative to Cu(I) activates the pendant dye for reaction with proximal biological nucleophiles and concomitant metal ion release, thus avoiding fluorescence quenching. Copper-directed acyl imidazole 649 for Cu(II) (CD649.2) provides foundational information on the existence and regulation of labile Cu(II) pools, including identifying divalent metal transporter 1 (DMT1) as a Cu(II) importer, labile Cu(II) increases in response to oxidative stress induced by depleting total glutathione levels, and reciprocal increases in labile Cu(II) accompanied by decreases in labile Cu(I) induced by oncogenic mutations that promote oxidative stress.

The rapid development of responsive fluorescent probes has advanced optical imaging for biological research and biomedical applications. Among different sensing strategies, activity-based sensing, which exploits the unique reactivity of the target chemical species to achieve high chemoselectivity, has emerged as a promising paradigm for the development of responsive probes for selective molecular imaging. Luminescent transition metal complexes have received considerable attention for bioimaging and biosensing applications over the last decade due to their remarkable photophysical behavior including intense emission with large Stokes\' shifts, long emission lifetimes, strong two-photon absorption, and high photostability. In this Review, we summarize the design strategies and applications of luminescent complexes of rhenium(I), ruthenium(II), and iridium(III) polypyridines as activity-based probes for the detection of various chemical species and bioactive molecules in live cells and organisms. The current challenges and future prospects of these complexes as activatable reagents for disease diagnosis and treatment are also discussed.

Obesity is a chronic health condition characterized by the accumulation of excessive body fat which can lead to and exacerbate cardiovascular disease, type-II diabetes, high blood pressure, and cancer through systemic inflammation. Unfortunately, visualizing key mediators of the inflammatory response, such as monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), in a selective manner is a profound challenge owing to an overlapping substrate scope that involves arachidonic acid (AA). Specifically, these enzymes work in concert to generate AA, which in the context of obesity, has been implicated to control appetite and energy metabolism. In this study, we developed the first selective activity-based sensing probes to detect MGL (PA-HD-MGL) and FAAH (PA-HD-FAAH) activity via photoacoustic imaging. Activation of PA-HD-MGL and PA-HD-FAAH by their target enzymes resulted in 1.74-fold and 1.59-fold signal enhancements, respectively. Due to their exceptional selectivity profiles and deep-tissue photoacoustic imaging capabilities, these probes were employed to measure MGL and FAAH activity in a murine model of obesity. Contrary to conflicting reports suggesting levels of MGL can be attenuated or elevated, our results support the latter. Indeed, we discovered a marked increase of both targets in the gastrointestinal tract. These key findings set the stage to uncover the role of the endocannabinoid pathway in obesity-mediated inflammation.

Peroxynitrite (ONOO- ) plays a critical role in Alzheimer\'s disease (AD). To reveal the ONOO- influx in AD brains, an activatable activity-based fluorescence probe Rd-DPA3 was designed by a structure-modulated strategy. Taking advantage of ONOO- -initiated two-step cascade reactions of a novel chemical trigger, Rd-DPA3 specifically responds to ONOO- in 0.3 mM of other reactive oxygen species (ROS) and varied proteins, and gives an intensive fluorescence enhancement (F/F0 =50). Moreover, with its mitochondria-targeting ability, Rd-DPA3 can be used to efficiently monitor the alternations of intracellular ONOO- levels in cerebral cells during oxidative stress. Significantly, due to NIR emission and good blood-brain barrier (BBB) crossing ability, Rd-DPA3 is suitable for in vivo imaging of cerebral ONOO- influx and illustrating an age-dependent accumulation of ONOO- in AD mice brains.