Porphyrins, known as the "pigments of life", have evolved from their natural roles into versatile tools for biomedical applications. The development of activatable porphyrins has significantly expanded their utility, enabling precise responses to a carefully selected target analyte. These advances have broadened their use in imaging, diagnosis, and therapy. These capabilities are driven by activity-based sensing (ABS), which enhances the selectivity and sensitivity to various disease biomarkers. However, their design and implementation are intrinsically complex. This perspective provides an easy-to-follow roadmap that details how such molecules can be developed. Furthermore, we highlight recent progress in ABS-modified porphyrins, focusing on how specific modifications achieve these remarkable properties across various biomedical platforms. The ongoing evolution of activatable porphyrins holds great promise for the development of sophisticated, responsive systems, offering more effective diagnostic and therapeutic tools.

Type-I photosensitizers (PSs) have attracted great attention in recent years as they minimally rely on the tissue oxygen (3O2) to generate highly cytotoxic reactive oxygen species (ROS) in the scope of photodynamic therapy (PDT). Thus, they hold great promise for effective treatment of hypoxic cancer cells, which is a challenging task for type-II PSs. However, compared to conventional type-II PSs, the number of cancer cell selective type-I PSs is quite low. Thus, there is still a need for type-I PSs that can induce photocytotoxicity only in cancer cells without causing damage to normal tissues even under light irradiation. Additionally, targeting PSs to specific organelles has lately appeared to be a promising approach to improve the therapeutic outcome of PDT. Although a few examples of organelle-targeted type-I PS cores have emerged recently, activity-based and organelle-targeted type-I PSs have remained scarce. In this study, we report two organelle-targeted and hydrogen sulfide (H2S) responsive type-I PSs (HEHM and HEH) based on a highly modular and easily accessible heavy atom decorated hemicyanine core. HEHM localizes to mitochondria due to its cationic structure, whereas HEH targets endoplasmic reticulum (ER) as it bears an ER-targeting sulfonamide moiety, and it marks the first example of an activity-based and ER-targeted type-I PS based on a hemicyanine core. Both PSs can be selectively activated in neuroblastoma cells (SH-SY5Y) upon reacting with high levels of endogenous H2S and induce similar photocytotoxicity through a type-I PDT mechanism under both normoxic (20% O2) and hypoxic (1% O2) conditions. HEHM is shown to cause PDT-induced mitochondria stress, while HEH triggers ER stress upon LED irradiation (640 nm, 66.7 mW cm-2). Additionally, HEH is shown to induce immunogenic cell death (ICD) followed by PDT action. In contrast, negligible ROS generation and cell death are observed in normal cells, which is a critical and challenging task for any type of therapeutic modality. They also allow fluorescence imaging of cancer cells due to their emissive nature, suggesting that they function as phototheranostic agents. This study introduces a rational approach to develop new generation activity-based and organelle-targeted type-I PDT agents towards effective and selective treatment of hypoxic tumors.

Cancer cells with high expression of aldehyde dehydrogenase 1A1 (ALDH1A1) are more resistant to chemotherapy, contribute to tumor progression, and are associated with poor clinical outcomes. ALDH1A1 plays a critical role in protecting cells from reactive aldehydes and, in the case of stem cells, regulates their differentiation through the retinoic acid signaling pathway. Despite the importance of this enzyme, methods to study ALDH1A1 high-expressing cancer cells in vivo remain limited. In this work, we developed AlDeLuc, the first logic-gated bioluminescence probe designed to selectively evaluate ALDH1A1 activity in tumor cells. The probe is sequentially activated by acidic intracellular compartments (i.e., endosomes) and ALDH1A1, ensuring precise detection of ALDH1A1 high-expressing cells and minimizing off-target detection of non-ALDH1A1 cells. Beyond demonstrating efficacy in multiple cancer cell lines and a murine model of breast cancer, we employed AlDeLuc to investigate how the population of ALDH1A1 high-expressing cells is influenced by the inflammatory status of a tumor in the context of a high-fat diet. These findings establish a molecular link between obesity, inflammation, and tumor progression.

Butyrylcholinesterase (BChE), a member of the human serine hydrolase family, is an essential enzyme for cholinergic neurotransmission as it catalyzes the hydrolysis of acetylcholine. It also plays central roles in apoptosis, lipid metabolism, and xenobiotic detoxification. On the other side, abnormal levels of BChE are directly associated with the formation of pathogenic states such as neurodegenerative diseases, psychiatric and cardiovascular disorders, liver damage, diabetes, and cancer. Thus, selective and sensitive detection of BChE level in living organisms is highly crucial and is of great importance to further understand the roles of BChE in both physiological and pathological processes. However, it is a very complicated task due to the potential interference of acetylcholinesterase (AChE), the other human cholinesterase, as these two enzymes share a very similar substrate scope. To this end, optical imaging probes have attracted immense attention in recent years as they have modular structures, which can be tuned precisely to satisfy high selectivity toward BChE, and at the same time they offer real time and nondestructive imaging opportunities with a high spatial and temporal resolution. Here, we summarize BChE selective imaging probes by discussing the critical milestones achieved during the development process of these molecular sensors over the years. We put a special emphasis on design principles and biological applications of highly promising new generation activity-based probes. We also give a comprehensive outlook for the future of BChE-responsive probes and highlight the ongoing challenges. This collection marks the first review article on BChE-responsive imaging agents.

As one of the self-luminescence imaging approaches that require pre-illumination instead of real-time light excitation, afterglow luminescence imaging has attracted increasing enthusiasm to circumvent tissue autofluorescence. In this work, we developed organic afterglow luminescent nanoprobe (nanotorch), which could emit persistent luminescence more than 10 days upon single light excitation. More importantly, the nanotorch could be remote charged by 660 nm light in a non-invasive manner, which showed great potential for real-time tracing the location of macrophage cell-based microrobots.

Chromenoxanthenes are colorful dyes which do not suffer from any fluorescence turn-off induced by spirocyclic forms as usually observed for conventional rhodamine and fluorescein xanthene analogues. In that way, wide variety of applications in particular in bioimaging can be foreseen for those underexplored fluorophores. Herein, a one-pot synthesis of rhodol-like chromenoxanthene dyes is reported, which are further modified at the periphery of the core structure to tune the optical properties. Resulting neutral derivatives are bright fluorophores in the orange-red spectral window, while cationic counterparts are almost non-emissive. These optical properties are fully characterized and are also rationalized via first-principles calculations. In particular, the occurrence of twisted intramolecular charge transfers is shown to account for the dark-state quenching occurring in the charged compounds. This feature is harvested in the construction of an activity-based fluorescent probe for mitochondria imaging. Excellent organelle localization is achieved, while the emission signal is proportional to the hydrogen peroxide concentration in the mitochondria. Furthermore, the absence of cytotoxicity bestows rhodol-like chromenoxanthene dyes a bright future in cellular imaging applications.

Photodynamic therapy (PDT) is a long known, FDA approved, and highly promising therapeutic modality, which is still developing. Photosensitizers (PSs) are at the core of PDT action and a worldwide effort has been put to develop PSs having multiple functionalities. In the design of new generation PSs, an attractive approach is to target organelles to get an exceptional therapeutic outcome as damaging these vital subcellular compartments improve the photocytotoxic effect of the PSs. To this end, small molecule organic PSs are suitable candidates due to ease of chemical modification, which allows simple implementation of the organelle targeting units on the PS structure. Furthermore, they hold unique characteristics such as low dark toxicity, high reactive oxygen species (ROS) generation capacity, biocompatibility, and tunable photophysical and photochemical properties. Accordingly, a wide variety of organelle-targeted organic PSs have been developed and proved to be highly successful both in therapeutic action and bioimaging applications. In this review article, we have summarized the recent advances in organelle localizing organic-based PSs by discussing the design principles, organelle targeting strategies and related bio-applications in detail. PSs targeting seven different organelles have been included. We have also highlighted the current challenges and gave a comprehensive outlook for the future of organelle-targeted PSs.

Butyrylcholinesterase (BChE), one of the critical human cholinesterases, plays crucial roles in numerous physiological and pathological processes. Accordingly, it is a striking and at the same time challenging target for bioimaging studies. Herein, we developed the first ever example of a 1,2-dixoetane-based chemiluminescent probe (BCC) for monitoring BChE activity in native biological contexts such as living cells and animals. BCC was initially shown to exhibit a highly selective and sensitive turn-on response in its luminescence signal upon reacting with BChE in aqueous solutions. Later, BCC was utilized to image endogenous BChE activity in normal and cancer cell lines. It was also shown through inhibition experiments that BChE can detect fluctuations of BChE levels successfully. In vivo imaging ability of BCC was demonstrated in healthy and tumor-bearing mice models. BCC enabled us to visualize the BChE activity in different regions of the body. Furthermore, it was successfully employed to monitor tumors derived from neuroblastoma cells with a very high signal to noise ratio. Thus, BCC appears as a highly promising chemiluminescent probe, which can be used to further understand the contribution of BChE to regular cellular processes and the formation of diseased states.

Hydrogen sulfide (H2S) as a critical messenger molecule plays vital roles in regular cell function. However, abnormal levels of H2S, especially mitochondrial H2S, are directly correlated with the formation of pathological states including neurodegenerative diseases, cardiovascular disorders, and cancer. Thus, monitoring fluxes of mitochondrial H2S concentrations both in vitro and in vivo with high selectivity and sensitivity is crucial. In this direction, herein we developed the first ever example of a mitochondria-targeted and H2S-responsive new generation 1,2-dioxetane-based chemiluminescent probe (MCH). Chemiluminescent probes offer unique advantages compared to conventional fluorophores as they do not require external light irradiation to emit light. MCH exhibited a dramatic turn-on response in its luminescence signal upon reacting with H2S with high selectivity. It was used to detect H2S activity in different biological systems ranging from cancerous cells to human serum and tumor-bearing mice. We anticipate that MCH will pave the way for development of new organelle-targeted chemiluminescence agents towards imaging of different analytes in various biological models.

A mitochondrion targeted and leucine aminopeptidase (LAP) activatable 1,2-dioxatane based chemiluminescent probe (MCL) for detection of LAP activity in living cancer cells and tumor bearing mice was reported. MCL displayed a selective and sensitive turn-on response in aqueous solutions upon reacting with the LAP enzyme. In cell culture studies, a selective luminescence intensity increase was observed in cancer cell lines, suggesting that MCL can differentiate between cancer and normal cells and allows detection of varying endogenous LAP concentrations. Using fluorescence imaging with a commercial Mitotracker dye, MCL was also shown to localize mitochondria in cancer cell lines. Furthermore, MCL was used to image tumors in mice models. MCL marks not only the first ever example of a mitochondria targeted chemiluminescent probe, but also the first ever example of an organelle targeted 1,2-dioxetane derivative.

Activity-based photosensitizers (aPSs) are highly attractive as they offer improved selectivity and better therapeutic outcome in the scope of photodynamic therapy (PDT). Here, a hydrogen sulfide (H2S) responsive iodinated resorufin-based PS (RHS) was developed to treat neuroblastoma cancer cells selectively. RHS was shown to be a phototheranostic agent as it turned on its fluorescence signal and singlet oxygen (O-1(2)) generation capability after reacting with H2S. RHS exhibited remarkable sensitivity towards H2S and proved to be highly cytotoxic in H2S rich SH-SY5Y human neuroblastoma cells upon light irradiation. In contrast, no photocytotoxicity was observed in H2S deficient nonmalignant fibroblast L929 cells. RHS marks the first example of a resorufin-based H2S activatable photo-theranostic agent, which paves the way for effective treatment of neuroblastoma through PDT modality.

Hydrogen sulfide (H2S) is one of the criticalgasotransmitters,which play important roles in regular physiological processes, especiallyin vital signaling pathways. However, fluctuations in endogenous H2S concentration can be linked to serious health problems,such as neurodegenerative diseases, cancer, diabetes, inflammation,cardiovascular diseases, and hypertension. Thus, it has attracteda great deal of attention in therapeutic applications, specificallyin the field of phototherapy. Photodynamic therapy (PDT) and photothermaltherapy (PTT) are two subclasses of phototherapy, which utilize eitherreactive oxygen species (ROS) or local temperature increase upon irradiationof a photosensitizer (PS) to realize the therapeutic action. Phototherapiesoffer unique advantages compared to conventional methods; thus, theyare highly promising and popular. One of the design principles followedin new generation PSs is to build activity-based PSs, which stay inactivebefore getting activated by disease-associated stimuli. These activatablePSs dramatically improve the selectivity and efficacy of the therapy.In this review, we summarize small molecule and nanomaterial-basedPDT and PTT agents that are activated selectively by H2S to initiate their cytotoxic effect. We incorporate single modePDT and PTT agents along with synergistic and/or multimodal photosensitizersthat can combine more than one therapeutic approach. Additionally,H2S-responsive theranostic agents, which offer therapyand imaging at the same time, are highlighted. Design approaches,working principles, and biological applications for each example arediscussed in detail.

A resorufin-based dual-locked fluorescent probe (RHT) was introduced to image melanoma cells selectively. RHT was shown to function as an AND molecular logic gate as it emitted a signal only in the presence of both hydrogen sulfide (H2S) and tyrosinase (Tyr), which are known to be overexpressed in melanoma cells. In vitro cell culture studies revealed that RHT can be activated with endogenous H2S and Tyr and allows selective imaging of B16-F10 cancer cells under confocal microscopy. RHT marks the first ever example of a fluorescent probe that is sequentially activated by H2S and Tyr.

Photodynamic therapy (PDT) is a clinically approved treatment modality used for a wide range of medical conditions, including malignant cancers. It employs cytotoxic reactive oxygen species (ROS), particularly singlet oxygen (1O2), to kill cells of interest and has attracted immense attention during the last decades. Molecular design of triplet photosensitizers is no doubt at the core of successful PDT action. Spatiotemporal control of ROS generation and consequent cancer cell selectivity is one of the highly sought characteristics of new-generation photosensitizers, to minimize severe adverse effects as well as to enhance the therapeutic outcome. Activatable photosensitizers have appeared to be a good candidate in this respect as they tend to stay in their “off” state prior to activation with various tumor-associated intracellular stimuli. In this chapter, we summarize the recent advances in the field of activatable photosensitizers by focusing on the design principles and biologically relevant activators.

An aggregation-induced emission (AIE) active light up fluorescent probe (TCFPB-AChE) was developed for selective in vitro and in vivo imaging of acetylcholinesterase activity.

Solid sampling high resolution continuum source molecule absorption spectrometry (SS-HR-CS MAS) was applied for the determination of chlorine in plastic using the strontium monochloride (SrCl) molecule. For this purpose, 10 mu L of 20 g L-1 strontium (prepared from Sr(NO3)(2)) solution were pipetted with aqueous Cl standards or 0.05 to 4 mg of slivered plastic samples on a platform and introduced into the furnace. Chlorine was determined with the molecular absorption of SrCl at 635.862 nm using 1100 degrees C and 2200 degrees C for the pyrolysis and vaporization temperatures, respectively. Aqueous standards were used for calibration. The accuracy of the method was evaluated using a certified polyethylene reference material. The limit of detection and characteristic mass values of the method were 2.5 ng and 0.4 ng, respectively. The chlorine concentrations in various polyethylene beverage containers were determined.