Direct Real‐Time Monitoring of Prodrug Activation by Chemiluminescence

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal‐to‐noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β‐galactosidase. The activation of the prodrug in the presence of β‐galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β‐galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β‐galactosidase‐overexpressing tumor‐bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.     

A Renal‐Clearable Macromolecular Reporter for Near‐Infrared Fluorescence Imaging of Bladder Cancer

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer‐specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real‐time near‐infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post‐injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non‐invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.     

Renal‐clearable Molecular Semiconductor for Second Near‐Infrared Fluorescence Imaging of Kidney Dysfunction

Real‐time imaging of kidney function is important to assess the nephrotoxicity of drugs and monitor the progression of renal diseases; however, it remains challenging because of the lack of optical agents with high renal clearance and high signal‐to‐background ratio (SBR). Herein, a second near‐infrared (NIR‐II) fluorescent molecular semiconductor (CDIR2) is synthesized for real‐time imaging of kidney dysfunction in living mice. CDIR2 not only has a high renal clearance efficiency (≈90 % injection dosage at 24 h post‐injection), but also solely undergoes glomerular filtration into urine without being reabsorbed and secreted in renal tubules. Such a unidirectional renal clearance pathway of CDIR2 permits real‐time monitoring of kidney dysfunction in living mice upon nephrotoxic exposure. Thus, this study not only introduces a molecular renal probe but also provides useful molecular guidelines to increase the renal clearance efficiency of NIR‐II fluorescent agents.     

A Photoexcitation‐Induced Twisted Intramolecular Charge Shuttle

Charge transfer and separation are important processes governing numerous chemical reactions. Fundamental understanding of these processes and the underlying mechanisms is critical for photochemistry. Herein, we report the discovery of a new charge‐transfer and separation process, namely the twisted intramolecular charge shuttle (TICS). In TICS systems, the donor and acceptor moieties dynamically switch roles in the excited state because of an approximately 90° intramolecular rotation. TICS systems thus exhibit charge shuttling. TICSs exist in several chemical families of fluorophores (such as coumarin, BODIPY, and oxygen/carbon/silicon–rhodamine), and could be utilized to construct functional fluorescent probes (i.e., viscosity‐ or biomolecule‐sensing probes). The discovery of the TICS process expands the current perspectives of charge‐transfer processes and will inspire future applications.     

Noninvasive Staging of Kidney Dysfunction Enabled by Renal‐Clearable Luminescent Gold Nanoparticles

As a “silent killer”, kidney disease is often hardly detected at an early stage but can cause lethal kidney failure later on. Thus, a preclinical imaging technique that can readily differentiate between the stages of kidney dysfunction is highly desired for improving our fundamental understanding of kidney disease progression. Herein, we report that in vivo fluorescence imaging, enabled by renal‐clearable near‐infrared‐emitting gold nanoparticles, can noninvasively detect kidney dysfunction, report on the dysfunctional stages, and even reveal adaptive function in a mouse model of unilateral obstructive nephropathy, which cannot be diagnosed with routine kidney function markers. These results demonstrate that low‐cost fluorescence kidney functional imaging is highly sensitive and useful for the longitudinal, noninvasive monitoring of kidney dysfunction progression in preclinical research.     

A Synthetic Fluorescent Mitochondria‐Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells

Ca²⁺ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca²⁺ concentration in living cells is of pivotal interest. Genetically‐encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria‐targeted synthetic Ca²⁺ indicators are plagued by several drawbacks, for example, non‐specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca²⁺ sensor, named mt‐fura‐2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca²⁺ indicator fura‐2. Mt‐fura‐2 binds Ca²⁺ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca²⁺] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt‐fura‐2 to cell types where the delivery of genetically‐encoded indicators is troublesome.     

A Sequential Dual-Lock Strategy for Photoactivatable Chemiluminescent Probes Enabling Bright Duplex Optical Imaging

Chemiluminescence (CL)-based technologies have revolutionized in vivo monitoring of biomolecules. However, significant technical hurdles have limited the achievement of trigger-controlled, bright, and enriched CL signal. Herein, a dual-lock strategy uses sequence-dependent triggers for bright optical imaging with real-time fluorescent signal and ultra-sensitive CL signal. These probes can obtain an analyte-triggered accumulation of stable pre-chemiluminophore with aggregation-induced emission (AIE), and then the pre-chemiluminophore exhibits a rapid photooxidation process (1,2-dioxetane generation) by TICT-based free-radical addition, thereby achieving an enrichment and bright CL signal. The dual-lock strategy expands the in vivo toolbox for highly accurate analysis and has for the first time allowed access to accurately sense and trace biomolecules with high-resolution, dual-mode of chemo-fluoro-luminescence, and three-dimensional (3D) imaging in living animals.     

AgHalo: A Facile Fluorogenic Sensor to Detect Drug‐Induced Proteome Stress

Drug‐induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug‐induced proteome stress prior to cell death. An aggregation prone Halo‐tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.     

Rational Design of a Dual‐Reactivity‐Based Fluorescent Probe for Visualizing Intracellular HSNO

Thionitrous acid (HSNO), the smallest S‐nitrosothiol, is emerging as a potential key intermediate in cellular redox regulation linking two signaling molecules H₂S and NO. However, the chemical biology of HSNO remains poorly understood. A major hurdle is the lack of methods for selective detection of HSNO in biological systems. Herein, we report the rational design, synthesis, and evaluation of the first fluorescent probe TAP‐1 for HSNO detection. TAP‐1 showed high selectivity and sensitivity to HSNO in aqueous media and cells, providing a useful tool for understanding the functions of HSNO in biology.     

Near‐Infrared Afterglow Semiconducting Nano‐Polycomplexes for the Multiplex Differentiation of Cancer Exosomes

The detection of exosomes is promising for the early diagnosis of cancer. However, the development of suitable optical sensors remains challenging. We have developed the first luminescent nanosensor for the multiplex differentiation of cancer exosomes that bypasses real‐time light excitation. The sensor is composed of a near‐infrared semiconducting polyelectrolyte (ASPN) that forms a complex with a quencher‐tagged aptamer. The afterglow signal of the nanocomplex (ASPNC), being initially quenched, is turned on in the presence of aptamer‐targeted exosomes. Because detection of the afterglow takes place after the excitation, background signals are minimized, leading to an improved limit of detection that is nearly two orders of magnitude lower than that of fluorescence detection in cell culture media. Also, ASPNC can be easily tailored to detect different exosomal proteins by changing the aptamer sequence. This enables an orthogonal analysis of multiple exosome samples, potentially permitting an accurate identification of the cellular origin of exosomes for cancer diagnosis.     

A Freezing‐Induced Turn‐On Imaging Modality for Real‐Time Monitoring of Cancer Cells in Cryosurgery

Cryosurgery has attracted much attention for the treatment of tumors owing to its clear advantages. However, determining the volume of frozen tissues in real‐time remains a challenge, which greatly lowers the therapeutic efficacy of cryosurgery and hinders its broad application for the treatment of cancers. Herein, we report a freezing‐induced turn‐on strategy for the selective real‐time imaging of frozen cancer cells. As a type of aggregation‐induced emission (AIE) fluorogen, TABD‐Py molecules interact specifically with ice crystals and form aggregates at the ice/water interface. Consequently, bright fluorescent emission appears upon freezing. TABD‐Py molecules are enriched mostly in the cancer cells and exhibit high biocompatibility as well as low cytotoxicity; therefore, a freezing‐induced turn‐on imaging modality for cryosurgery is developed, which will certainly maximize the therapeutic efficacy of cryosurgery in treating tumors.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

Harnessing Intramolecular Rotation To Enhance Two‐photon Imaging of Aβ Plaques through Minimizing Background Fluorescence

The aggregation of amyloid beta (Aβ) proteins in senile plaques is a critical event during the development of Alzheimer's disease, and the postmortem detection of Aβ‐rich proteinaceous deposits through fluorescent staining remains one of the most robust diagnostic tools. In animal models, fluorescence imaging can be employed to follow the progression of the disease, and among the different imaging methods, two‐photon microscopy (TPM) has emerged as one of the most powerful. To date, several near‐infrared‐emissive two‐photon dyes with a high affinity for Aβ fibrils have been developed, but there has often been a tradeoff between excellent two‐photon cross‐sections and large fluorescence signal‐to‐background ratios. In the current work, we introduced a twisted intramolecular charge state (TICT)‐based de‐excitation pathway, which results in a remarkable fluorescence increase of around 167‐fold in the presence of Aβ fibrils, while maintaining an excellent two‐photon cross section, thereby enabling high‐contrast ex vivo and in vivo TPM imaging. Overall, the results suggest that adopting TICT de‐excitation in two‐photon fluorophores may represent a general method to overcome the tradeoff between probe brightness and signal‐to‐background ratio.     

The Fundamentals of Real‐Time Surface Plasmon Resonance/Electrogenerated Chemiluminescence

We report the integration of surface plasmon resonance (SPR), cyclic voltammetry and electrochemiluminescence (ECL) responses to survey the interfacial adsorption and energy transfer processes involved in ECL on a plasmonic substrate. It was observed that a Tween 80/tripropylamine nonionic layer formed on the gold electrode of the SPR sensor, while enhancing the ECL emission process, affects the electron transfer process to the luminophore, Ru(bpy)₃²⁺, which in turn has an impact on the plasmon resonance. Concomitantly, the surface plasmon modulated the ECL intensity, which decreased by about 40 %, due to an interaction between the excited state of Ru(bpy)₃²⁺ and the plasmon. This occurred only when the plasmon was excited, demonstrating that the optically excited surface plasmon leads to lower plasmon‐mediated luminescence and that the plasmon interacts with the excited state of Ru(bpy)₃²⁺ within a very thin layer.