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Discerning and quantifying the critical biothiols cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are vital for understanding their synergistic roles in biological systems. In this study, we synthesized a series of phenylethynylcoumarin fluorescent probes with varied structures to investigate the mechanisms underlying biothiol detection. We found that different substituents (−OCH3, −H, −CN) at the para-position of the phenylacetylene, combined with an aldehyde group at the 3-position of the coumarin, significantly affected the probes’ reactivity and produced distinct response patterns toward biothiols. These insights enable the strategic development of fluorescent probes tailored to provide the personalized and discriminative detection of these biothiols. Additionally, probes CPOMeand CPCNwere specifically evaluated for their efficacy in physiological environments, demonstrating their ability to accurately distinguish between Cys, Hcy, and GSH in living cells through unique fluorescent signals.

Mitochondrial DNA (mtDNA) damage is a prevalent phenomenon that has been proven to be implicated in a wide spectrum of diseases. However, the progressive attenuation of probe signals in response to mtDNA damage within living cells inherently limits the sensitivity and precision of current probes for detecting mtDNA damage. Herein, we employ an innovative organelle signal ratio imaging approach, utilizing the mitochondria–nucleus migration probe MCQ, to achieve unparalleled sensitivity in detecting mtDNA damage in living cells. MCQ exhibited an initial preferential binding to mtDNA, facilitated by its cationic quinolinium moiety, but migrated to the nucleus upon mtDNA damage. This unique migration behavior not only enhanced the spatial identifiability of mtDNA damage but also amplified detection sensitivity and precision significantly by harnessing the intensified nucleus signal against the attenuated mitochondrial signal. This innovative approach established a positive correlation between the signal and mtDNA damage, enabling the detection of even subtle mtDNA damage at the early stage of apoptosis with a remarkable 23-fold enhancement following just 5 min H2O2induction in living cells, whereas conventional methods relying solely on the fading of mitochondrial signals proved insufficient. Furthermore, MCQ’s ability to monitor the occurrence of mtDNA damage achieved the intricate differentiation between apoptosis and ferroptosis. By monitoring mtDNA damage, drug-induced apoptosis in cancer cells was further conducted using MCQ to evaluate the therapeutic efficacy of four anticancer drugs at very low concentrations. This innovative strategy not only paves the way for ultrasensitive detection of mtDNA damage but also holds immense promise for early monitoring of mtDNA damage-associated diseases.

To answer the call for effective and timely intervention in cardiovascular diseases (CVDs), the development of fluorescent probes that can precisely identify atherosclerotic plaques, the root cause of various fatal CVDs, is highly desirable but remains a great challenge. Herein, by integrating bis(trifluoromethyl)benzyl and phenothiazine into the coumarin matrix, a robust fluorescent probe, NOR1, has been developed. NOR1 responds sequentially to lipid droplets (LDs) and HClO via fluorescence turn-on and ratiometric readouts, respectively, with a fast response rate (within 70 s for LDs and 80 s for HClO), excellent sensitivity (detection limit: 0.41 μg/mL for LDs and 23.38 nM for HClO), and high selectivity. Based on these impressive features, NOR1 was successfully applied to discriminate foam cells from others by simultaneously monitoring two hallmark events, lipid accumulation and oxidative stress, in foam cells. Furthermore, the use of NOR1 to monitor in real time the transformation process of A7r5 cells into foam cells under high LDL/glucose conditions was successfully realized for the first time. Importantly, we further demonstrate the ability of NOR1 to precisely identify atherosclerotic plaques with clear margin delineation, highlighting its potential utility in elucidating the pathological mechanism and clinical diagnosis of atherosclerosis.

Dynamic self-assembly of iridium complexes in water-soluble nanocontainers is an important bottom-up process for fabricating electrochemiluminescence (ECL) bioprobes. PEGylated apoferritin (PEG-apoHSF) as the host offers a confined space to alter and modify the self-assembly of trans-bis(2-phenylpyridine)(acetylacetonate)iridium(III) [Ir(ppy)2(acac)] based on a pH-dependent depolymerization/reassembly pathway, allowing the formation of ECL-active iridium cores in PEG-apoHSF cavities (Ir@PEG-apoHSF). With an improved encapsulation ratio in PEG-apoHSF, the coreactant ECL behavior of the fabricated Ir@PEG-apoHSF nanodots with tri-n-propylamine (TPrA) was further demonstrated, exhibiting maximum ECL emission at 530 nm that was theoretically dominated by the band gap transition. The application of Ir@PEG-apoHSF as a bioprobe in a “signal-on” ECL immunosensing system was developed based on electroactive Ti3C2TxMXenes/TiO2nanosheet (Ti3C2Tx/TiO2) hybrids. Combining with the efficiently catalyzed electro-oxidation of TPrA and Ir(ppy)2(acac) by Ti3C2Tx/TiO2hybrids, the developed immunosensor showed dramatically amplified ECL responses toward the target analyte of neuron-specific enolase (NSE). Under experimental conditions, linear quantification of NSE from 100 fg/mL to 50 ng/mL was well established by this assay, achieving a limit of detection (LOD) of 35 fg/mL. The results showcased the capability of PEGylated apoHSF to host and stabilize water-insoluble iridium complexes as ECL emitters for aqueous biosensing and immunoassays.

The ratiometric detection of cysteine (Cys) and homocysteine (Hcy) is very challenging because of their highly similar chemical structures and properties. By introducing the phenylethynyl group into a coumarin dye as the sensing group, the ratiometric fluorescent probe CPwas developed to selectively and rapidly discriminate between Cys and Hcy. With a single-wavelength excitation, the presence of Cys or Hcy induced the probe to produce distinct ratiometric fluorescence changes: from red (λmaxem= 608 nm) to blue (λmaxem= 485 nm) toward Cys and from red to mixed red/blue toward Hcy. Moreover, the probe was capable of visualizing and discriminating between intracellular Cys and Hcy in HeLa cells and zebrafish by exhibiting different ratiometric fluorescence signals.

Electrocheluminescence (ECL) immunoassay amplified by coreaction accelerators has experienced major breakthroughs in ultrasensitive detection of biomarkers. Herein, CeO2/SnS2heterostructures were synthesized and applied as a novel coreaction accelerator to enhance the ECL efficiency of the luminol–dissolved O2system for the first time. Benefiting from the well-matched lattice spacing, ultrafine CeO2nanoparticles (NPs) were grown in situ on layered SnS2nanosheets (NSs) with improved dispersion. CeO2/SnS2as an electroactive substrate can remarkably accelerate the generation of abundant superoxide anion radicals (O2•–) to react with luminol anion radical (L•–), achieving about 2-fold stronger ECL intensity than that of pure CeO2NPs. To avoid harsh chemical synthesis of conventional ECL labels and simplify the antibody conjugation process, ferritin (Ft) was served as a natural nanocarrier to immobilize luminol molecules (Lum@Ft) via a one-step linkage, whose protein nanocage can easily connect with the detection antibody. Moreover, a robust site-oriented immobilization strategy using HWRGWVC heptapeptide as specific capturer was further adopted to maintain the bioactivity of the capture antibody on the amine-functionalized CeO2/SnS2surface, which promoted the incubation efficiency markedly. On account of this advanced sensing strategy, a brand new biosensor was constructed for the accurate detection of heart failure biomarkers, which performed with favorable linearity in the range of 0.0001–50 ng/mL and achieved the detection limit of 36 fg/mL.

Mitochondria play a pivotal role in maintaining normal physiological functions. Mitochondrial autophagy, namely, mitophagy, is a selective catabolic disposal of impaired mitochondria through an autophagic mechanism during episodes of mitochondrial harm. This selective removal, e.g., mitophagy, is essential for mitochondrial quality control and is closely related to the pathogenesis of many diseases. The abnormal buildup of defective mitochondria in vivo was used as a target to prevent the development of cancer. The mitochondrial autophagy process of disease-related cells is usually accompanied by a decrease in polarity and pH, and the fluorescence sensing effects caused by these two factors are usually contradictory. Here, we propose a reinventing strategy to develop a dual-channel and dual-responsive fluorescent probe HDTVBthat is capable of tracking mitochondrial autophagy by monitoring fluctuations in mitochondrial pH and polarity. Based on the aggregation-induced emission (AIE) moiety and hemicarpine moiety push–pull system with activated near-infrared (NIR) emission and pH-activatable cyclization reaction, HDTVBwas able to differentiate tumors from normal sites via polarity- and acidity-triggered structural changes of the probe in the course of mitochondrial autophagy. HDTVBis expected to be applied to clinical diagnosis and tumor excision guided by fluorescence, offering a new route in physiological and biochemical research.

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-HClOon the surface of LDs verified its excellent performance in detecting HClO in the foam cells. SFL-HClOcan 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-HClOcan 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.