Fluorescence Signal Research Articles

A Dual-Channel Fluorescence Probe for Early Diagnosis and Treatment Monitoring of Acute Kidney Injury by Detecting HOCl and Cys with Different Fluorescence Signals.

The pathogenesis of acute kidney injury (AKI) is a multifaceted process involving various mechanisms, with oxidative stress playing a crucial role in its development. Hypochlorite (HOCl) and cysteine (Cys) are indicators of oxidative stress in AKI pathophysiology, directly reflecting the degree of oxidative stress and disease severity. However, their exact mechanism of action in AKI pathophysiology remains unknown. Herein, we developed a dual-channel fluorescent probe, MB-NAP, which allowed for the simultaneous detection of HOCl and Cys. The probe exhibited distinct fluorescence responses in the green channel (λex = 450 nm, λem = 560 nm) and red channel (λex = 610 nm, λem = 690 nm), without any spectral crosstalk, allowing for accurate measurement of both analytes. We successfully applied MB-NAP to monitor the levels of HOCl and Cys in cellular and in vivo models of AKI, revealing a significant increase in their concentrations compared to normal models. Furthermore, MB-NAP was demonstrated to exhibit outstanding capabilities for drug screening by effectively real-time monitoring HOCl and Cys. This study not only provides a more sensitive and reliable method/tool for tracking AKI-related pathological processes but also offers a potential breakthrough in the early diagnosis and identification of therapeutic agents aimed at mitigating oxidative stress-induced damage in AKI.

A Dual-Mode Colorimetric and Fluorescence Biosensor Based on a Nucleic Acid Multiplexing Platform for the Detection of Listeria monocytogenes.

Listeria monocytogenes (L. monocytogenes) is one of the most prevalent threats, capable of inducing diverse illnesses and presenting a serious threat to public health. Herein, we demonstrate a novel dual-mode colorimetric/fluorescence biosensor based on the exponential amplification reaction and strand displacement reactions (EASDR), which has multiplexing capability that significantly promotes the anchoring and trapping of Pt nanoparticles (Pt NPs) and fluorescent dyes for sensitive detection of Listeria monocytogenes (L. monocytogenes). The method works by targeting specific bacteria with aptamers and promoting repeated EASDR to affect the immobilization of Pt NPs and fluorescent dyes in the orifice plate, which could produce changes in fluorescence and colorimetric signals. The assay achieves a detection limit of 38 CFU/mL in colorimetric and 10 CFU/mL in fluorescence. Furthermore, the developed biosensor can be applied to the analysis of raw food samples (milk), and good recoveries were obtained in spiked food samples. In summary, the dual-mode biosensor improves the accuracy of detection by simultaneously outputting two signals and shows great potential in the specific identification and detection of foodborne pathogens.

A Paper-Based Sensor for the Detection of Gastroesophageal Reflux Disease Utilizing a Cleavable Fluorescent Polymer.

Nowadays, gastroesophageal reflux disease (GERD) has emerged as one of the major hazards to the health of the upper gastrointestinal tract, and there is an urgent need for a low-cost, user-friendly, and non-invasive detection method. Herein, a paper-based sensor (CP sensor) for the non-invasive screening of GERD is proposed. The sensor is structured as a specially shaped cellulose paper strip embedded with fluorescent colloids, which are self-assembled from a cleavable synthetic fluorescent polymer (P4). Benefiting from the introduction of amide bonds and the unique assembled structure of the nanocolloids, the pepsin in the sample solution will hydrolyze the water-soluble branches in the micellar shell during detection, resulting in a corresponding output of the fluorescent signal. This responsiveness, which can be observed by the naked eye, is so sensitive with a minimum detectable concentration for pepsin as low as 0.3 ng·mL-1. Clinical trials have further demonstrates that the designed paper sensor is capable of providing improved accuracy in the early diagnosis of GERD.

Design of triplex-forming peptide nucleic acid-based fluorescent probes for forced intercalation sensing of double-stranded RNA structures.

The diverse functional roles of RNA within cells have led to a growing interest in developing RNA-binding fluorescent probes to investigate RNA functions. In particular, the probes for double-stranded RNA (dsRNA) structures are of significant value given the importance of the secondary and tertiary RNA structures on their biologic functions. This review highlights our recent efforts on the development of triplex-forming peptide nucleic acid (TFP)-based probes for fluorescence sensing of dsRNA structures. We demonstrated that the forced intercalation of asymmetric cyanine dyes integrated as base surrogate within the probes was useful for achieving significant light-up response toward target dsRNAs. We also showed that the TFP probes conjugated with small RNA-binding molecules facilitated the fluorescence sensing of biologic relevant dsRNAs containing unpaired nucleobases. The binding and fluorescence signaling functions of such probes were discussed, emphasizing their potential as analytical tools for studying dsRNA structures.

Polydopamine-encapsulated carbon dots to boost analytical performance for microplastics detection in fluorescence mode.

Akind of sulfur-doped carbon dots was preparedwhich were encapsulated with polydopamine (S-CDs@PDA) that has fluorescence response on polyethylene (PE) microplastics (MPs). Modified membranes were constructed using S-CDs@PDA for MP detection. Through heating and vacuum filtration process, yellow emission from the modified membrane appeared because of the combination between S-CDs@PDA and PE MPs. Notably, the fluorescence signal value of PE MPs detected by S-CDs@PDA-modified membrane was 21.3% higher than that of unmodifiedS-CDs membrane, and the detection efficiency was 8% higher than that of S-CDs membrane. The minimum detection limit for modified membranes was 4mg. Due to the good adhesion of polydopamine (PDA), S-CDs@PDA-modified membrane was more easily adhered to PE MPs, showing its excellent detection ability. The rapid quantitative detection of PE MPs in 10min was realized with a linear equation of y = 3081x + 3686.1 in a linear range of 4-14mg. Such modified membrane exhibited excellent anti-photobleaching using continuous 365-nm excitation and its sensing performance was further confirmed in sea and river water samples. S-CDs@PDA could detect solid MP powders as well as MPs dispersed in liquids. The detection method constructed with a modified glass fiber filter membrane enables rapid identification and quantitative detection of PE MPs without relying on large-scale instrumentation. This study provided research ideas for fluorescent tracing of PE MPs and paved the way for quantitative detection of micron-sized plastics with smaller particle sizes.

Logic Circuits for Intelligent Microcystin Monitoring Based on Aptamer Recognition and Toehold-Mediated Hairpin DNA Self-Assembly.

A sensitive fluorescence biosensor was developed for microcystin-LR (MC-LR) detection using H1, H2, and H3 DNA probes as sensing elements. The aptamer in H1 can recognize the target. H2 was labeled with FAM and BHQ. The MC-LR and H1 binding will activate the H2 and H3 self-assemblies through toehold-mediated strand displacement. In the formed products (MC-LR/H1/nH2/nH3), FAM and BHQ will be separated and a high FAM fluorescence signal can be observed for the MC-LR assay. The biosensor is sensitive with a detection limit of 53 fM. We further constructed several logic circuits (AND-AND cascaded circuit, feedforward circuit, and resource allocation circuit) using MC-LR, MC-LA, and MC-YR as the three inputs. The numbers 0 and 1 are used to code the input and output signals. The AND-AND cascade circuit can produce a high output signal only in the (111) input combination. In the feedforward circuit, MC-LR and MC-LA can activate the logic circuit to produce high signals, and MC-YR will inhibit the self-assembly and execute the negative feedforward operation. Through the rational design of the DNA probe hybridizations on four different magnetic beads (MBs), the resource allocation circuit can achieve an intelligent allocation of input information. Our proposed fluorescence biosensor can not only provide a sensitive platform for microcystin detection but also serve as a smart and intelligent logic system for microcystin sensing.

Ultra-sensitive fluorescence-activated droplet single-cell sorting based on Tetramer-HCR-EvaGreen amplification

The current single-cell analysis technologies such as fluorescence-activated cell sorting (FACS) and fluorescence-activated droplet sorting (FADS) could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability. Here, an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen (THE) fluorescence signal amplification. The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification. Upon application to target MCF-7 cells, the platform exhibited high efficacy and selectivity while maintaining more than 95% cell viability. The THE-FADS achieved sorting efficiencies of 55.5% and 50.3% with purities of 91% and 85% for MCF-7 cells in PBS solutions and simulated serum samples, respectively. The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells. The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.

Dual-Emissive Iridium(III) Complex with Aggregation-Induced Emission: Mechanistic Insights into Electron Transfer for Enhanced Hypoxia Detection in 3D Tumor Models.

Accurate oxygen detection and measurement of its concentration is vital in biological and industrial applications, necessitating highly sensitive and reliable sensors. Optical sensors, valued for their real-time monitoring, nondestructive analysis, and exceptional sensitivity, are particularly suited for precise oxygen measurements. Here, we report a dual-emissive iridium(III) complex, IrNPh2, featuring "aggregation-induced emission" (AIE) properties and used for sensitive oxygen sensing. IrNPh2 exhibits dual emissions at 450 and 515 nm, with 515 nm triplet-state emission demonstrating remarkable oxygen sensitivity due to its long-lived excited state (12.12 μs) and high quantum yield (68%). Stern-Volmer analysis reveals a notable quenching constant (Ksv = 12.44%-1) and an ultralow detection limit of 0.0397%, emphasizing its superior performance. The oxygen quenching mechanism is driven by electron transfer (ET), supported by computational studies showing the lowest-unoccupied molecular orbital (LUMO) alignment of IrNPh2 with the πg* orbitals of triplet oxygen, leading to superoxide radical (O2•-) formation. Electron paramagnetic resonance (EPR) studies further confirm this pathway. Biological evaluations using a three-dimensional (3D) U87-MG glioma spheroid model highlight the ability of IrNPh2 to detect hypoxic regions, with significant fluorescence enhancement under hypoxia and minimal cytotoxicity (>80% viability at 100 μM). With high sensitivity, low detection limits, and biocompatibility, IrNPh2 emerges as a promising candidate for oxygen sensing in environmental and biomedical applications, especially tumor hypoxia detection.

Aptamer-Driven Multifunctional Nanoplatform for Near-Infrared Fluorescence Imaging and Rapid In Situ Inactivation of Salmonella typhimurium.

Salmonella typhimurium (S. typhimurium) is a prominent pathogen responsible for intestinal infections, primarily transmitted through contaminated food and water. This underscores the critical need for precise and biocompatible technologies enabling early detection and intervention of bacterial colonization in vivo. Herein, a multifunctional nanoplatform (IR808-Au@ZIF-90-Apt) was designed, utilizing an S. typhimurium-specific aptamer to initiate cascade responses triggered by intracellular ATP and GSH. The nanoplatform precisely targets S. typhimuriumvia aptamer recognition, promoting bacterial aggregation through nanoparticle sedimentation in an oscillatory system. Furthermore, the intelligent nanoplatform significantly enhances the sensitivity of S. typhimurium detection based on near-infrared (NIR) fluorescence signals, achieving a detection limit as low as 2 CFU mL-1. Additionally, in situ NIR irradiation was applied at the 30 min peak of fluorescence detection, enabling rapid and irreversible inactivation of S. typhimurium through the synergistic effects of photothermal and photodynamic effects. Importantly, in a mouse model of intestinal infection, the nanoplatform successfully detected early S. typhimurium colonization and achieved highly efficient in situ inactivation without adversely affecting the major organs. In conclusion, the nanoplatform achieved precise localized detection and in situ inactivation of S. typhimurium, offering valuable insights for disease surveillance and epidemiological studies, with promising implications for food safety and public health.

A Dual-Cycle Isothermal Amplification Method for microRNA Detection: Combination of a Duplex-Specific Nuclease Enzyme-Driven DNA Walker with Improved Catalytic Hairpin Assembly

The association between microRNAs and various diseases, especially cancer, has been established in recent years, indicating that miRNAs can potentially serve as biomarkers for these diseases. Determining miRNA concentrations in biological samples is crucial for disease diagnosis. Nevertheless, the stem-loop reverse transcription quantitative PCR method, the gold standard for detecting miRNA, has great challenges in terms of high costs and enzyme limitations when applied to clinical biological samples. In this study, an isothermal signal amplification method based on a duplex-specific nuclease (DSN) enzyme-driven DNA walker and an improved catalytic hairpin assembly (CHA) was designed for miRNA detection. First, biotin–triethylene glycol-modified trigger-releasable DNA probes were conjugated to the streptavidin-coated magnetic beads for recognizing the target miRNA. The DSN enzyme specifically hydrolyzes DNA strands when the DNA probe hybridizes with the targeted miRNA. This recycling process converts the input miRNA into short trigger fragments (catalysts). Finally, three hairpins of improved CHA are driven by this catalyst, resulting in the three-armed CHA products and a fluorescence signal as the output. This dual-cycle biosensor shows a good linear relationship in the detection of miR-21 and miR-141 over the final concentration range of 250 fM to 50 nM, presenting an excellent limit of detection (2.95 amol). This system was used to detect miR-21 and miR-141 in MCF-7 and 22RV1 cells, as well as in 1% human serum. This system can be used to evaluate the expression levels of miRNAs in different biological matrices for the clinical diagnosis and prognosis of different cancers.

A High-Efficiency Autocatalysis-Oriented Cascade Circuit via Reciprocal Hug-Amplification for Assay-to-Treat Application.

Developing a DNA autocatalysis-oriented cascade circuit (AOCC) via reciprocal navigation of two enzyme-free hug-amplifiers might be desirable for constructing a rapid, efficient, and sensitive assay-to-treat platform. In response to a specific trigger (T), seven functional DNA hairpins were designed to execute three-branched assembly (TBA) and three isotropic hybridization chain reaction (3HCR) events for operating the AOCC. This was because three new inducers were reconstructed in TBA arms to initiate 3HCR (TBA-to-3HCR) and periodic T repeats were resultantly reassembled in the tandem nicks of polymeric nanowires to rapidly activate TBA in the opposite direction (3HCR-to-TBA) without steric hindrance, thereby cooperatively manipulating sustainable AOCC progress for exponential hug-amplification (1:3Nn). Our experimental verifications manifested that the T-dependent AOCC amplifier achieved fast input transduction and efficient fluorescence readout. As predicted, the flexible programming of reactive hairpin species endowed the repeating nicks in productive 3HCR nanowires with great possibilities and accessibilities to graft tailored modular elements, such as G-rich AS1411 aptamers capable of adopting G-quadruplex conformations (G4) that readily facilitated the embedding of zinc(II) protoporphyrin IX (ZnPPIX), a kind of heme oxygenase-1 enzyme inhibitor. Thus, the cascading ZnPPIX/G4 entities acted as fluorescent signal reporters, photosensitizers and anticancer drugs, thereby creating an updated AOCC-based assay-to-treat platform for ultrasensitive biosensing, discernible cell imaging and efficient photodynamic therapy of cancer cells. This would offer a new paradigm to advance the rational integration of dynamic DNA assembly and amplifiable recycling circuits for applicable bioassay and theranostics.

APE1-Activated and NIR-II Photothermal-Enhanced Chemodynamic Therapy Guided by Amplified Fluorescence Imaging.

The development of intelligent nanotheranostic technology that integrates diagnostic and therapeutic functions holds great promise for personalized nanomedicine. However, most of the nanotheranostic agents exhibit "always-on" properties and do not involve an amplification step, which may largely limit imaging contrast and restrict therapeutic efficacy. Herein, we construct a novel nanotheranostic platform (Hemin/DHPs/PDA@CuS nanocomposite) by assembling DNA hairpin probes (DHPs) and hemin on the surface of PDA@CuS nanosheets that enables amplified fluorescence imaging and activatable chemodynamic therapy (CDT) of tumors. The cancer-relevant APE1 triggers nucleic acid amplification with DHPs to generate activatable and amplified fluorescence signals for discriminating cancer cells from normal cells. Meanwhile, excessive G-quadruplex/hemin-based DNAzyme are also activated, and they function as Fenton-like catalysts to catalyze the production of highly toxic hydroxyl radicals (•OH) for CDT. Moreover, owing to the excellent photothermal conversion efficiency in the near-infrared-II (NIR-II) window, the PDA@CuS not only improves the catalytic performance of CDT but also furnishes PTT. A remarkable antitumor therapeutic effect is demonstrated both in vitro and in vivo. Therefore, the Hemin/DHPs/PDA@CuS nanocomposite is expected to provide a promising avenue for precise imaging-guided antitumor therapy.

Two cellular models for analyzing mitochondrial heteroplasmy

Aim: Mitochondria are essential for brain development, and the presence of different mitochondrial types is called mitochondrial heteroplasmy. Mitochondrial dysfunction is a central aspect of many people’s neurological diseases. Heteroplasmy is commonly observed in eukaryotes due to mitochondrial genome (mtDNA) mutation, paternal leakage, mitochondria transplantation/mitotherapy, and somatic cell nuclear transfer (SCNT). In this study, we developed two novel approaches to construct mitochondrial heteroplasmy cellular models. Methods: Model 1: the yak cell line (Bos grunniens) was transfected with p-eGFP-neo plasmid while mammary alveolar cell-T (MAC-T) cell line from cattle cells (Bos taurus) was stained with MitoTracker Deep Red FM. The yak cell line was used as recipient cells which fused with enucleated cattle cells. Model 2: The cattle cell line was stained with MitoTracker Green FM while yak cells were stained with MitoTracker Deep Red FM. Cattle cells were used as recipient cells which fused with enucleated yak cells. Following fusions, the single cells exhibiting dual positive fluorescence signals were sorted into 96-well plate by fluorescence-activated cell sorting. Confocal fluorescence examination confirmed that the cells with mitochondrial heteroplasmy were sorted. Results: The two methods can generate a variety of mitochondrial heteroplasmy cells of interest which can aid in understanding the patterns and influencing factors underlying heteroplasmy changes. Conclusions: The mitochondrial heteroplasmy cellular model contributes to managing heteroplasmy mitochondrial changes and preventing the development of mitochondrial declines.

A Prototype of Ultrasensitive Time-Resolved Fluoroimmunoassay with Enhanced Fluorescence System for the Trace Determination of Urinary 8-Hydroxy-2`-Deoxyguanosine, the DNA Oxidative Stress Biomarker.

This study presents a new highly sensitive and specific time-resolved fluoroimmunoassay (TRFIA) for the measurement of trace amounts of the urinary 8-hydroxy-2`-deoxyguanosine (8-OHdG) which is a biomarker for oxidative stress on DNA. The assay relied on a competitive binding approach and a mouse monoclonal antibody which recognized 8-OHdG with high specificity. In this assay, 8-OHdG conjugated with bovine serum albumin protein (8-OHdG-BSA) was employed as a solid phase antigen. The competition occurred between the 8-OHdG present in the sample solutions and the assay plate-coated 8-OHdG-BSA for a limited quantity of the anti-8-OHdG antibody labeled with a chelate of europium of ethylenediaminetetraacetic acid. The fluorescence signal of the europium chelate-labeled antibody was enhanced by a solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The validation demonstrated a working range of 10-600 pg mL-1 and a limit of quantitation of 10 pg mL-1. When applied to urine samples, the assay exhibited satisfactory accuracy and precision in quantifying 8-OHdG. In summary, this study introduces the first TRFIA capable of detecting urinary levels of 8-OHdG at picogram levels. The assay outperforms existing analytical techniques for 8-OHdG in terms of sensitivity, convenience, and analysis throughput.

Pegylated NIR Fluorophore-Conjugated OBHSA Prodrug for ERα-Targeted Theranostics with Enhanced Imaging and Long-Term Retention

In recent years, the near-infrared (NIR) fluorescence theranostic system has garnered increasing attention for its advantages in the simultaneous diagnosis- and imaging-guided delivery of therapeutic drugs. However, challenges such as strong background fluorescence signals and rapid metabolism have hindered the achievement of sufficient contrast between tumors and surrounding tissues, limiting the system’s applicability. This study aims to integrate the pegylation strategy with a tumor microenvironment-responsive approach. A novel esterase-activated EPR strategy prodrug, OBHSA-PEG-DCM, was designed. This prodrug links OBHSA, a protein degrader capable of efficient ERα protein degradation, to the PEG-modified fluorescent group (dicyanomethylene-4H-pyran, DCM) via an ester bond. This integration facilitates targeted drug delivery and enhances the retention of the fluorescent group within the tumor, allowing distinct in vivo tumor imaging periods. Experimental results show that, benefiting from overexpressed esterase in cancer cells, OBHSA-PEG-DCM can be efficiently hydrolyzed, releasing OBHSA and pegylated DCM. OBHSA demonstrated potent inhibition against MCF-7 cells (IC50 = 1.09 μM). Simultaneously, pegylated DCM exhibited remarkable in vivo imaging capabilities, lasting up to 12 days in mice, due to the enhanced permeability and retention (EPR) effect. OBHSA-PEG-DCM holds promise as a theranostic agent for ERα-positive breast cancer, offering both therapeutic and diagnostic capabilities. Importantly, this study highlights the utility of pegylated NIR fluorophores for long-circulating drug delivery systems, addressing current challenges in achieving high-contrast tumor imaging and effective targeted drug release.

Deep imaging of LepR+ stromal cells in optically cleared murine bone hemisections

Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional (3D) architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions. Deciphering the spatial interaction of leptin receptor-expressing (LepR+) stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue. In this study, we introduce an optimized protocol for the 3D analysis of skeletal tissues, enabling the visualization of hematopoietic and stromal cells, especially LepR+ stromal cells, within optically cleared bone hemisections. Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae. The protocol entails tissue fixation, decalcification, and cryosectioning to reveal the marrow cavity. Completed within approximately 12 days, this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals. The bone hemisections are compatible with diverse antibody labeling strategies. Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software, assessing a spectrum of parameters. With proper storage, the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months. This protocol is robust, straightforward to implement, and highly reproducible, offering a valuable tool for tissue architecture and cellular interaction studies.

A Small‐Molecule Drug for the Self‐Checking of Mitophagy

Mitophagy that disrupt mitochondrial membrane potential (MMP), represents a critical focus in pharmacology. However, the discovery and evaluation of MMP‐disrupting drugs are often hampered using commercially available marker molecules that target similar or identical zones. These markers can significantly interfere with, obscure, or amplify the functional effects of MMP‐targeting drugs, frequently leading to clinical failures. In response to this challenge, we propose a “one‐two punch” drug design strategy that integrates both target‐zone drug functionality and non‐target zone biological reporting within a single small‐molecule drug. We have developed a novel mitophagy self‐check drug (MitoSC) that exhibits dual‐color and dual‐localization properties. The functional component of this system is a variable MitoSC that disrupts MMP homeostasis, thereby inducing mitophagy. Upon activation, this component transforms into a blue‐fluorescent monomer (MitoSC‐fun) specifically within the mitochondrial target zone. The biological reporting component is represented by a red‐fluorescent monomer (MitoSC‐rep) that localizes to lysosomes, the non‐target zone. As mitophagy progresses, the fluorescent signals from MitoSC‐rep (lysosomes) and MitoSC‐fun (mitochondria) converge, enabling real‐time monitoring of the mitophagy process. Our findings underscore the potential of a single‐molecule drug to exert target‐zone specific actions while simultaneously providing non‐target zone self‐checking, offering a new perspective for drug design.

A Small-Molecule Drug for the Self-Checking of Mitophagy.

Mitophagy that disrupt mitochondrial membrane potential (MMP), represents a critical focus in pharmacology. However, the discovery and evaluation of MMP-disrupting drugs are often hampered using commercially available marker molecules that target similar or identical zones. These markers can significantly interfere with, obscure, or amplify the functional effects of MMP-targeting drugs, frequently leading to clinical failures. In response to this challenge, we propose a "one-two punch" drug design strategy that integrates both target-zone drug functionality and non-target zone biological reporting within a single small-molecule drug. We have developed a novel mitophagy self-check drug (MitoSC) that exhibits dual-color and dual-localization properties. The functional component of this system is a variableMitoSCthat disrupts MMP homeostasis, thereby inducing mitophagy. Upon activation, this component transforms into a blue-fluorescent monomer (MitoSC-fun) specifically within the mitochondrial target zone. The biological reporting component is represented by a red-fluorescent monomer (MitoSC-rep) that localizes to lysosomes, the non-target zone. As mitophagy progresses, the fluorescent signals fromMitoSC-rep(lysosomes) andMitoSC-fun(mitochondria) converge, enabling real-time monitoring of the mitophagy process. Our findings underscore the potential of a single-molecule drug to exert target-zone specific actions while simultaneously providing non-target zone self-checking, offering a new perspective for drug design.

A ratiometric fluorescent probe with dual near infrared emission for in vivo ratio imaging of cysteine.

Accurately detecting cysteine (Cys) in vivo is crucial for diagnosing Cys-related diseases. A novel ratiometric fluorescent probe featuring dual near-infrared emission is developed in this study for the in vivo ratio imaging of Cys. The probe comprises a hemicyanine organic small-molecule dye (HCy-CYS) with specific Cys recognition capabilities covalently coupled with carbon dots (CDs) synthesized using glutathione (GSH) as the carbon source (GCDs), forming a unique composite nanofluorescent probe (GCDs@CYS). The probe undergoes a specific reaction with acrylate upon the addition of Cys, converting HCy-CYS to HCy-OH. Consequently, the GCD fluorescence intensity at 685nm gradually decreases, whereas that of HCy-OH at 720nm progressively increases, yielding a ratiometric fluorescence signal. Notably, both emission wavelengths of the probe exceed 650nm, thereby effectively mitigating the interference from background signals during cellular and in vivo imaging. Furthermore, the probe demonstrates high specificity for Cys, enabling its differentiation from homocysteine and GSH. The Cys concentration and fluorescence ratiometric intensity exhibit a strong linear correlation at 10-150μM with a detection limit of 0.95μM. These results indicate that the ratiometric fluorescent probe can serve as a valuable tool for monitoring Cys-related physiological or pathological processes.

Harmonized technical standard test methods for quality evaluation of medical fluorescence endoscopic imaging systems

Fluorescence endoscopy technology utilizes a light source of a specific wavelength to excite the fluorescence signals of biological tissues. This capability is extremely valuable for the early detection and precise diagnosis of pathological changes. Identifying a suitable experimental approach and metric for objectively and quantitatively assessing the imaging quality of fluorescence endoscopy is imperative to enhance the image evaluation criteria of fluorescence imaging technology. In this study, we propose a new set of standards for fluorescence endoscopy technology to evaluate the optical performance and image quality of fluorescence imaging objectively and quantitatively. This comprehensive set of standards encompasses fluorescence test models and imaging quality assessment protocols to ensure that the performance of fluorescence endoscopy systems meets the required standards. In addition, it aims to enhance the accuracy and uniformity of the results by standardizing testing procedures. The formulation of pivotal metrics and testing methodologies is anticipated to facilitate direct quantitative comparisons of the performance of fluorescence endoscopy devices. This advancement is expected to foster the harmonization of clinical and preclinical evaluations using fluorescence endoscopy imaging systems, thereby improving diagnostic precision and efficiency.