Ulinastatin (UTI) is a glycoprotein extracted and purified from human urine. As a protease inhibitor, it mainly functions to inhibit the activity of various proteases such as trypsin and elastase, while also exerting anti-inflammatory effects and protecting tissues and organs. Therefore, the detection of UTI is of great significance in guiding clinical medication, evaluating disease severity, monitoring treatment effects, and judging prognosis. Based on CeO2:Eu3+ nanomaterials and luminol molecules, a dual-mode biosensor integrating electrochemiluminescence (ECL) and fluorescence (FL) was constructed for the sensitive detection of UTI. CeO2:Eu3+ significantly enhanced the ECL intensity of luminol; combined with a sandwich-type immunoreaction, this enabled sensitive and selective ECL detection of UTI. Meanwhile, FL detection of UTI was realized by leveraging the change in the intensity of luminol's characteristic emission peak in FL. Moreover, this dual-mode detection strategy can effectively improve detection accuracy, enabling the detection of UTI with high sensitivity, accuracy, and selectivity. Experimental results showed that the ECL sensor exhibited good linearity for UTI with a limit of detection (LOD) of 8.28 pM in the concentration range of 10-11-10-5 M. The FL sensor exhibited good linearity for UTI with a LOD of 0.026 nM in the linear range of 10-10 - 10-4 M. When this dual-mode biosensor was applied to the detection of UTI in serum samples, the results of both detection modes were satisfactory.
A sensitive fluorescence (FL) biosensor was developed for the detection of Staphylococcus aureus (S. aureus) using DNA tetrahedrons (DTNs) based stimuli-responsive DNA microcapsules. FAM-labeled DTNs were initially encapsulated within the inner pores of DNA microcapsules. Owing to the physical shielding effect of the DNA shell, no FL signal was detected, maintaining the biosensor in a "fluorescence off" state. Upon exposure to S. aureus, the aptamer specifically bound to the target bacteria, triggering the disassembly of the DTN-loaded DNA microcapsules. This led to the controlled release of FAM-labeled DTNs and the subsequent recovery of the FL signal. Based on this, sensitive and specific detection of S. aureus was achieved with a detection limit of 0.36 CFU∙mL- 1. Moreover, satisfactory results were obtained for S. aureus detection in spinach leaves, milk and human serum, demonstrating that the proposed sensing system held promising application prospects for the detection of S. aureus in food and related complex matrices.
Eggerthella lenta (E. lenta) is an opportunistic anaerobic pathogen associated with severe systemic infections, yet rapid and accurate diagnostic tools remain limited. To address this challenge, we developed a highly sensitive and specific dual-readout diagnostic platform integrating recombinase-aided amplification (RAA) with the CRISPR/Cas13a system, targeting the highly conserved rsmG gene of E. lenta. The assay offers two detection modalities: a real-time fluorescence readout and a visually interpretable lateral flow strip. Analytical evaluation demonstrated that the fluorescence-based assay achieved a limit of detection (LOD) of 4.4 copies per reaction (95% CI: 3.7-5.6 copies/reaction), while the instrument-free lateral flow assay yielded an LOD of 10⁴ copies per reaction. The platform exhibited exceptional specificity, showing no cross-reactivity with 10 common non-target bacterial species. Clinical validation was performed using 24 synovial fluid samples, all confirmed positive for E. lenta by Sanger sequencing. The fluorescence assay successfully detected all 24 samples, achieving a detection rate of 100% (24/24). In parallel, the lateral flow assay detected 21 of the 24 positive samples, yielding a detection rate of 87.5% (21/24). The three samples undetected by the lateral flow strip were verified as true positives by sequencing, indicating that the discrepancy was due to the lower analytical sensitivity of the strip format rather than a lack of specificity. In conclusion, this dual-mode RAA-CRISPR/Cas13a platform serves as a robust and practical tool for rapid clinical diagnosis and point-of-care (POC) triaging of E. lenta infections. The fluorescence format is optimal for high-sensitivity laboratory testing, whereas the lateral flow variant provides a deployable alternative for rapid, point-of-care screening in resource-limited environments.
A simple and sensitive fluorescence sensing method was developed for ciprofloxacin (CIP) determination based on manganese-doped carbon dots (Mn-CDs). The Mn-CDs were synthesized through a one-step hydrothermal method using anhydrous citric acid and manganese chloride tetrahydrate as precursors. The prepared Mn-CDs exhibited good dispersibility, uniform nanoscale morphology, abundant surface functional groups and favorable fluorescence properties. The incorporation of Mn was designed to introduce coordination-related binding sites for CIP, thereby enhancing the interaction between Mn-CDs and CIP. Under excitation at 330 nm, the Mn-CDs showed a pronounced fluorescence enhancement response toward CIP, enabling their use as fluorescent probes for quantitative detection. Under the optimized conditions, the fluorescence intensity increased linearly with CIP concentration over the range of 20 nM-10 μM, with a detection limit of 1.12 nM. The proposed sensing system exhibited satisfactory selectivity toward CIP over various potentially interfering substances and good storage stability. The practicality of the method was further verified by analysis of pond water samples, affording recoveries of 86-118% with relative standard deviations below 5%. In addition, the method showed acceptable applicability for CIP determination in different pharmaceutical formulations. These results indicate that the Mn-CD-based fluorescent probe provides a convenient, sensitive and promising platform for CIP determination in environmental and pharmaceutical samples.
A novel "signal-on" fluorescence sensing strategy was carried out based on GR5 DNAzyme and covalent organic framework (COF) to detect lead ions (Pb2+). The COF was synthesized at room temperature, with excellent structural stability and high fluorescence quenching efficiency, enabling low background signal and reliable detection. The GR5 DNAzyme that responds to Pb2+ contains a large single-stranded loop structure, which can be adsorbed onto the COF, resulting in fluorescence quenching of the attached FAM fluorophore. In the presence of Pb2+, the GR5 DNAzmye is specifically activated and cleaved, releasing a short FAM-labeled fragment of 5 bases. Because this short fragment has weak affinity for the COF, it is released from the COF surface, leading to fluorescent recovery. The sensor based on COF/DNA enzyme shows high selectivity and sensitivity, with a detection limit of 0.067 nM. In addition, the method has been successfully applied to the detection of Pb2+ in real water samples, with recoveries ranging from 89.58% to 112.70%. This work provides a simple and sensitive platform for Pb2+ detection and further demonstrates the potential of COFs as high-efficient fluorescence quenchers in functional nucleic acid-based biosensing.
This study introduces the first reported stepwise fluorimetric strategy for the sensitive and rapid determination of the ultra-short-acting β₁-blocker, landiolol hydrochloride. Initially, an environmentally benign method was developed by exploiting the drug's native fluorescence in water (λex/λem = 217/298 nm). While this cost-effective approach successfully utilized water as a green solvent, it demonstrated limited LOD of 32.57 ng/mL. To enhance fluorescence performance, various organic solvents were investigated, with acetonitrile providing the highest signal at λex/λem = 222/300 nm and improving the LOD to 16.31 ng/mL. To enable ultra-trace clinical monitoring, a third "turn-on" method was developed using silver nanoparticles (AgNPs). By measuring fluorescence enhancement at λex/λem = 260/524.6 nm upon interaction with landiolol, this platform amplifies the signal, achieving an outstanding LOD of 3.10 ng/mL. AgNPs were prepared using a green synthesis approach employing Aloe vera extract as a natural reducing and stabilizing agent in an aqueous medium avoiding using hazardous chemicals. Water was used throughout both nanoparticle preparation and the AgNP-enhanced determination, reinforcing the eco-friendly profile of the method. Unlike native fluorescence, the AgNP-assisted method enhances sensitivity through nanoparticle-mediated surface passivation, with a possible auxiliary contribution from resonance energy transfer (RET)-like processes. Selectivity is improved through preferential adsorption of landiolol on the AgNP surface and a large Stokes shift that minimizes interference from UV-absorbing matrix components. The resulting enhanced sensitivity and reduced matrix interference make this method a powerful tool for ultra-trace, selective, and sustainable determination of landiolol in pharmaceutical formulations and human plasma, demonstrating its suitability for routine quality control and bioanalytical applications.
The co-administration of Dapagliflozin and Sitagliptin has attracted considerable interest in the management of type 2 diabetes mellitus due to their complementary therapeutic effects. However, their simultaneous determination is analytically challenging because of the significant overlap in their native fluorescence spectra. In this study, a selective and sensitive first-derivative synchronous spectrofluorimetric method was developed for the simultaneous determination of both drugs without prior separation. The proposed approach enabled efficient spectral resolution through zero-crossing points at 348 nm and 289 nm for dapagliflozin and sitagliptin, respectively, using a constant wavelength difference (Δλ = 30 nm). The method exhibited excellent linearity over the concentration ranges of 50-1000 ng/mL and 100-2000 ng/mL for dapagliflozin and sitagliptin, respectively, covering concentration levels relevant to their reported maximum plasma concentrations (Cmax), with low limits of detection (16.02 and 31.07 ng/mL, respectively), indicating high sensitivity. The proposed method demonstrated satisfactory accuracy (mean recoveries of 100.67% and 99.86%) and precision (%RSD < 2%). The method was successfully applied to the analysis of pharmaceutical dosage forms and spiked human plasma, showing reliable recoveries. To the best of our knowledge, this is the first validated spectrofluorimetric method for the simultaneous determination of these co-administered drugs, offering a simple, cost-effective, and efficient alternative to conventional analytical techniques.
A new Schiff-base fluorescent probe namely methyl 2-(((2-hydroxypyridin-3-yl) methylene) amino) pyrimidine-5-carboxylate (HL) has been designed and synthesized. Structural characterization of the purely synthesized compound was acquired by IR spectroscopy, 1H NMR, 13C NMR and Mass spectra. The Schiff base compound was explored as chemosensor for various transition as well as alkali metal ions viz. (Na+, K+, Ca2+, Ba2+, Sr2+, Cd2+, Ni2+, Co2+, Cu2+, Mn2+, Mg2+, Zn2+ and Fe3+). The study clearly reveals that the probe HL can rapidly and selectively recognize Mg2+, with the detection limit (LOD) of 7.2 × 10- 7 M. The results of fluorescence spectroscopic studies clearly reveals that a remarkable enhancement of the fluorescence intensity of the probe HL was observed in presence of Mg2+ ions, which was barely impacted by other metal cations. The stoichiometric ratio of HL to Mg²⁺ was determined to be 1:1 from Job's plot, B-H plot, 1H NMR titration, Mass spectra and the binding constant was calculated as 3.7 × 10⁴ M⁻¹ using the Benesi-Hildebrand method. Reversibility nature of the complex has been studied in presence of EDTA, which in turn regenerated free ligand for further Mg2+ sensing. HL further exhibits INHIBIT logic gate behaviour with Mg²⁺ and EDTA as inputs, as observed from the emission response at 414 nm. Over a broad pH range (9-12), this chemosensor shows excellent fluorescence sensing abilities to Mg2+. The probe HL has also been utilized for the detection of Mg2+ in real water samples. In addition, HL showed moderate antibacterial activity against Escherichia coli and Staphylococcus aureus, highlighting its bioactive potential.
Ratiometric fluorescence sensing provides built-in self-calibration against fluctuations in excitation intensity and environmental conditions, yet it commonly demands complex design of dual-emission materials. Herein, we present an exceptionally simple strategy: physically mixing separately synthesized blue-emitting carbon dots and yellow-emitting carbon dots to yield a white-light-emitting composite (CA/OPD-CDs). Under single excitation at 256 nm, the mixture displays three well-resolved emission peaks at 340, 440, and 560 nm. The 560 nm peak is selectively and efficiently quenched by Ag⁺ ions, whereas the 440 nm peak remains stable, enabling self-calibrated ratiometric detection via the intensity ratio F₅₆₀/F₄₄₀. The probe exhibits a linear response to Ag⁺ from 0 to 11 µM (R² = 0.996) with a detection limit of 80 nM, well below the WHO drinking water guideline. Outstanding selectivity over 13 other metal ions is demonstrated. UV-Vis absorption and fluorescence lifetime measurements confirm a static quenching mechanism through ground-state complex formation. The method was successfully applied to Ag⁺ determination in tap water, achieving recoveries of 97.9-106.5%. This work demonstrates that simple physical blending of distinct carbon dots can replace intricate one-pot syntheses to generate powerful self-calibrating ratiometric sensors, offering a facile and generalizable platform for analytical applications.
Rapid, species-level identification of harmful algal blooms (HAB) remains constrained by laboratory-based molecular workflows and bulky fluorescence instrumentation. We report an integrated portable optoelectronic biosensing system for post-extraction quantification of Heterosigma akashiwo 18S rDNA. The key innovation is not the GO-based recognition chemistry itself, but the analytical signal-reading and conversion strategy: GO-regulated fluorescence recovery is captured by a sealed lens-filter-PIN photodiode module and converted through a resistor-based current-to-voltage circuit into an amplified photovoltage output. FITC-labelled ssDNA probes are quenched by GO nanosheets and recover fluorescence upon target hybridization. The recovered fluorescence is excited by a fixed-wavelength 488 nm laser, filtered at 520 ± 10 nm, collected by a sealed optical detection head, converted from photocurrent to photovoltage across a 10 kΩ sense resistor, amplified 500-fold, and digitized for quantitative readout. This voltage-mode architecture eliminates reliance on benchtop fluorimeters, microscope-camera assemblies, or picoampere-level current instrumentation, thereby improving the portability and practical usability of GO-based fluorescence assays. Using a 12-well double-layer microfluidic cartridge and a modified Stern-Volmer linearization strategy, the system quantified target DNA over 10- 4-105 pM with a limit of detection of 79.90 aM. After extracted DNA was introduced into the cartridge, the assay delivered results within 30 min. Specificity was verified using one- and two-base mismatch sequences and non-complementary DNA from co-occurring HAB species. Mixed-sample tests at both gene and cell levels further demonstrated sequence-specific quantification. This work advances GO-regulated fluorescence HAB sensing toward a portable post-extraction voltage-output platform for rapid microalgal monitoring.
A rapid and sensitive fluorescence-based method for antioxidant capacity assessment was developed using a lanthanide-based metal-organic framework (LVMOF-1). The Eu3+-MOF, incorporating an electron-deficient viologen ligand, enables host-guest interactions with phenolic antioxidants, leading to a quantitative quenching of a composite optical signal driven by a resonance convolution of native Eu3+ photoluminescence and second-order scattering, inner filter effect and colloidal modulation. Using gallic acid as a model compound, the system exhibited a non-linear Langmuir-type response that was successfully linearized through logarithmic transformation, enabling reliable quantification of total antioxidant capacity over an extended concentration range. The method showed excellent analytical performance, with a limit of detection of 0.0077 mg L-1, good linearity (R2 = 0.992), and reproducibility below 7% RSD. Compared to conventional assays (Folin-Ciocalteu, DPPH, ABTS, and FRAP), it provided higher sensitivity, shorter analysis time (≤ 2 min), and improved robustness in complex matrices, inherently minimizing color-induced spectral interferences due to the high sensitivity of fluorescence detection. Application to honey, tea, and wine samples showed very strong correlations with established methods (Pearson r = 0.947-0.996). These results were further supported by Bland-Altman analysis, which confirmed good overall agreement with method-dependent differences, and principal component analysis (PCA), which revealed a common underlying antioxidant capacity dimension. Additionally, the LVMOF-1 assay showed the best greenness (AGREE score 0.59) and lowest cost (0.63 € per sample), highlighting its potential as a sustainable alternative for routine antioxidant screening.
Green carbon quantum dots (CQDs) with minimal environmental damage can be used as a fluorescent probe. Owing to their non-toxicity and high solubility in water, they can play a significant role in environmental monitoring and water safety. Here, a straightforward hydrothermal process using a home microwave was used to create CQDs using chicory leaves known as Cichorium Intybus. The probe was used for the determination of cefdinir (CFD) in various samples such as biological and pharmaceuticals to verify the practicality of the proposed method. The CQDs showed a fluorescence with the wavelength varying from 375 to 500 nm and the maximum emission wavelength being at 419 nm. The linear range was 10 nmol L-1 to 700 nmol L-1 with a detection limit of CFD of 3.00 nmol L-1. Based on the ideas of click chemistry principles, the "Click Analytical Chemistry Index" (CACI) is a tool for evaluating and contrasting the usefulness and application of analytical techniques. This work broadens the use of CQDs in pharmaceutical analysis, spiked plasma and synthetic urine samples. CACI tool guaranteed that upcoming analytical techniques using the CQDs fluorescence probe satisfy the changing needs of both industrial applications and scientific research.
Severe fever with thrombocytopenia syndrome (SFTS), caused by the severe fever with thrombocytopenia syndrome virus (SFTSV), poses a serious public health threat due to its high fatality rate, and delayed diagnosis is strongly associated with disease progression and increased mortality. Conventional laboratory diagnosis still relies largely on quantitative real-time PCR (qPCR), which requires specialized instruments, trained personnel, and centralized testing facilities. These challenges underscore the urgent need for rapid, sensitive, and portable diagnostic methods for SFTSV detection in clinical and on-site settings. Here, we developed an integrated isothermal amplification-based point-of-care testing (IAPOCT) platform that incorporates a recombinase polymerase amplification (RPA)-based sensing and interpretation strategy with a smartphone-interfaced portable device. This platform demonstrated low-copy analytical detection capability, achieving a limit of detection (LOD) as low as 10 copies/μL. The core amplification and fluorescence readout steps can be completed within 20 min, while the complete serum testing workflow from thermal lysis to result interpretation requires approximately 25 min. Preliminary evaluation in a cohort of 24 clinical serum specimens showed complete qualitative agreement with qPCR, correctly identifying 20 positive and 4 negative samples. Endpoint fluorescence signals were analyzed through the smartphone-based interface, enabling portable and objective qualitative interpretation of clinical samples. The lyophilized reagent format was incorporated to facilitate handling and integration with the portable testing workflow for routine on-site molecular testing in resource-limited settings. This work provides a practical platform for rapid SFTSV detection with portable fluorescence readout, with potential applications in clinical screening and on-site epidemiological surveillance. By integrating RPA-based amplification, lyophilized reagents, smartphone-assisted interpretation, and a portable device, the platform offers a compact analytical strategy for point-of-care molecular testing of infectious diseases.
In this paper, a D-π-A fluorescent probe, (E)-2-(5,5-dimethyl-3-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yl)vinyl)cyclohex-2-en-1-ylidene)malononitrile (DFP) was synthesized via the condensation of dicyanoisophorone and 9-formyljulolidine. The structure was characterized by NMR and ESI-HR-MS, and the structure optimization and energy level analysis were carried out by density functional theory (DFT) calculation. Additionally, its metal ion recognition performance was assessed by UV-vis absorption spectroscopy and fluorescence spectroscopy. The results showed that the probe could specifically recognize Fe3+ in CH3OH/H2O (1:1, v/v, pH = 7.2) buffer system. Upon the addition of Fe3+, the probe solution changed from blue-purple to colorless, accompanied by a significant fluorescence quenching of over 90%, exhibiting high selectivity without obvious interference from other metal ions. Job's plot and ESI-MS titration experiments revealed a 1:1 stoichiometry for the complex between the probe and Fe3+, with a binding constant of 1.4 × 104 M-1. Furthermore, the probe exhibited a low detection limit of 0.33 µM for Fe3+, and the optimal pH range for the response was 5-9. In terms of practicality, the probe has been successfully used for visual detection of test paper and imaging of Fe3+ in living cells.
Chikungunya virus (CHIKV) is a mosquito-borne pathogen posing a growing global threat, yet existing nucleic acid detection methods remain difficult to implement in resource-limited settings. In this study, we developed complementary catalytic hairpin assembly (CHA)-based lateral flow immunochromatography assay platforms for CHIKV nucleic acid detection by coupling CHA with time-resolved fluorescence immunochromatographic assay (TRFIA) and colloidal gold immunochromatographic assay (GICA). After systematic optimization of reaction conditions to reduce background leakage, CHA-TRFIA achieved a detection limit of 100 fM with a linear range of 100 fM to 1 μM (R2 = 0.976), whereas CHA-GICA showed a visual detection limit of 1 pM with a linear range of 1 pM to 1 μM (R2 = 0.947). Both platforms effectively discriminated CHIKV from mutant sequences and non-target viral sequences (dengue virus, Zika virus, Mayaro virus, Ross River virus, and Sindbis virus; P < 0.0001). In blinded trials using 100 pseudovirus-spiked human serum samples (50 positive and 50 negative), at 35 °C, CHA-TRFIA and CHA-GICA achieved sensitivities of 94% and 92%, with AUCs of 0.970 and 0.984, respectively; at room temperature, the corresponding sensitivities were 92% and 88%, with AUCs of 0.980 and 0.917, respectively. Specificity was 100% for both platforms under both conditions. Both platforms completed detection within 30 min. CHA-TRFIA is suitable for quantitative analysis, whereas CHA-GICA is more appropriate for rapid equipment-free visual screening, together providing a practical option for CHIKV testing in resource-limited settings.
The complex multi-layered security signals about fluorescence-based anti-counterfeiting technology with smartphone-based readers are getting a lot of attention. The blue/red biobased fluorescent carbon dots (CDs) with an average particle size of 2.73 nm are prepared using reed, O-Phenylenediamine (OPD) and Phosphoric acid (H3PO4) as precursors by a facile hydrothermal method without organic solvents. To prevent carbon dot aggregation and experience self-quenching in water or organic solvents, CDs are embedded into the high-performance porous silica material (MCM-41) by another hydrothermal treatment. In contrast, the silica framework of MCM-41 provides essential physical protection for the CDs, allowing for their uniform distribution within the material. This leads to improved fluorescence intensity, increased fluorescence lifetime of 2.02 ns, and a quantum yield of 33.29%. The matrices on filter paper, hydrogels (cellulose), and membranes (polyvinyl alcohol) with CDs@MCM-41 demonstrate distinct color variations under daylight, UV light, and a filter, this capability facilitates simple yet effective multi-layer security features for anti-counterfeiting applications, with accurate and fast color analysis can be achieved through the RGB software on a smartphone.
Gamma-ray interaction with matter is fundamental to radiation protection, nuclear technology, and dosimetric applications. In this study, the gamma-ray attenuation characteristics and thermoluminescent (TL) behaviour of transition metal (Fe3+, Mn2+) and rare-earth (Eu3+) doped yttrium orthoaluminate (YAlO3) phosphors were systematically investigated. The phosphors with composition (Y1-xMx)AlO3 were synthesized using the solution combustion method. Gamma-ray attenuation parameters were evaluated over the photon energy range of 356-1330 keV. Important radiation interaction parameters such as mass attenuation coefficient (MAC), linear attenuation coefficient (LAC), half-value layer (HVL), tenth-value layer (TVL), and mean free path (MFP) were experimentally determined using a narrow-beam transmission setup with a NaI(Tl) detector and compared with theoretical values obtained from XCOM, Phy-X/PSD, and EpiXS software. The results reveal a strong energy dependence of attenuation parameters. The MAC decreases from about 0.105 cm2 g- 1 at 356 keV to 0.052 cm2 g- 1 at 1330 keV, while HVL increases from approximately 4.6-5.0 mm to 9.8-10.2 mm, indicating higher photon penetration at elevated energies. Eu3+- doped YAlO3 shows about 2-3% higher attenuation efficiency than Fe3+ and Mn2+ doped samples due to its higher atomic number. Experimental results agree well with predictions within ± 5%. TL measurements indicate a maximum intensity of about 473 a.u. at 5 mol % Eu-doping, representing an ~ 83% enhancement. The findings demonstrate a clear relationship between photon absorption efficiency and TL response. Although thermoluminescent properties and radiation attenuation characteristics of phosphor materials have been extensively investigated independently, studies exploring the possible correlation between photon interaction parameters and thermoluminescent response in doped YAlO3 phosphors remain limited. This work addresses this gap by systematically correlating gamma-ray interaction parameters with thermoluminescent behaviour. The novelty of the present work lies in the combined experimental and computational evaluation of both thermoluminescent and photon attenuation characteristics within the same YAlO3 host matrix and in assessing the influence of different activator ions on these complementary radiation response mechanisms.
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder in which cutaneous neurofibromas (cNFs) represent one of the most common and burdensome manifestations. No approved pharmacological treatment exists. Preclinical studies are essential to evaluate candidate therapies, but reliable outcome and endpoint measures for cNFs in animal models remain limited. We developed and validated a standardized methodology to assess drug efficacy in the Prss56Cre Nf1-KO mouse model which recapitulates key features of cNFs. In this model, Nf1 inactivation and tdTomato (Tom) reporter expression were specifically targeted to Schwann cells (SCs) responsible for cNF development. This approach enables real-time monitoring, isolation, and manipulation of tumor SCs at any time. We defined macroscopic (tumor count, total Tom+ fluorescent surface area, fluorescence intensity) and microscopic (cell-type composition defined by immunolabeling with a panel of specific markers, area quantification) endpoints, developed dedicated ImageJ scripts for automated image analysis, and compared the results with those obtained using the conventional manual method. Both automated measurements showed excellent reproducibility (ICC = 1) and strong correlation with manual analysis (Spearman's coefficient > 0.90), while significantly reducing analysis time (up to 100-fold faster). Bland-Altman analyses confirmed the absence of systematic bias compared with manual scoring. The standardized image naming and metadata integration further facilitated data consolidation and statistical analysis. This validated approach provides a reliable, reproducible, and time-efficient framework for evaluating drug effects on cNFs in preclinical studies. It establishes a foundation for robust efficacy testing of candidate therapies, facilitates cross-study comparability, and accelerates therapeutic development and clinical translation.
Dysregulation of chromatin remodeling is a key driver of malignant progression. The SWI/SNF ATPase subunits SMARCA2 and SMARCA4 are essential for chromatin dynamics, yet the clinicopathological and microenvironmental landscape of SMARCA2/4-deficient esophageal adenocarcinoma (EAC) remains insufficiently defined. We analyzed 722 resected EACs from a large Western cohort. SMARCA2 and SMARCA4 status were assessed by immunohistochemistry, and tumors were classified as SMARCA-intact or SMARCA-deficient (complete loss of nuclear expression in tumor cells with internal controls). Molecular co-alterations were evaluated by immunohistochemistry and fluorescence in situ hybridization, including amplifications of MET, ERBB2 (HER2), EGFR, PIK3CA, MYC, MDM2, TERT, and Y-chromosome loss (LOY). Several markers were available from prior works. Digital pathology workflows quantified CAF markers (SMA, PDGFRβ, FAP, Periostin, Tenascin) and immune infiltrates (including CD4, CD8, FOXP3, CD20, MUM1, mast cell tryptase). Overall survival was examined using Kaplan-Meier estimates and Cox regression models. SMARCA2/4-deficient tumors accounted for 11.2% of the cohort (81/722) and were enriched among patients aged ≥ 65 years. SMARCA-deficient EACs showed a significantly higher frequency of MET amplification (16.9%), particularly after neoadjuvant therapy. Within SMARCA-deficient tumors, PDGFRβ-positive CAFs, increased plasma cell (MUM1+) and mast cell infiltrates correlated with a favorable outcome, whereas loss of Y-chromosome (LOY) identified an adverse-risk subgroup with particularly poor prognosis. This largest-to-date study defines SMARCA-deficient EAC as a distinct subtype characterized by frequent MET amplification and high-risk interaction with LOY, alongside prognostically relevant stromal-immune features, supporting refined biomarker-based risk stratification and therapeutic exploration.
Coal occupies a dominant position in China's energy structure and provides crucial support for the rapid development of the national economy. Coal and its derivatives possess advantages such as high carbon content, abundant reserves, and low cost, making them ideal carbon sources for the preparation of coal-based carbon dots (C-CDs). C-CDs not only inherit the excellent properties of traditional carbon dots, including tunable fluorescence emission, stable optoelectronic performance, good water solubility, and biocompatibility, but also possess advantages such as broad availability of raw materials and low preparation costs. This paper focuses on the application development of C-CDs in various fields, reviewing the research progress on carbon dots prepared from coal and coal derivatives as precursors. It systematically summarizes their main synthesis routes, performance characteristics, and application studies in areas such as analytical detection, optoelectronic devices, energy, and catalysis. This study aims to provide solid theoretical support for the directional preparation of high-performance C-CDs. By rationally selecting precursors and optimizing the preparation process, it achieves precise directional control over the structure and properties of C-CDs, promoting their development toward large-scale production, environmental friendliness, and enhanced performance. Simultaneously, it provides innovative technological impetus for the green transformation and upgrading of the coal industry.