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Quantifying the fission product ¹⁴⁷Nd in nuclear debris samples is an important component of post-detonation nuclear forensics. The most accurate quantifications are obtained when Nd is purified from all other fission products, actinides, activation products, and environmental matrix contained within the debris. In this study, a recently developed method for Nd purification was tested, purifying ¹⁴⁷Nd from solutions of mixed fission products using high-speed counter-current chromatography (HSCCC). Importantly, the new method allowed for faster elution of Nd from the column as compared with established high performance liquid chromatography (HPLC) methods, and resulted in accurate/precise ¹⁴⁷Nd quantification by gamma-ray spectrometry. While the up-front equipment costs associated with HSCCC may be higher, its operational costs are on par with those of HPLC (solvents, extractants, power). Gas-flow proportional beta decay counting revealed contamination from the nearest neighbor lanthanide ¹⁴³Pr (a gamma-silent radioisotope) in the HSCCC-purified samples, but the activity contribution from ¹⁴⁷Nd could still be quantified. Remarkably consistent elution profiles were observed for the HSCCC method, spanning rare earth element (REE) loadings of more than 10 orders of magnitude (tracer to mmol quantities). The reliability and speed of the new method suggest utility for the rapid separation and quantification of ¹⁴⁷Nd in unknown samples.

MicroRNAs (miRNAs) are promising molecular markers for early-stage cancer, enabling advancements in early diagnosis, precision treatment, and prognosis evaluation. However, their detection remains challenging due to low abundance, driving the demand for highly sensitive and accurate sensing platforms. Herein, we developed a cascade amplification strategy integrating catalytic hairpin assembly (CHA) with a light-initiated chemiluminescent assay (LiCA) for ultrasensitive detection of serum miRNA-21. The platform employs H1-functionalized chemibeads (¹O₂-luminescent acceptors) and H2-conjugated sensibeads (photosensitizer donors), wherein target miRNA triggers ternary complex formation and initiates autonomous CHA cycling. This process facilitates femtomolar-sensitive, low-background detection without RNA extraction by utilizing proximity-driven (<200 nm) singlet oxygen transfer to generate a collective chemiluminescence signal. The assay exhibits high sensitivity (LOD: 0.21 pM) and excellent linearity (R² = 0.9958), coupled with strong matrix tolerance in 2 % human serum, as evidenced by accurate and consistent recoveries (98.8-112 %) with <8 % deviation at spiked concentrations from 0.1 to 5 pM. When applied to clinical serum samples (n = 78), the platform demonstrated perfect diagnostic discrimination (AUC = 1.00) with 100 % sensitivity and specificity at a cutoff of 0.667 pM across breast, gastric, and colorectal cancers. A strong correlation with qPCR (r = 0.78) was observed, and miRNA-21 levels were significantly elevated in cancer patients compared to healthy controls. This platform combines the programmability of CHA with the anti-interference advantages of LiCA, offering a robust tool for low-abundance miRNA analysis in complex matrices.

Bone defects, commonly associated with osteoporosis, result in fragile bones prone to fractures in both men and women. Osteoporotic fractures often lead to prolonged healing due to impaired cell differentiation. Understanding cellular crosstalk during bone regeneration is crucial for developing effective treatments. Periosteum-derived progenitor cells (PDPCs) and adipose-derived stem cells (ADSCs) play essential roles in bone formation because ADSCs secret key growth factors, such as bone morphogenetic protein-2, Wnt family member 1, Periostin, and platelet-derived growth factor subunit A, that promote osteogenesis. To investigate cellular interactions in bone healing process in the natural bone microenvironment, we developed a biomimetic folding paper co-culture system that mimics the inflamed three-dimensional bone structure. This system enables co-culture of PDPCs and ADSCs, allowing the study of their protein crosstalk and osteogenic potential. Moreover, neutralizing assays were conducted to inhibit specific cytokines and evaluate their influence on osteogenesis. Our findings confirm that ADSCs promote osteogenesis through their secreted growth factors that enhance the differentiation and activity of PDPCs. Disrupting these cellular interactions through cytokine inhibition led to a significant reduction in osteogenic potential. It was evidenced by decreased protein expression and gene activation associated with bone formation. This biomimetic folding paper co-culture system effectively mimics the natural bone microenvironment and provides a novel platform to study bone healing mechanisms. This approach may lead to the development of more effective treatments for bone fractures.

Acetoacetate (AcAc) and β-hydroxybutyrate (βOHB) are ketone bodies involved in energy metabolism, particularly during physiological states of glucose scarcity, such as fasting, exercise, and the implementation of a ketogenic diet. The production (ketogenesis) and utilization (ketolysis) of ketone bodies are dynamic processes that can be quantified using stable isotope-labeled tracers in metabolic tracing studies, necessitating precise and sensitive analytical methods for accurately measuring both labeled and unlabeled pools. Although UHPLC-MS/MS has recently emerged as a reliable tool for quantifying ketone bodies, its dependence on ¹³C-labeled internal standards limits its utility in ¹³C-based tracer studies. AcAc, in particular, poses challenges due to its chemical instability and the scarcity of authentic, stable, isotopically labeled internal standards. While the chemical reduction of AcAc to βOHB provides a solution, this necessitates a cumbersome desalting step. To overcome these limitations, we developed a novel approach using deuterated AcAc (d₃-AcAc) and [3,4,4,4-d₄]βOHB as internal standards for the simultaneous quantification of ¹³C-labeled and unlabeled ketone bodies in biological samples. We optimized the synthesis of AcAc from ethyl-AcAc via base-catalyzed hydrolysis, achieving 99.2 ± 0.2 % purity at 60 °C for 3 h, as confirmed by ¹H NMR. Stability assessments in the extraction buffer and post-extraction serum samples confirmed the robustness of newly synthesized d₃-AcAc for at least 5 h. A comparative analysis against the labor-intensive conventional method demonstrated superior precision, accuracy, and ease of application, enabling high-throughput metabolic and clinical studies. The optimized UHPLC-MS/MS method substantially improves metabolic tracing capabilities, enabling rapid and accurate investigation of ketone body tracing studies across various physiological and pathological conditions.

Microplastic pollution, particularly from polystyrene (PS), poses increasing risks to human health due to its persistence in the environment, tendency to bioaccumulate, and ability to induce oxidative stress and hepatic injury. However, tools for dynamic imaging that elucidate the mechanistic link between microplastic exposure and liver toxicity remain lacking. Herein, we report a far-red fluorescent probe (emission maximum at 674 nm), DDAO-CT, derived from the hydroxyl-substituted red-emitting fluorophore DDAO (9,9-dimethylacridin-2(9H)-one derivative), designed for in vivo visualization of chymotrypsin activity, a key enzyme marker known to be upregulated during microplastic-induced liver damage. DDAO-CT is constructed by conjugating a 4-bromobutyryl recognition group to the red-emissive fluorophore DDAO, which quenches fluorescence by disrupting intramolecular charge transfer (ICT). Upon enzymatic hydrolysis by chymotrypsin, the ICT pathway is restored, resulting in a 14-fold enhancement in fluorescence at 674 nm with a detection limit of 3.5 ng/mL. The probe had high selectivity, low cytotoxicity, and excellent responsiveness to enzyme activity both in vitro and in PS-exposed hepatocytes. Notably, in vivo imaging in mouse models revealed dose-dependent fluorescence signals in the liver, which correlated closely with histopathological damage and elevated serum markers of liver injury. These results show that chymotrypsin activation is a downstream event of PS bioaccumulation, establishing DDAO-CT as an effective tool for visualizing pollutant-induced hepatic dysfunction. This study presents a novel chemical biology platform for noninvasive assessment of environmental hepatotoxins and offers mechanistic insights into microplastic-induced liver injury at the enzymatic level.

In this work, a new environmentally friendly natural deep eutectic solvent based on two natural compounds, camphor and citral, was proposed for the first time. The solvent exhibits favorable physicochemical properties, including stability in the presence of water, low viscosity, and good solubilizing capacity. The proposed solvent was investigated by FT-IR and ¹H NMR spectroscopy. The solvent was successfully applied for the extraction and determination of 11 polycyclic aromatic hydrocarbons in food samples (cucumber, salad, and tea infusion) by high-performance liquid chromatography with fluorescence detection. The formation of hydrogen bonds between camphor (ketone) and citral (aldehyde) resulted in the effective extraction solvent capable of interacting with non-polar polycyclic aromatic hydrocarbons via lipophilic and π-π stacking interactions. The proposed microextraction procedure provided high enrichment factors (49-68) and satisfactory extraction recoveries (71-98 %). Under optimized experimental conditions, the limits of detection ranged from 0.1 to 0.3 μg kg^-1, while typical maximum residue limits for polycyclic aromatic hydrocarbons in foodstuffs range from 1.0 to 5.0 μg kg^-1.

A three-dimensional DNA walker offers an efficient strategy for sensitive detection of analytes with signal amplification. In this study, we report a target-silent, self-driven DNA walker for detecting small molecules (SMs). The DNA walker is composed of the Mg²⁺-dependent 8-17E DNAzyme walking strand conjugated with an SM (W-SM) and the three-dimensional walking track on gold nanoparticle (AuNP). The AuNP surface is functionalized with monoclonal antibody (mAb) and fluorescently labeled substrate of DNAzyme. Without target molecules, the W-SM is attached to the surface of AuNP via the antigen-antibody interaction. The DNAzyme catalytically cleaves the substrate, driving the W-SM autonomously moving along the walking track and generating high fluorescence. In the presence of SM target, the SM target competes with the W-SM in binding with the antibody on AuNP, and the DNA walker becomes inactive, causing fluorescence decline. This DNA walker enabled detection of digoxin and folic acid at concentrations as low as 0.2nM and 1 nM, respectively. It also performed well in diluted serum samples in responding to targets. This proposed strategy provides a new approach for constructing a DNA walker with a simple design for sensitive detection of small molecules in solution phase, showing promise in applications.

In this work, a potential-resolved electrochemiluminescence (ECL) multiplex immunoassay (MIA) was developed using Ag-doping methionine-stabilized Au nanoclusters (Met-AuAgNCs) with immobilized co-reactant as the anodic ECL tag and nanocomposite of gold nanoparticles/graphene oxide/N, N'-dicaproate sodium-3,4,9,10-perylene-dicarboximide (AuNPs/GO/PDI) as the cathodic ECL tag. Compared with methionine-stabilized Au nanoclusters (Met-AuNCs), the ECL of Met-AuAgNCs was enhanced 5.61-fold. When anodic co-reactant of N,N-diethylethylenediamine (DEDA) was connected to Met-AuAgNCs, the ECL of DEDA-Met-AuAgNCs was 11.3-fold of that of Met-AuAgNCs in DEDA solution due to the shorter charge transfer distance between Met-AuAgNCs and DEDA. After a pre-oxidation at 0.95 V for 60 s, the ECL of DEDA-Met-AuAgNCs was further enhanced by 10.6- and 27.9-fold in the cyclic voltammetric and potential step modes, respectively. The pre-oxidation ECL enhancement was demonstrated by an immobilized co-reactant promoters mechanism. In a potential-resolved ECL-MIA, carbohydrate antigen 125 and carbohydrate antigen 19-9 were adopted as model analytes, with the detection limits of 0.029 and 0.076 mU mL^-1, respectively. The work provides a proof of concept using self-ECL luminophores with immobilized co-reactant promoters in situ formed for potential-resolved ECL-MIAs with isolated anodic and cathodic co-reactants.

Precise localization of carbon-carbon double bonds in unsaturated lipids is essential for elucidating lipid functions and disease mechanisms, yet conventional liquid chromatography-mass spectrometry workflows are hampered by labor-intensive pretreatment, solvent consumption, and insufficient sensitivity. Here, we presented an online supercritical fluid derivative extraction-pressure change focusing-supercritical fluid chromatography-mass spectrometry platform that integrated derivatization, extraction, purification, separation, and detection into a single automated workflow. The supercritical fluid derivative extraction strategy enabled simultaneous in situ epoxidation and cleanup, while the pressure change focusing strategy effectively mitigated chromatographic band broadening, yielding sharper peaks and enhanced sensitivity. Systematic optimization established robust operating conditions, enabling comprehensive lipid analysis to be accomplished within 24 min using only 2.5 μL of sample. The validated method achieved excellent linearity, trueness, and recovery, showing coefficients of determination (R²) > 0.9930, recoveries of 73.8-111.8%, and trueness of 82.1-116.4% with precision better than 13.7% (RSD). The method was applied to plasma samples from schizophrenia mouse models. A total of 56 unsaturated fatty acids were identified with fully resolved positions of carbon-carbon double bonds, of which eight species exhibited significant abundance changes. Moreover, isomer ratio analysis revealed disease-associated remodeling of desaturation patterns, providing new insights into lipid metabolic dysregulation in schizophrenia. Overall, the established online platform represents a rapid, sensitive, and environmentally friendly strategy for structural lipidomics, offering strong potential for biomarker discovery and broader applications in biomedical and clinical research.

The study of drug metabolites is essential for evaluating safety and optimizing drug design. Recent classes of high-molecular-weight drugs, such as PROTACs and LYTACs, present challenges for traditional metabolite identification approaches due to their complex structures. To address these limitations, we developed Drug Metabolite Finder (DMetFinder), a novel mass spectrometry-based tool designed to enhance metabolite identification. DMetFinder employs cosine similarity algorithms to filter compounds with similar structures, minimizing the risk of overlooking metabolites with large fragment losses. It also efficiently detects multiply charged ions and incorporates isotope abundance and adduct ion scoring to refine identification accuracy. By calculating a total weighted score, DMetFinder reduces false positives associated with single-filter strategies. Experimental validation demonstrates that DMetFinder significantly improves the identification of metabolites from PROTACs, providing valuable insights for future drug development.