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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.

Diethylamine (DEA) is a known precursor to some of the most toxic chemical warfare agents (CWA) as both V-agents and Fourth Generation Agents (FGAs) contain the dialkylamine functionality. DEA is also a readily available commercial substance with extensive use in the chemical industry for legitimate purposes. Because of this potential dual use, it is desirable to develop methods to trace the origin of dialkylamines if they are used for illicit purposes. We herein demonstrate that it is possible to differentiate six commercial batches of DEA using position-specific isotope analysis (PSIA) by both ²H and ¹³C NMR. Using a high-field NMR spectrometer together with a cryogenic ²H probe, we have produced ²H-{¹H} NMR data with high accuracy and precision. The PSIA by NMR results show that the intramolecular ²H ratios of all six DEAs are significantly different while two of the six DEAs have unique ¹³C ratios. Further, the intramolecular isotopic variations can be used to link the DEAs to different suppliers. The two nuclei separately contribute to isotopic profiles of the DEAs. However, combining the two techniques provides a higher-resolved isotopic profile that can differentiate all DEAs and thus be useful in forensic investigations of illegal use of chemical weapons.

This study focuses on the development of an integrated analytical platform with in situ surface-enhanced Raman spectroscopy (SERS) monitoring capabilities, achieved through the design of a dual-functional, two-dimensional microporous covalent organic framework (COF). The hydrazone-functionalized COF not only exhibits selective recognition for Au(III)/Pt(IV) ions but also achieves efficient recovery of Au(III) from the leachate of waste electronic components, with a recovery efficiency of 95.73 %. By harnessing the enzyme-like catalytic activity of the COF@Au composite and the synergistic combination of COF's confinement effects with the localized surface plasmon resonance of Au nanoparticles, we created enhanced electromagnetic field regions on the material surface. This configuration enables real-time SERS tracking of reactant-product evolution during the reduction of 4-nitrothiophenol (4-NTP), with a reaction rate constant of 0.0181 s^-1. This integrated platform combines precious metal recovery reactions, catalytic processes and in-situ SERS monitoring (" recovery - catalysis - monitoring "), establishing a new paradigm of green and real-time tracking analysis.

Four-dimensional printing technologies are accelerating the development and fabrication of stimuli-responsive devices for analytical applications. Herein, we employed the multi-material fused deposition modeling printing technique, iron(II,III) oxide nanoparticle-incorporated polylactic acid filaments, and acrylonitrile butadiene styrene filaments to fabricate an all-in-one solid-phase extraction (SPE) device, which featured the flow manifolds connecting a monolithic packing and four temperature-controlled and magnetically actuated switching valves that were actuated by a hammer-shaped cantilever. When the inner chamber of the cantilever was filled with warm water (above the glass transition temperature of the polylactic acid), the applied external magnetic field induced the bending of the cantilever to switch the flow direction. When removing the external magnetic field, the cantilever returned to its original position, allowing recovery of the flow direction for magnetically actuated fluid control. The optimized SPE device manipulated the sample and eluent streams to pass through the monolithic packing and enabled an automated SPE scheme coupled with inductively coupled plasma mass spectrometry for determination of Mn, Co, Ni, Cu, Zn, Cd, and Pb ions, with the method's detection limits ranging from 0.3 to 2.8 ng L^-1. We validated the reliability and applicability of this analytical method through determining the metal ions in reference materials (CASS-4, SLRS-5, 1643f, and Seronorm™ Trace Elements Urine L-2) and real samples (seawater, river water, ground water, and human urine). Our results suggest that four-dimensional printing technologies can effectively fabricate thermo-magneto-responsive analytical devices and enhance the practical applicability of conventional SPE schemes for multiple trace metal analysis.