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Per- and polyfluoroalkyl substances (PFASs) are persistent pollutants with shifting pollution characteristics toward short-chain and structurally diverse variants, posing great challenges for precise analysis. Pretreatment, as a rate-limiting step, lacks systematic guidance for technology selection. This review makes a unique contribution by constructing a "target substance characteristics-sample matrix-detection requirements" three-dimensional matching framework, systematically categorizing pretreatment technologies into solvent-based, adsorption-based, and advanced types, and dissecting their mechanisms, advantages, limitations, and applicability. Solvent-based technologies excel in long-chain PFASs analysis in simple matrices, while adsorption-based methods enable efficient enrichment of short-chain PFASs via functional materials, and advanced technologies meet green and complex matrix demands. Background contamination control strategies are also emphasized. Current challenges include inefficient short-chain enrichment, high material costs, and underdeveloped on-site devices. Critical needs involve the development of functional solvents, artificial intelligence-driven adsorbents, standardized protocols, and full-process PFASs-free systems. This review provides actionable references for PFASs analysis optimization.

Mass spectrometry combined with stable isotope labeling is a powerful technique for detecting disease-related changes in glycosylation patterns and identifying potential biomarkers. However, stable isotope labeling reagents that simultaneously offer high sensitivity, low cost, and stable sialic acid modifications remain scarce. In this study, we developed a convenient and cost-effective microwave-assisted method for synthesizing a stable isotopic quaternary phosphonium hydrazide labeling reagent pair, ¹⁴N/¹⁵N-P₄HZD, for the quantitation difference analysis of N-glycans using HPLC-ESI-HRMS with high sensitivity and convenience. This strategy features high labeling efficiency, excellent reproducibility, and strong linearity (R² = 0.9984) within a dynamic range spanning two orders of magnitude. The reagent pair is compatible with multiple ion source mass spectrometers and front-end chromatographic separation technologies. In particular, it enhances the ionization efficiency of sialylated N-glycans and facilitates their detection. The relative quantification method has been effectively applied to analyze the variations in N-glycomic profiles from two muscular atrophy models induced by simulated microgravity, specifically the C2C12 cell and hindlimb unloading mouse serum. We discover that these variations display characteristic relevance in both models. N-Glycans Man₃GlcNAc₃Fuc₁ and Man₃GlcNAc₄Gal₁Fuc₁Sia₁ exhibit their potential as biomarkers for the early diagnosis of muscular atrophy. The mass spectrometry method based on the ¹⁴N/¹⁵N-P₄HZD reagent pair offers a convenient and feasible strategy for the difference analysis of N-glycomics, demonstrating significant potential for application in the discovery of clinical biomarkers.

Potato early blight, caused by Alternaria solani, presents a significant threat to the potato industry. Existing detection methods for A. solani often fail to simultaneously achieve simplicity and accuracy. A gold-platinum (AuPt) nanozyme-assisted CRISPR/Cas12a system, termed the nanoparticle enzyme-assisted CRISPR detection (NACD assay) was developed. By integrating the precise target recognition of CRISPR with the enzyme-like activity of AuPt nanozymes, this system achieves simple, sensitive, and visual detection of A. solani. The NACD assay provided visual results through a distinct color change produced by the substrate catalyzed by the AuPt nanozyme. It can detect 100 copies/μL of the target dsDNA (A. solani 5.8S rRNA gene) and 10⁻³ ng/μL A. solani genomic DNA. This detection method demonstrates high specificity, with no cross-reactivity observed with three other pathogens. Moreover, the incorporation of a filter paper-based readout enables straightforward visual detection by the naked eye, making it particularly suitable for on-site testing. Overall, these features make it an effective on-site diagnostic tool, allowing the potato industry to manage early diseases more efficiently.

Chronic hepatitis B (CHB) is a global health threat caused by the hepatitis B virus (HBV). Nucleos(t)ide analogs (NUCs) are the standard treatment for CHB patients with HBV replication. However, NUCs cannot eliminate covalently closed circular DNA (cccDNA), the template for HBV replication, from the nuclei of hepatocytes; hence, patients with CHB need life-long treatment with NUCs because HBV relapses easily upon cessation of NUCs because of the residual cccDNA. However, NUCs can be terminated by accurately monitoring the activity of the cccDNA. Despite the recent development of highly sensitive detection systems for HBV biomarkers, the detection accuracy remains poor, especially at lower concentrations. Therefore, it is desirable to develop more sensitive and reliable biomarkers that have a good correlation with cccDNA, even at lower concentrations. In this study, we developed a digital enzyme-linked immunosorbent assay (ELISA) system for detecting hepatitis B core antigen (HBcAg) using a pair of monoclonal antibodies. The digital ELISA system successfully detected 2.9 pg/mL, which can be converted to 58 aM HBcAg, and the sensitivity was approximately 1000 times higher than that of conventional ELISA. Furthermore, the influence of plasma and serum matrices on the sensitivity can be ignored if the concentrations of the matrices are 1% or less. In addition, the antibody-blocking assay indicated that the digital ELISA system reacted specifically with HBcAg, even in the presence of serum or plasma. Collectively, our highly sensitive digital ELISA system for detecting HBcAg is a potentially new approach for evaluating therapeutic strategies for CHB.

Examining the changes in microRNA expression in vivo enhances the understanding of their tumor-associated functions and facilitates clinical assays based on their roles as disease markers. However, due to the low abundance and high homology, their detection remains challenging. Herein, we developed an exponential amplification method by combining a symmetric dumbbell-type probe (SDTP) and toehold-initiated hyperbranched rolling circle amplification (HRCA) to obtain high sensitivity and selectivity. The toehold region of the dumbbell-type probe specifically binds to the target, triggering a toehold-mediated strand displacement reaction and thus activating SDTP into a circular structure. In the presence of phi29 DNA polymerase, primer P2, and dNTPs, the HRCA reaction proceeds continuously using the circular structure as a template, enabling exponential amplification of let-7a. When the concentration of let-7a ranges from 1.00 fmol·L^-1 to 1.00 nmol·L^-1, the fluorescence intensity obtained by this method exhibits a linear correlation with the logarithm of let-7a concentration (unit: pmol·L^-1), with a detection limit of 214 amol·L^-1. This method can distinguish homologous miRNAs with single-base mismatches. Serum spiking recovery experiments verified the accuracy of the method in actual serum sample detection; meanwhile, the simplicity was demonstrated by the detection of let-7a in different cell lysates without RNA extraction. Therefore, our work exhibited a simple, sensitive, and specific detection of miRNAs in biological samples, which holds promise as a potential tool for miRNA analysis in clinical testing.

Globoid cell leukodystrophy (GLD) is a genetic neurodegenerative disease caused by mutations in galactosylceramide β-galactosidase (GALC) that results in the accumulation of the cytotoxic sphingolipid, psychosine. As psychosine is a biomarker specific to GLD, identifying the most afflicted regions of the nervous system can assist in better understanding the disease mechanism. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) and parallel reaction monitoring were utilized to elucidate the spatial distribution of the psychosine analyte and confirm the identity of the ion in a sagittal section of a GALC-deficient mouse brain. The presence of the psychosine was increased in specific anatomical regions of the brain responsible for the bodily functions that are impaired by GLD (cerebellum and brain stem). Several electrospray solvent additives (dopants) have enhanced the detection of various analyte types but with little success in enhancing the detection of sphingolipids. This study investigates the usefulness of ammonium fluoride electrospray doping in the positive ion mode for lipidomic IR-MALDESI MSI analysis.

Upconversion nanoparticles (UCNPs)-based fluorescence lateral flow immunoassay (FLFIA) has significant application prospects in clinical diagnosis, but it still suffers from relatively low sensitivity in detecting trace amounts of biomarkers. Here, we report for the first time a strong fluorescence-emitting core-shell-shell NaLuF₄@NaLuF₄:Yb,Er@NaLuF₄ UCNP as the FLFIA probe. The UCNPs-based FLFIA platform enables point-of-care detection of cardiac troponin I (cTnI) with high sensitivity, accuracy, and resistance to autofluorescence interference. NaLuF₄:Yb,Er encapsulated in the intermediate shell layer can effectively reduce the cross-relaxation and surface quenching of the luminescence center, resulting in a higher quantum yield. Compared with the conventional core-shell NaLuF₄:Yb,Er@NaLuF₄ UCNPs, the fluorescence intensity increases by 2.4 times. The UCNPs-based FLFIA was further integrated with a portable smartphone platform for rapid, on-site detection. The UCNPs probe enabled sensitive cTnI detection, with a detection limit of 0.035 ng/mL in buffer and 0.059 ng/mL in serum, and a detection range of 0.05-100 ng/mL. In testing clinical samples, the results from the platform showed excellent correlation with hospital results. We anticipate that the NaLuF₄@NaLuF₄:Yb,Er@NaLuF₄ UCNPs can serve as a promising immunolabeling nanoprobe for highly sensitive and accurate FLFIA detection.

Solid-contact ion-selective electrodes (SC-ISEs) have gained prominence as versatile tools for monitoring ionic species in complex agricultural and food matrices, offering low-cost, miniaturizable, and field-deployable alternatives to conventional laboratory assays. This review critically examines electrochemical transduction techniques for SC-ISEs, including potentiometry, coulometry, amperometry, voltammetry, and electrochemical impedance spectroscopy. By evaluating these techniques based on their detection mechanisms, sensitivity ranges, selectivity characteristics, and application potential, we highlight how dynamic electrochemical modes can overcome potentiometry limitations. Emphasis is placed on the role of solid-contact design, nanostructured materials (e.g., conducting polymers, carbon nanomaterials, and laser-induced graphene), and integrated readout strategies that enhance sensor performance in real-world applications. We also analyze state-of-the-art configurations for ion detection in soil, water, and food products. Finally, we discuss current challenges and offer perspectives on SC-ISEs integration into cyber-physical systems, where real-time, connected, and autonomous sensing will be central for sustainable agriculture and food systems, while also addressing regulatory considerations.

Capillary electrophoresis (CE) is primarily a technique for separating and analyzing samples based on their differing physicochemical properties. CE is renowned for its rapid analytical capability and minimal sample requirements, rendering it widely applicable in the biomedical field. However, the silica hydroxyl groups (Si-OH) present on the capillary wall can adversely affect protein separation. Modifying capillary walls through physical adsorption or chemical bonding can suppress protein adsorption, thereby enhancing separation efficiency. This review examines the causes and solutions for protein adsorption, types of capillary coatings, and the application of coated capillaries in protein separation. Finally, it outlines future trends for coated capillaries in protein separation.