Rapid molecular diagnostics are essential for effective surveillance of infectious diseases in swine production systems. Here, we report SHARP (Single-step Hybrid RPA-CRISPR/EsCas13d Platform), a one-pot CRISPR-based platform for detection of porcine circovirus type 3 (PCV3). By integrating rapid nucleic acid release with CRISPR/Cas13d detection, a single-step one-pot detection system was established, enabling detection within 30 min under simplified reaction conditions. In the visual readout mode, the detection limit was 50 copies/μL, with no cross-reactivity observed against common swine viruses. After lyophilization and rehydration, the SHARP system showed consistent detection performance across 50 clinical samples, in agreement with qPCR results. These results indicate that SHARP provides a simplified workflow for rapid PCV3 detection, with potential for decentralized molecular surveillance in livestock production systems.

Glycoproteins are widely involved in physiological processes of eukaryotic cells, and their expression and glycosylation modifications often become aberrant during the early stages of malignant transformation, making them highly promising molecular biomarkers for the early detection and monitoring of cancer. However, the efficient recognition of glycan structures and their translation into sensitive analytical signals remain technical challenges.

Foodborne pathogens remain a major threat to public health and food safety, among which Salmonella typhimurium is a leading cause of bacterial foodborne illness. Conventional culture-, immunoassay-, and nucleic-acid-based methods are reliable but often require laboratory facilities, trained personnel, and relatively long processing times, limiting their use for rapid on-site screening. Lateral flow immunoassays are attractive for field detection but commonly suffer from limited sensitivity and subjective visual interpretation. This study addresses the need for a rapid, specific, and more sensitive on-site assay for S. typhimurium.

Zinc ions (Zn[2+]) are essential for human health, and their accurate detection in food is critical for dietary safety and nutritional assessment. Although near-infrared fluorescent probes have been widely developed for Zn[2+] detection, their application in complex food matrices remains underexplored. Herein, we report DCMB, a novel near-infrared probe based on a dicyanomethylene-4H-pyran (DCM) derivative, exhibiting high selectivity for Zn[2+] with an emission maximum at 640 nm. Anti-interference was systematically evaluated against metal ions, food-relevant small molecules, and biomolecules. DCMB enabled accurate Zn[2+] quantification in diverse food matrices: fruits (apples, bananas), dairy products (milk), cereals (oats), and pharmaceuticals (zinc gluconate tablets), with results consistent with inductively coupled plasma mass spectrometry. DCMB-loaded test strips allowed rapid and portable naked-eye visual detection and smartphone-based RGB analysis of Zn[2+] in food and supplement samples. Moreover, DCMB facilitated real-time tracking of endogenous and exogenous Zn[2+] in 4T1 cells and supported in vivo imaging in mice, with effective suppression of background autofluorescence. Together, DCMB enriches the repertoire of near-infrared fluorescent probes for Zn[2+] detection in complex food matrices.

Commercial poloxamers exhibit compositional heterogeneity, yet the minor species contributing to this heterogeneity are often incompletely resolved and therefore treated as a single impurity fraction. This limits accurate analytical characterization of pharmaceutical-grade materials and complicates the analysis of structure-property relationships. In this study, we establish an analytical approach that combines mixed-mode liquid chromatography, reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) with multiangle light scattering and refractive index detection, complemented by MALDI-TOF mass spectrometry and [1]H NMR spectroscopy, to separate, identify, and quantify minor side products in commercial high-molar-mass poloxamers. We identified poly(propylene oxide) (PPO)-poly(ethylene oxide) (PEO) diblock copolymers and PEO homopolymer as the principal side products, and determined the amount of PEO homopolymer at levels as low as ∼0.7 wt percent. Additionally, RPLC further resolved the compositional substructure within the PPO-PEO diblock population, revealing a distribution in the PPO block length that was not resolved by SEC alone. Comparison of commercial poloxamer 407 samples from different manufacturers showed that, among the heterogeneity parameters resolved here, differences in the PPO-rich character of the main PEO-PPO-PEO triblock population were most plausibly associated with the observed variations in critical micelle concentration and hydrodynamic radius of the micelles, whereas the quantified amount of PEO homopolymer had little effect under the conditions studied. Overall, the results show that combining analytical techniques enables comprehensive characterization of poloxamers by separating, identifying, and quantifying synthesis-derived side products, while also revealing the heterogeneity parameters most plausibly associated with micellization behavior.

Although nanozymes are promising substitutes for natural enzymes due to their exceptional stability, low cost, and activity tunability, peroxidase (POD)-mimicking metal oxides like Co3O4 are often limited by its moderate intrinsic activity. In this study, phosphorus-doped Co3O4 nanofibers (P-Co3O4 NFs) are synthesized via electrospinning and calcination. Optimizing the P incorporation level yields 21% P-Co3O4 NFs with significantly enhanced peroxidase-like activity. Experimental and theoretical results reveal that P incorporation modulates the local electronic structure of Co sites, promoting H2O2 adsorption, interfacial electron transfer, and Co[2+]/Co[3+] redox cycling, while the nanofibrous morphology ensures abundant accessible surfaces. A dual-mode colorimetric platform is constructed for ascorbic acid (AA) detection, yielding detection limits of 0.57 μM for solution-based assays and 1.66 μM for paper-based formats. Accurate total antioxidant capacity (TAC) determination in real samples (vitamin C tablets, fruits, beverages) is also demonstrated, with excellent recovery rates and assay time of only 140 s (solution) or 2 min (paper). This work provides mechanistic insights into heteroatom-doped nanozymes and offers a rapid, reliable sensing strategy for food quality monitoring.

Canine distemper virus (CDV) is a highly contagious RNA virus that causes severe disease in domestic dogs and diverse wildlife species, including endangered giant pandas. Current CDV diagnosis mainly relies on laboratory-based RT-qPCR, which requires nucleic acid extraction, trained personnel, and temperature-controlled instruments, limiting its use in field surveillance and resource-limited settings. Although CRISPR-based diagnostics offer promising alternatives, chemically defined extraction-free workflows compatible with one-pot Cas13d detection remain limited. This study addresses the need for a rapid, visual, extraction-free, and low-infrastructure method for CDV detection.

Chronic kidney disease (CKD) is a major global health concern, requiring timely and accurate assessment of kidney function for effective patient management. Creatinine and Cystatin-C (CysC) are well-established biomarkers for estimating glomerular filtration rate (eGFR), a fundamental parameter for CKD monitoring. However, routine clinical assessment relies on invasive blood sampling and analytical methods with limited molecular specificity, particularly for CysC, which is predominantly measured using immunoassays. Sweat has recently emerged as a promising non-invasive biofluid that enables repeated sampling without patient discomfort; however, analytical strategies capable of selectively quantifying CysC and its clinically relevant proteoforms are still lacking. Here, we present the first validated, non-invasive, and patient-friendly protocol for the simultaneous determination of creatinine, intact CysC, and its most abundant circulating proteoform, 3-proline hydroxylated cystatin-C (3prOH-CysC), in human sweat. The workflow integrates iontophoresis-based sweat induction using pilocarpine or carbachol as stimulating agent, sweat collection on bovine serum albumin-pretreated filter paper to minimize analyte adsorption, micro-extraction by packed sorbent sample clean-up and analyte preconcentration, and proteoform-resolved analysis by liquid chromatography-tandem mass spectrometry. The protocol showed near-quantitative recovery (about 85% for native CysC and 3prOH-CysC and about 95% for creatinine), excellent linearity (R[2] > 0.995), low LOD (0.01 mg/dL for creatinine, 0.4 μg/L for native CysC, and 0.3 μg/L for 3prOH-CysC), and good overall intra- and inter-day precision (RSD <15%). Application of the method to a clinical pilot cohort of stage 3-5 CKD patients demonstrated for the first time the presence of native CysC and 3prOH-CysC as well as two oxidized CysC proteoforms, namely Ox(M14)-CysC and Ox(M41)-CysC. Moreover, significant sweat-blood correlations for creatinine (ρ = 0.62, p = 0.003), native CysC (ρ = 0.56, p = 0.017), and 3prOH-CysC (ρ = 0.75, p < 0.001) were observed. These relationships were independent of age and body mass index. Overall, this study provides proof-of-concept evidence supporting the feasibility of proteoform-resolved LC-MS/MS analysis of sweat for kidney function assessment. The molecular specificity afforded by this approach, beyond that of conventional immunoassays, highlights its potential for biomarkers characterization and warrants further investigation toward patient-centered and longitudinal CKD assessment strategies.

Paper-based analytical devices constitute one of several complementary approaches to enabling portable, decentralized chemical analysis. Despite their low cost, simplicity, and compatibility with on-site testing, their analytical performance is often limited by uncontrolled wetting, reagent leaching, evaporation-driven signal drift, limited stability of recognition elements, and insufficient selectivity in complex matrices. This review critically examines deep eutectic solvents (DESs) not only as sustainable replacements for conventional solvents but also as functional components of paper analytical platforms. Particular attention is paid to how DESs can transform cellulose-based substrates by providing chemically tunable microenvironments through hydrogen-bonding and ionic interactions with cellulose hydroxyl groups, thereby regulating wettability and capillary transport, improving reagent retention, stabilizing biomolecules and nanomaterials, enabling ion-conductive domains for electrochemical transduction, and serving as immobilized sorptive phases for sample preparation. Current strategies for integrating DESs into paper substrates, sensing zones, and porous supports are discussed across colorimetric, optical, electrochemical, and extraction-based workflows. The review also evaluates key limitations, including high viscosity, incomplete understanding of DES-cellulose interactions, residual DES effects, matrix-dependent responses, leaching, long-term stability, and manufacturability. Concluding recommendations highlight key future directions, such as standardized reporting of DES composition and retention, device-relevant characterization of capillary flow and signal stability, quantitative evaluation of matrix effects, and scalable strategies for designing reproducible DES-functionalized paper analytical platforms.