Probing non-covalent membrane protein-lipid interactions remains challenging due to the high hydrophobicity and aggregation propensity of membrane proteins. Here, we present a sheath liquid capillary electrophoresis-native top-down mass spectrometry (CE-nTDMS) platform that enables reproducible infusion of aquaporin Z-cardiolipin assemblies and preserves their non-covalent electrostatic interactions during top-down fragmentation. This workflow generated a diagnostic fragment ion that localizes a lipid-binding region within the protein backbone. Notably, charge-reduced precursor fragment ions of the lipid-bound assembly exhibited asymmetrical isotopic distributions following collisional activation, while corresponding apo precursors displayed symmetrical isotopic distributions. These findings demonstrate the utility of CE-nTDMS for reliable analysis of membrane protein-lipid assemblies and provide new insight into the behavior of lipid-bound fragments during collisional activation.

This study presents a sensing strategy based on reduction-boosted fluorescence of carbon dots for ALP detection, which exhibits a low limit of detection (LOD) of 0.65 U L-1 and a linear range of 5-70 U L-1. This assay can be used to detect ALP in human serum, showing great potential for early diagnosis.

Fat, oil, and grease (FOG) deposits present significant challenges in wastewater systems, yet the stability of their fatty acid (FA) components prior to analysis remains poorly defined. Given the lack of a validated sample-handling and preservation method for wastewater FOG, this study quantified the degradation of FAs in synthetic FOG stored at -20 °C, -10 °C, 4 °C, and 25 °C over 15 months and established a temperature-specific degradation threshold. FA degradation was modelled using zero- and first-order kinetics and temperature-dependent models, including the Williams-Landel-Ferry, reciprocal polynomial, Power-law, and Vogel-Tamman-Fulcher models. Results showed that FAs degraded under all storage conditions, with cumulative losses exceeding 50% at every temperature after 15 months. After one month, degradation ranged from only 1.9% at -20 °C to 15.1% at 25 °C. Kinetic analysis indicated first-order behaviour at -20 °C, 4 °C, and 25 °C, whereas zero-order kinetics were observed at -10 °C, reflecting distinct mechanisms between complete and partial freezing. Temperature-dependent modelling indicated that frozen storage slowed degradation, but FA loss at higher temperatures exceeded model predictions due to concurrent oxidative and hydrolytic reactions. Storage duration at 30% and 50% loss thresholds confirmed that only deep-freezing extended preservation for nearly one year, whereas partial freezing, refrigeration, and ambient storage substantially reduced stability. These findings demonstrate that common storage conditions alter FOG composition prior to analysis and establish a quantitative basis for standardised sample handling and more reliable wastewater research.

Microplastics (MPs) released from contact lenses during wear can persistently contact the ocular surface through tear circulation, thereby posing a threat to ocular health. Combining thermal analysis with mass spectrometry enables accurate quantitative detection of low-concentration MPs from contact lenses, supporting exposure risk assessment. In this study, a hyphenated device consisting of a mini-pyrolyzer and a time-of-flight mass spectrometer (Py-TOFMS) was developed to enable rapid quantitative detection of MPs released from contact lenses. Compared with conventional Py-GC/MS, which typically requires approximately 30 min per sample, the proposed Py-TOFMS method eliminates the GC separation step and completes the analysis in 15 min, thereby simplifying the detection workflow and reducing the risk of sample loss and contamination. Py-TOFMS exhibited good limit of detection (LOD = 0.09 μg) and limit of quantification (LOQ = 0.27 μg), along with good linearity (R2 ≥ 0.98). This study systematically simulated daily use conditions, including soaking, heating, and ultraviolet (UV) radiation. All tested contact lenses were found to release MPs, with the most significant release occurring under UV exposure. Furthermore, by combining optical microscopy and infrared spectroscopy to observe the morphological distribution of MPs and the aging effect of contact lenses, a preliminary systematic assessment was conducted on the potential ocular health risks associated with the release of MPs from contact lenses. This study reveals that UV exposure poses the most significant risk of MPs release from contact lenses, underscoring the urgency of assessing the potential health risks associated with ocular exposure to MPs.

This study presents the design and implementation of an innovative optical sensor based on Chemiluminescence Resonance Energy Transfer (CRET) utilising luminol, green carbon dots, and K3Fe(CN)6 for the specific detection of calcium ions (Ca2+) in human saliva. The sensor exploits the unique photoluminescent properties of green carbon dots, which serve as the acceptor in the CRET mechanism, while luminol acts as a chemiluminescent donor. Upon binding of calcium ions, a significant change in the luminescence intensity was observed, indicating a successful interaction between the target ion and the sensor components. Experimental results demonstrated a high sensitivity with a limit of detection (LOD) of 0.71 mg L-1, showcasing a linear response within the concentration range of 10.02-240.04 mg L-1. The sensor exhibited excellent selectivity for calcium ions over other common ions present in saliva.

Smartphones are promising platforms for portable molecular diagnostic assays, particularly for non-urban communities and low-resource settings that lack laboratory facilities. Engineering of peripheral components around smartphones, and the adoption of advanced light-emitting materials like semiconductor quantum dots (QDs), have enabled sensitive fluorescence detection with smartphone cameras. Here, we investigated two complementary strategies for enhancing fluorescence signals and signal-to-background ratios with smartphone-based devices: adoption of "supra-QD" and "super-QD" colloidal assemblies of QDs, and a simple and robust implementation of laser-scanning illumination. The latter has been unexplored to date, with nearly all smartphone-based devices based on conventional epi-illumination for excitation of fluorescence. The laser scanning increased signal-to-background ratios (up to 8-fold) for imaging microwell chips with fluorescent solutions, was suitable for Förster resonance energy transfer (FRET)-based sensing in this format, and was far superior to epi-illumination for imaging lateral flow membranes (14-fold increase in signal-to-background ratio). Compared to individual QDs, the supra-QD and super-QD assemblies lowered detection limits by orders of magnitude, both in bulk solution and in lateral flow binding assays. The combined benefits of laser scanning and super-QDs is projected to provide nanogram per millilitre or better limits of detection in lateral flow immunochromatographic assays. As a head-to-head comparison that demonstrates and quantifies the potential gains from an atypical illumination approach and emerging ultra-bright materials, this work informs strategies toward realizing globally accessible point-of-care diagnostic technologies based on smartphones.

The prolonged or excessive use of berberine (Ber) can induce drug resistance and adverse reactions, necessitating the development of simple, sensitive, and rapid on-site detection methods. In contrast to conventional sequence truncation, we rationally extended the stem region of the aptamer to elevate its melting temperature, complemented by loop-region base mutagenesis. The obtained aptamer (BBRE7M9) exhibits substantially enhanced thermal stability, which is 6 °C and 10 °C higher than those of BBR38S-3A10C and BBR4S3, respectively. Its dissociation equilibrium constant remained stable at 1.42-1.86 μM across 22-40 °C. Circular dichroism, molecular docking, site-directed mutagenesis, and displacement assays collectively indicated that Ber binds within a hydrophobic minor groove constituted by C8, A9, T10, T17, and T18. This confined microenvironment restricts intramolecular motions and suppresses non-radiative transitions, triggering a fluorescence turn-on response. Upon integration with a smartphone-based portable dark-box platform, the sensor exhibited a linear response over 0-800 nM with a detection limit of 53.9 nM and satisfactory selectivity. This stem-elongation approach demonstrates a practical route to enhancing aptamer thermal stability, and the mechanistic elucidation of the key binding sites provides a structural basis for designing berberine-aptamer-based turn-on fluorescent probes. More broadly, this strategy may also serve as a reference for optimizing other aptamer sensors that suffer from similar temperature-induced signal attenuation.

A coumarin-based fluorescent ligand, 4-hydroxy-2-oxo-2H-chromene-3-carbaldehyde (HCC), was synthesized and evaluated for the selective detection of tryptophan (Trp) among the nine essential amino acids. The probe exhibited remarkable selectivity toward Trp through an excited-state intramolecular proton transfer (ESIPT) mechanism facilitated by intramolecular hydrogen transfer. Upon interaction with Trp, HCC forms an HCC-Trp complex (HCP), which undergoes a transient tautomeric transformation between enol (E) and keto (K) forms via an excited-state intramolecular proton transfer (ESIPT) process. This results in an approximately 11-fold enhancement in fluorescence intensity at 385 nm, accompanied by a bathochromic shift in both the absorption and emission spectra. The probe HCC exhibited a low detection limit of 1.53 μM and a binding constant of 1.6 × 10[4] M[-1] towards Trp. The sensing behaviour and binding mechanism were systematically confirmed using UV-Vis and fluorescence spectroscopy, [1]H NMR, mass spectrometry, Job's plot analysis, and density functional theory (DFT) calculations. Additionally, HCC exhibited notable binding affinity toward biomacromolecules such as calf thymus DNA (ct-DNA), bovine serum albumin (BSA), and pig liver esterase (PLE), supported by spectroscopic studies and molecular docking analysis. The binding constant for ct-DNA, BSA and PLE was found to be 5.7 × 10[4] M[-1], 5.6 × 10[4] M[-1] and 1.19 × 10[5] M[-1], respectively. Fluorescence imaging experiments further confirmed the applicability of HCC for detecting Trp in biological samples along with in vitro anticancer studies. Thus, this study demonstrates that a simple coumarin-based small-molecule probe can selectively recognize tryptophan through an ESIPT-driven mechanism. The probe HCC exhibits high sensitivity, strong binding affinity, and compatibility with biological systems, enabling fluorescence-based detection and imaging of Trp. The findings highlight the potential of HCC as a multifunctional probe for biochemical sensing and provide a promising platform for developing small-molecule fluorophores for amino acid recognition.

This study presents a simple and sensitive fluorescence-based sensing platform for arsenic (As) detection in water using cadmium sulfide (CdS) nanostructures. Three morphologically distinct CdS nanostructures (nanorods, nanoparticles, and nanocubes) were synthesized via thermal decomposition of cadmium(II) bis(dibutylcarbamodithioate) and evaluated as fluorescent probes. Among them, CdS nanocubes exhibited the highest fluorescence quenching efficiency (86%), attributed to their high surface energy and enhanced interaction with arsenic species. The sensing system showed a linear response over the concentration range of 0-20 mg L[-1], with a limit of detection of 0.012 mg L[-1], a limit of quantification of 0.041 mg L[-1], and a relative standard deviation of 2.11%, demonstrating excellent sensitivity and precision. Practical applicability was validated using arsenic-spiked water samples, with results in close agreement with those obtained using the commercial QUICK™ arsenic field test kit. Density functional theory (DFT) calculations further revealed favorable adsorption of arsenic species on the CdS (001) surface, providing atomic-level insight into the fluorescence quenching mechanism. These findings demonstrate a cost-effective, robust, and mechanistically informed sensing strategy for arsenic monitoring in environmental waters.

Bacteraemia is a severe infection associated with high risks of complications and mortality. Diagnosis relies mainly on blood cultures, followed by culture-based methods, which remain the gold-standard but are time-consuming. Isothermal bacterial DNA amplification provides a rapid, reliable and cost-effective alternative for identifying and characterising bacteria directly from positive blood cultures, enabling a timelier diagnosis, particularly in resources-limited settings. However, routine implementation has been hindered by sample processing requirements and its limited multiplexing capacity. Here, we developed an on-chip loop-mediated isothermal amplification (LAMP) workflow, ALADIM (accessible and integrated LAMP analysis for multiplex detection of infectious agents in microfluidics), that integrates bacterial extraction and multiplex LAMP, followed by instrument-free amplicons detection using lateral flow immunoassay (LFIA), all within two hours (vs. 24-48 hours for the gold-standard). To demonstrate the capabilities of our device, we targeted the most common bacteria found in bacteraemia, E. coli, the co-amplification of two highly prevalent antimicrobial resistance genes, blaCTX-M-15 and blaOXA-48, and the detection of a positive control. Multiplex LAMP followed by LFIA detection was first evaluated using 14 strains cultured in LB. On-chip results were comparable to those obtained with our reference method (tube-based LAMP in a thermocycler followed by LFIA detection), showing 100% sensitivity, >84% specificity for the detection of malF and blaOXA-48, and 68% sensitivity, 83% specificity for blaCTX-M-15. As a proof of concept for the applicability of the complete ALADIM workflow, we analysed positive overnight blood cultures spiked with 15 strains resulting in 61 independent experiments (DNA extraction/amplification/LFA readout). In this complex matrix, ALADIM specificity remained above 77% for all targets, while sensitivity exceeded 94% for malF and blaOXA-48 but decreased to 56% for blaCTX-M-15. A similar decrease in overall sensitivity was observed for the in-tube method (>68%), highlighting the challenges associated with direct whole-blood analysis. This feasibility study shows the integration of multiplex DNA amplification, microfluidic processing, and LFIA detection in a single platform. Future work will focus on improving sample preparation, assay performance in complex matrices, and expanding multiplexing capabilities of ALADIM towards additional targets, to deliver a clinically reliable point-of-care testing device for low- and middle-income countries.

Non-invasive glucose monitoring plays a key role in the diagnosis and management of diabetes and related diseases. However, the application of conventional SERS glucose sensors in complex biofluids is often limited by spectral overlap between Raman signals from the reporter molecules and those of endogenous biomolecules. To overcome this issue, we developed a SERS nanoprobe capable of interference-free glucose detection. The probe comprises a gold-core silver-shell nanostructure (Au@Ag NPs), with 4-mercaptobenzonitrile (MBN) as the Raman reporter embedded between the core and the shell, and glucose oxidase (GOx) immobilized on the outer silver surface via electrostatic adsorption. Glucose is oxidized under GOx catalysis to generate H2O2, which etches the silver shell and consequently attenuates the SERS signal intensity. Accordingly, the intensity of the characteristic peak at 2226 cm[-1] decreases as the glucose concentration increases from 0 to 2 mM, enabling quantitative detection. The excellent interference-free capability of the nanoprobe originates from two structural and molecular designs. First, MBN produces exclusive Raman signals within the biological Raman-silent window (1800-2800 cm[-1]), completely eliminating spectral overlap with endogenous biomolecules. Second, the core-shell structure physically isolates the internal Raman reporter from external biological matrices, effectively maintaining signal stability and accuracy. These design features endow the probe with outstanding selectivity for glucose over its structural analogues (e.g., fructose and lactose). The probe was further applied to glucose detection in human urine samples, supporting its potential utility in clinical urinalysis.

DNA methylation is a key epigenetic modification regulating gene expression without altering the DNA sequence, and its aberrant patterns serve as important biomarkers for cancer and cardiovascular diseases. Electrochemical biosensors have emerged as powerful platforms for site-specific methylation analysis due to their high sensitivity, simplicity, low cost, and compatibility with miniaturized devices. More importantly, electrochemical interfaces enable direct coupling between molecular recognition and nanoscale signal amplification, allowing detection limits to be pushed beyond intrinsic electrochemical constraints. This review presents a mechanism-oriented framework for electrochemical DNA methylation sensing, integrating advances in interfacial signal transduction and nanomaterial-enabled amplification. Emphasis is placed on classifying representative material platforms, including carbon-based nanomaterials, metallic nanostructures, metal-organic frameworks (MOFs)/covalent organic frameworks (COFs) and DNA nanostructures, elucidating their roles in regulating electrochemical responses. In particular, we highlight how these materials modulate interfacial electron-transfer kinetics, adsorption, conductivity, and catalytic activity, governing the translation of methylation events into amplified signals. Finally, we outline emerging directions toward more quantitative and systematic design principles for interfacial biosensing architectures.

Transmission X-ray microscopy combined with X-ray absorption near-edge structure (TXM-XANES) spectroscopy enables nanoscale chemical state imaging of transition metal oxides, yet the low signal-to-noise ratio (SNR) of single-pixel spectra undermines the reliability of conventional edge-energy (E0) quantification. Herein, we present an analytical framework based on white-line peak fitting that integrates skewed Gaussian fitting for precise peak extraction, area normalization for spectral standardization, and average spectrum analysis for chemical state referencing. Compared with the E0 method, our approach reduces the full width at half maximum (FWHM) of the energy distribution to one-third (0.28 eV versus 0.83 eV) and achieves an average coefficient of determination (R[2]) of 0.997 across the entire particle, confirming superior accuracy under low-SNR conditions. Using a high-nickel Ni90 cathode as a model system, we establish a linear correlation between white-line peak energy and cell voltage. In battery cycling, 45 nm chemical-state mapping reveals interparticle reaction heterogeneity, improved uniformity after the first activation cycle, and a non-radial reaction pattern with spatially random heterogeneity originating from intragranular defects rather than Li[+] diffusion limitations. These observations are consistent with a kinetically trapped state model in which some regions remain inactive during the first charge, gradually overcome the activation barrier, and homogenize, though the activation is not fully reversible. This strategy enhances the quantitative fidelity and throughput of nanoscale chemical-state imaging and offers an efficient analytical route for operando studies of battery materials and heterogeneous systems.

Understanding dynamic cellular processes requires technologies capable of probing living cells with high spatial and temporal precision while preserving viability. Conventional approaches, including bulk omics, microinjection, and electroporation, often provide only static snapshots or induce significant cellular perturbation. Recent advances in micro- and nanoscale probes are enabling minimally invasive intracellular delivery, sampling, sensing, and manipulation with unprecedented precision. This review examines the current state of the art in micro and nanoscale probe technologies, including fluidic force microscopy (FluidFM), nanopipettes, open-space microfluidic probes, and nanoneedle arrays. We discuss how these complementary platforms enable controlled delivery of biomolecules, longitudinal molecular sampling, intracellular electrophysiology, electrochemical sensing, and spatiotemporally resolved analyses from single cells to living tissues. We placed particular emphasis on their ability to overcome the limitations of destructive endpoint assays by facilitating repeated interrogation of the same cell, quantitative molecular delivery, and multimodal measurements with subcellular resolution. Finally, we discuss the remaining engineering challenges, including improving throughput, integrating complementary sensing modalities, and advancing these promising technologies into robust platforms for laboratory studies of cultured cells and tissues.

Plant leaves are not only vital for material exchange with the environment, but also act as primary sites for the deposition, permeation, and action of exogenous functional molecules in agricultural applications such as pest control, nutrient supplementation, and post-harvest processing. Visualizing the spatial distribution of exogenous molecules in plant leaves is of great importance. Traditional methods like chromatography and mass spectrometry may require destroying samples and make it difficult to obtain in situ distribution information. Typical Raman spectroscopy suffers from fluorescence interference and low sensitivity. This study uses tobacco leaves as a model and applies stimulated Raman Scattering (SRS) microscopy combined with Lasso spectral unmixing to achieve label-free, high-resolution, rapid visualization, and semi-quantitative analysis of propylene glycol and glycerol. The results show that SRS can clearly distinguish the characteristic spectra of tobacco, propylene glycol, and glycerol, visually presenting the distribution differences of polyols on the surface and inside the leaf. Besides, the co-localization of the two components and the analysis of their relatively mass ratios are also realized. This method provides a universal technical solution for detecting the distribution of exogenous small molecules in plant leaves.