Endotoxin detection is critical for the diagnosis and management of sepsis and for the safety evaluation of pharmaceutical products. Here, we report a peptide-based digital enzyme-linked immunosorbent assay (dELISA) that integrates two high-affinity, phage-displayed peptides into a single-molecule array platform, enabling highly sensitive and specific endotoxin detection without the use of antibodies. The carboxylated activation of magnetic beads was optimized using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at 10 °C, and L-lysine (Lys) was identified as an effective blocking agent that maintained bead monodispersity through electrostatic repulsion. Under optimized conditions, the assay achieved a detection limit of 0.2 pg mL[-1] with a linear range spanning 0-200 pg mL[-1]. The method exhibited strong accuracy (coefficient of variation (CV) < 15%) and interference resistance (recovery rates of 76.9%-110.1%) when applied to pharmaceutical formulations such as saline, 5% dextrose, and commercial ophthalmic solutions. In clinical validation, the platform successfully quantified endotoxin in plasma samples from septic and non-septic patients and demonstrated excellent recovery (78.0%) in whole blood spiked with exogenous endotoxin. By eliminating antibody-related limitations, this peptide-based dELISA offers a robust and translational tool for direct endotoxin detection in complex biological matrices.

Membrane-based adsorbents serve as core functional materials for the rapid analysis of trace substances. However, the inherent trade-off between permeability and adsorption capacity generally limits the analytical performance of membrane-based micro-solid phase extraction (M-μSPE) in trace detection. To address this limitation, a hydroxyl-functionalized microporous organic network (MON-OH) membrane was fabricated in this study. Its unique hollow porous structure and abundant hydroxyl active sites effectively balanced the permeability and adsorption performance during extraction. A highly sensitive method for the detection of endogenous salicylic acid (SA) in plants was established by coupling MON-OH-based M-μSPE with HPLC-MS/MS. The results showed that the method had excellent analytical performance, with a wide linear range (1.5-1000.0 pg mL-1), a good Pearson correlation coefficient (R ≥ 0.9993), a low limit of detection (0.5 pg mL-1), and good reproducibility (RSD ≤ 5.6%, n = 6). This method successfully detected trace levels of salicylic acid (SA) ranging from 1.5 to 50.1 ng g-1 in tomato roots, stems and leaves. The data revealed that SA in different plant tissues exhibited divergent response patterns under Cd stress, and an obvious concentration threshold existed for SA to respond to Cd exposure. This work demonstrates the outstanding application potential of MON membranes in sample pretreatment and offers a novel synthetic strategy for functionalized MON membranes.

The relentless pursuit of rapid, ultrasensitive, and real-time analytical tools has positioned evanescent wave-based biosensors at the forefront of modern diagnostics. These platforms, which exploit the surface-confined electromagnetic field generated during total internal reflection, enable label-free, highly sensitive detection of biomolecular interactions. This review critically examines the recent advances in evanescent-wave-based analytical approaches for nanobiosensing, providing a comprehensive overview of the current research landscape and future perspectives. We delve into the predominant analytical platforms, including Surface Plasmon Resonance (SPR), Localized Surface Plasmon Resonance (LSPR), and various optical waveguide and fiber-optic configurations. A central focus is placed on the synergistic integration of nanomaterials, such as metallic nanoparticles, graphene and other 2D materials, and quantum dots, to enhance analytical performance through signal amplification and novel surface functionalities. Furthermore, we highlight current trends shaping the field's trajectory, including convergence with microfluidics to create Lab-on-a-Chip (LOC) systems for point-of-care (POC) applications, the deployment of machine learning for advanced data interpretation, and the development of portable, smartphone-integrated readers. Finally, the review addresses the persistent analytical challenges, including matrix effects, nonspecific binding, and manufacturing scalability, and provides a forward-looking perspective on the emerging materials and technologies poised to overcome these hurdles and redefine the future of bioanalytical science.

5-Aminosalicylic acid (5-ASA), a common anti-inflammatory, is an emerging contaminant due to its widespread presence in aquatic environments and ecological risks. Developing sensing platforms for its trace detection is essential for environmental monitoring. This study developed a highly selective molecularly imprinted polymer (MIP) combined with reduced graphene oxide (rGO) for electrochemical detection of 5-ASA. A Deep Eutectic Solvent (DES), chosen for its eco-friendliness and excellent solvating ability, served as the functional medium to improve the imprinting process. The synthesis was suggested by molecular dynamics (MD) simulations and Quantum Theory of Atoms in Molecules (QTAIM) theory, which identified an optimal molar ratio of 3 : 1 : 3 for choline chloride : caffeic acid : ethylene glycol. The MIP/rGO sensor exhibited excellent analytical capabilities, including a wide linear range (0.3-700 μM) and a low detection limit (0.15 μM). It also demonstrated high selectivity against interferents and was effectively used on water samples, confirming its reliability as a sensor for pharmaceutical residues in water systems.

Exosomal piR-823 is a promising specific biomarker for colorectal cancer, yet its ultrasensitive detection remains challenging due to its extremely low abundance and high sequence complexity. Herein, we developed a cascaded CHA-CRISPR/Cas12a photoelectrochemical (PEC) sensing platform enabled by an S-scheme Bi2WO6/BiOBr heterojunction for the reliable detection of piR-823. The flower-like spherical Bi2WO6/BiOBr heterojunction exhibits enhanced visible-light absorption and efficient charge transfer arising from the S-scheme structure, which establishes a strong photoactive basis for the PEC platform. By integrating catalytic hairpin assembly with CRISPR/Cas12a trans-cleavage activity, a cascaded signal amplification strategy is established, which significantly improves the detection sensitivity. Under optimized conditions, the platform shows a distinct negative correlation between transient photocurrent and the logarithm of piR-823 concentration (1.0-1.0 × 10[6] fM), with an ultralow detection limit of 0.34 fM (S/N = 3). It also demonstrates high specificity, good reproducibility, and satisfactory recovery (96.87%-103.41%) in exosome lysate. This work not only presents an efficient PEC biosensing platform for piRNA analysis but also offers guidance for the rational construction of S-scheme heterojunctions in high-performance PEC biosensors, holding great promise for early colorectal cancer diagnosis and prognostic monitoring.

The application of tandem mass spectrometry (MS/MS) for detecting inborn errors of metabolism (IEM) is an important achievement in newborn screening (NBS). A single blood spot provides information on about 60 metabolites and related ratios that enable the screening of approximately 50 different diseases. A major limitation of newborn screening for IEM is the lack of matrix-matched dried blood spot (DBS) quality control (QC) materials in clinical laboratories. Herein, we developed DBS QC materials for IEM and investigated their recovery, homogeneity, and stability. Four concentration levels of DBS materials were prepared by spiking 9 amino acids, 6 acylcarnitines, and succinylacetone into a hemoglobin solution, which was dialyzed prior to preparing DBS. Hemoglobin solutions containing the predetermined quantities of metabolites were spotted, dried, and packaged. Isotope-dilution mass spectrometry was employed to estimate the endogenous contents and validate the final concentrations of alanine (Ala), proline (Pro), valine (Val), leucine (Leu), ornithine (Orn), methionine (Met), phenylalanine (Phe), arginine (Arg), citrulline (Cit), carnitine (C0), acetylcarnitine (C2), glutarylcarnitine (C5DC), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), and succinylacetone (SA). The recovery, homogeneity, and post-preparation short-term stability of the DBS materials were also evaluated using an MS/MS assay. The recovery for amino acids was 81.4%-121.7%, with the exception of 68.2% for Arg. The recovery for acylcarnitines and succinylacetone was 60.0%-97.9%. The homogeneity, evaluated as between-spot coefficient of variation (CV) for the multilevel DBS materials, was 4.4%-16.6% for amino acids and 3.3%-19.8% for acylcarnitines and succinylacetone. Furthermore, DBS materials stored at 4 °C showed minimal deviation over the 28-day evaluation period for all metabolites. Multilevel DBS QC materials based on a hemoglobin matrix for newborn screening of IEM were developed for the first time. These QC materials can be used to monitor the daily analytical performance and improve the technical proficiency in clinical laboratories.

The development of mixed-mode chromatography (MMC) is a research hotspot in the field of separation science, and carbon quantum dots (CQDs) show remarkable potential in the development of stationary phases for chromatographic columns due to their excellent properties. In this study, amphiphilic CQDs with both hydrophilic groups and hydrophobic long-chain alkyl groups were synthesized from mercaptopropionic acid and 1,12-diaminododecane, and bonded with silica gel to obtain the column stationary phase Sil-CQDs. The stationary phase effectively separated hydrophobic and hydrophilic compounds such as alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), anilines, nucleoside bases, sulfonamides, and thioureas in both reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) modes. The impacts of chromatographic conditions on the retention behaviour of analytes were investigated. Additionally, the Sil-CQDs column showed good separation results in comparison with the Sil-Cl column without CQDs. It also exhibited outstanding stability and reproducibility, satisfying the needs for practical analysis. This work offers a novel approach for the separation and analysis of complicated materials as well as an innovative idea for the use of CQDs in mixed-mode chromatography.

Recent studies have demonstrated that terahertz spectroscopy can identify starch absorption peaks in solid pellets containing powdered plant samples with minimal interference of other plant matrices. Here, we applied this analytical method to assess photosynthetic capacity in plant leaves with a relatively low starch content. To monitor starch produced through photosynthesis, rice leaves were measured in the frequency range of 3.0-9.5 THz using a Fourier-transform spectrometer equipped with a bolometer. A starch over-accumulation mutant (α-glucan water dikinase1) and wild type (cv. Nipponbare) were harvested after 2 and 3 months of cultivation. In the terahertz measurements, all leaves exhibited an absorption peak at 9.0 THz attributed to starch and the peak intensities were consistent with starch contents obtained by chemical analysis. Furthermore, correlations were found between second derivative signals and starch content (R[2] = 0.86-0.87). However, a higher accuracy model was developed using peak height calculated using the peak top at 9.0 THz and a baseline between 8.65 and 9.35 THz (R[2] = 0.91). These results indicate that just three frequencies are sufficient on behalf of the full range spectra, suggesting the feasibility of a starch sensor for future applications. Therefore, terahertz technology can serve as a valuable analytical method for assessing starch accumulation and photosynthetic capacity in unpurified plant leaves.

Molecular absorption spectroscopy provides a species-selective basis for quantitative gas sensing and compact optical diagnostics in combustion, industrial processes, and emission monitoring. However, in scanned semiconductor laser spectrometers operating without an online frequency reference, residual tuning nonlinearities introduce deviations in the wavenumber coordinate that affect spectral registration, baseline variation, and concentration retrieval. Here, we report a joint inversion method for CO2 direct absorption spectroscopy, in which the molecular absorption-band structure is used as an internal spectral constraint for effective wavenumber coordinate correction and concentration retrieval. Using the prior trajectory derived from the scan as the initial coordinate, the method estimates the residual wavenumber correction for each scan, a compact model of the non-absorbing intensity baseline, and the CO2 mole fraction within a unified forward model, without requiring an online Fabry-Perot etalon, reference absorption cell, or wavemeter. In experiments with a DFB laser operating near 4990 cm[-1], a 45.0 cm effective path length, and standard CO2/N2 gas mixtures with concentrations ranging from 1000 to 4500 ppm, the absorption-band prior method reduced the absorbance residual RMSE from 2.01 × 10[-3] to 9.60 × 10[-4] compared with that of single-peak retrieval. For concentration retrieval, the mean absolute relative error and concentration standard deviation decreased from 4.08% to 1.63% and from 92.3 ppm to 38.9 ppm for single-scan retrieval and from 2.32% to 1.39% and from 60.8 ppm to 27.1 ppm after averaging over nine scan periods, respectively. These results show that spectroscopic constraints applied across the absorption band provide an effective approach for improving spectral registration, concentration accuracy, and retrieval stability over short measurement intervals in DAS systems operating without an online frequency reference.

Jerantinines are natural indole alkaloids with promising anticancer activity in breast cancer cells via mechanisms of action involving tubulin binding and disruption of microtubule dynamics, with growth inhibition values, GI50 < 0.4 µM. However, metabolic responses of different breast cancer phenotypes are not yet understood. In this work, LC-MS-based untargeted metabolomics was used to investigate the response of four selected breast cancer cell lines representing different subtypes (MCF-7, BT-474, MDA-MB-231, and SKBR-3) to treatment with Jerantinine A acetate (JAa). Based on univariate, multivariate and pathway analyses, the results showed metabolic perturbations in amino-acid metabolism, fatty acid oxidation, and redox homeostasis. In breast cancer cells treated with 0.4 µM of JAa for 72 h, the elevated levels of oxidation-related metabolites, including 2-oxoglutarate, butyryl carnitine, propionyl carnitine, and 5-oxo-L-proline, indicated increased oxidative stress. Interestingly, only MCF-7 cells accumulated the antioxidants glutathione and taurine relative to untreated controls, and this response was accompanied by upregulation of wild-type p53 in this cell line. This p53-associated antioxidant capacity offers a mechanistic explanation for the difference in observed GI50 values: GI50 of 0.37 µM in MCF-7 compared to 0.19 µM and 0.14 µM in SKBR-3 and BT-474 cells, respectively, both of which harbour mutant p53 and did not mount an equivalent antioxidant response. These findings provide new insights into the metabolic effects of JAa on breast cancer cells and further support its potential as an anticancer agent. The observed metabolic differences between cell lines may inform future strategies for personalized therapy.

Biomass-based CDs constitute a rapidly developing, eco-friendly category of nanomaterials engineered mainly for ecological biosensing applications. By utilizing organic materials, including crop residues, botanical wastes and discarded food products, as the carbon source or precursor, these CDs offer a green synthesis method that avoids the risk of traditional chemical synthesis. As these green CDs are fabricated from natural precursors, these nanomaterials may contain surface-bound heteroatoms, like sulfur, nitrogen, and phosphorus, derived from their organic feedstocks. Therefore, these functionalized CDs facilitate the quick, easy and highly sensitive identification of environmental pollutants, such as pharmaceutical residues, toxic heavy metals, agricultural pesticides and novel contaminants. The presence of heteroatoms improves their fluorescence properties, selectivity, and interaction with target analytes. Consequently, these CDs exhibit rapid, user-friendly, and sensitive detection of environmental contaminants, including pesticides, heavy metals, antibiotics, and emerging pollutants. From this perspective, green-synthesised CDs are expected to have a substantial long-term impact on environmental monitoring. Their compatibility with portable sensing platforms, suitability for field-deployable platforms, and ability to detect contaminants at trace levels make them promising candidates for next-generation environmental monitoring systems. However, the transformation of these sustainable nanomaterials into practical environmental solutions will require further investigations into their biomass precursors, structural property correlations, and mechanism-driven sensing strategies. Instead of mainly focusing on lower detection limits, future research studies should focus on mechanistic insights, real environmental stability and real-world applicability to enable the practical utilisation of sustainable carbon dot sensing platforms.

Understanding the behaviour of lipids during digestion of food and formulations can enable more rational design of lipid-based materials to enhance the oral delivery of drugs and nutrients. Traditionally, light microscopy and electron microscopy have been used to 'watch' digestion at the droplet level, while small angle X-ray scattering has more recently given structural insights at the ensemble level, however none of these techniques provide information on local changes in chemical composition of a digesting fat droplet. In this study, we used synchrotron Fourier transform infrared microspectroscopy (s-FTIRM) as a chemical imaging technique to map the distributions of milk components and their digestion products in individual fat globules to obtain a better understanding of the interfacial characteristics and chemical specificity behind the structural changes that occur during digestion. The results showed localisation of fatty acids at the core and the oil/water interface of digesting fat globules and the presence of fatty acid-calcium soaps, which exist both as isolated aggregates and are partially accumulated at the interface. This proof-of-concept study highlights the novel application of s-FTIRM as a label-free, chemically-specific imaging tool to resolve the spatial distribution of digestion products, such as free fatty acids and fatty acid-calcium soaps, within and around individual milk fat globules. This approach provides new insights into the localised chemical changes during lipid digestion at the single droplet level.

The development of diagnostic tests that offer rapid and quantitative results can improve the health of patients, suffering from both chronic and acute diseases. Inflammatory bowel disease (IBD) is a chronic autoimmune condition, where a patient can undergo periods of sudden flare-ups and prolonged remission. The current biomarkers of interest are either detected using central laboratory methods, or, are non-specific to IBD. In this work, we develop a series of lateral flow immunoassays for the detection of IBD biomarkers lactoferrin and myeloperoxidase, in an artificial stool matrix. By utilising surface enhanced Raman scattering (SERS) active nanotags, we demonstrate the feasibility of these SERS-based lateral flow immunoassays for proof-of-concept singleplex and duplex detection via Raman mapping. Due to the variation in background caused by the stool matrix, we have investigated methods of assessing the SERS signal from the Raman maps, to transition the tests from giving qualitative to quantitative results. This has been achieved by extracting the SERS intensities from the maps associated with non-specific interactions, to define unique intensity thresholds for each lateral flow immunoassay, overall improving the sensitivity, reproducibility, and linearity in limit of detection studies. We achieved limits of detection of 3.2 ng mL[-1] for lactoferrin and 5.0 ng mL[-1] for myeloperoxidase, and limits of quantification of 18.8 ng mL[-1] for lactoferrin and 8.0 ng mL[-1] for myeloperoxidase. These values are well below the "normal" level of these biomarkers in stool, indicating this assay would be able to accurately monitor changing biomarker concentrations. Furthermore, we used a ratiometric approach to evaluate the maps from duplex lateral flows and detect each biomarker simultaneously on the same strip. Overall, this work demonstrates that it is possible to quantitatively detect IBD biomarkers in stool on a lateral flow with SERS, using improved Raman signal analysis methods to overcome complex sample background interference and enhance sensitivity.

The development of fluorescent probes that integrate spatiotemporal organelle targeting with analyte sensing remains a significant challenge. Herein, we report a modular library of four styryl pyridinium fluorophores (Py-Cz-CN, Py-Cz, Py-Am-CN, and Py-Am) based on a donor-π-acceptor (D-π-A) architecture for sequential organelle targeting and sulfite sensing. All four probes exhibited rapid and selective plasma membrane localization, with high colocalization coefficients (PCC = 0.81-0.88). Notably, Py-Cz underwent spontaneous time-dependent translocation from the plasma membrane to mitochondria over 6 h, with the Pearson's correlation coefficient increasing from 0.15 to 0.87, and this mitochondrial accumulation was confirmed to be ΔΨm-dependent by CCCP-induced membrane depolarization. In contrast, cyano substitution at the vinyl bridge preserved plasma membrane localization while introducing a Michael acceptor for selective reaction-based fluorescence turn-off sensing of bisulfite/sulfite. Py-Cz-CN exhibited high sensitivity toward bisulfite (LOD = 5.9 μM) and enabled both exogenous and endogenous sulfite imaging in live cells. DFT and TDDFT calculations further supported the excited-state characteristics and sensing mechanism. This work establishes a simple molecular design strategy for combining organelle-specific trafficking with analyte-responsive fluorescence in multifunctional small-molecule probes.

The toxicity of organophosphate (OP) insecticides and pesticides is increasing in the human population owing to their indiscriminate use globally. The need to achieve food security for the rising population is the main reason for the excessive use of such chemical compounds. The hazardous effects of OP pesticides can be mitigated only through conscientious efforts in detecting them in various food and water resources. In the present study, we developed carbon quantum dots (CQDs) and recombinant Nus-organophosphorus hydrolase (Nus-OPH) enzyme co-immobilized chitosan polymer film-based biosensors for the detection of OP compounds, such as coumaphos (CP), methyl parathion (MP) and ethyl paraoxon (EP), present in river water and household water resources using a fiber optic spectrophotometer. The E. coli strains were utilized for the expression of the Nus-OPH enzyme, purified and subsequently immobilized on the chitosan thin films containing CQDs that were synthesized from triethylene diamine and phthalic acid through microwave irradiation. The thin-film biosensors composed of CQDs-Nus-OPH were characterized utilizing several methods like XRD, XPS, TEM, CLSM, and enzyme activity measurements. The biosensing studies of CP, EP, and MP were performed by recording fluorescence changes using reflectance probe-mediated measurements in a fiber optic spectrophotometer. This study determined that the CQDs-Nus-OPH thin film-based biosensor has the potential to detect/analyze EP, CP, and MP in the 0-100 µM range with the limits of detection of 145 ppb, 178 ppb, and 188 ppb, respectively, within a time frame of 5 minutes. The accuracy of the biosensor in the OP-spiked water matrices was found to be 100%-102%, indicating that the CQDs-Nus-OPH thin-film biosensors can detect the analyte accurately. These results indicate that this biosensor platform shows potential for organophosphate detection, although further studies are needed to improve specificity and analyte discrimination.