Legionnaires' disease is a severe and often fatal pneumonia primarily caused by the inhalation of aerosols contaminated with the waterborne pathogen Legionella pneumophila serogroup 1. While conventional culture-based detection is often standard for monitoring evaporative cooling systems, the required 10-day cultivation period delays necessary risk assessments. To overcome this time barrier, we applied a novel approach to the flow-based chemiluminescence sandwich microarray immunoassay using cryopreserved Legionella pneumophila standards for rapid and culture-independent serotyping. The microarray features nine monoclonal and one polyclonal capture antibodies and successfully characterized the highly virulent Pontiac group at a concentration down to 10[5] total cells per 100 mL and specifically identified the highly infectious Philadelphia subtype at lower concentrations as a strain of the Pontiac group. However, some limitations of direct subtyping remain. To address the challenge, a novel workflow combining immunomagnetic separation and flow cytometry with the chemiluminescence sandwich microarray immunoassay was developed. Since viability is maintained after flow cytometric quantification, the cells can be subsequently cultured for a brief period, and single colonies can be used for subtyping. As before, we spiked real process water samples containing complex mixtures of four different subtypes using the cryopreserved Legionella pneumophila standards. This combined approach demonstrated diagnostic sensitivity, and successful reduction of the total processing time. The method enables both the precise, early quantification of Legionella pneumophila and detailed subtyping for demanded and exceeded action limits within real and complex process water matrices.

Protein-rich foods release biogenic amines (BAs) during prolonged storage, and these compounds serve as key biomarkers of food spoilage. Current separation-based methods for detecting BAs in food require sophisticated instrumentation and laborious sample pretreatment procedures. The array-sensing strategy, which mimics biological olfaction, provides a simple and rapid approach for the simultaneous detection of multiple BAs without the need for separation. Here, we constructed a multidimensional fluorescent sensor array using porphyrin-functionalized carbon dots (P-CDs) and their composites with metal ions (M-P-CDs), thereby incorporating the excitation wavelength as an additional signal dimension. This design reduces the number of physical sensors required while maintaining high discrimination capability, thereby overcoming a key limitation of conventional sensor arrays that depend solely on increasing the number of sensors. With the support of chemometric and machine learning methods, the sensor array successfully discriminated among ten BAs. We achieved quantitative detection of putrescine, histamine, and tyramine, determining their limits of detection (LODs) to be 2.37, 2.96, and 1.55 mg/L, respectively. The sensor array exhibited excellent stability (i.e., no significant fluorescence change over 30 days) and high selectivity against common interfering substances. Validation experiments on milk samples demonstrated the array's ability to reliably discriminate specimens at different spoilage stages. This work establishes a novel multidimensional array-sensing strategy for detecting BAs, an approach that holds great potential for food safety applications.

Amino acid metabolism disorders have strong links to various kidney diseases, and the kidney is essential for maintaining systemic amino acid homeostasis. Given the lack of obvious clinical symptoms in the early stages of kidney disease, searching for reliable metabolic biomarkers is essential for early diagnosis. Endogenous AAs are widely present in human biological matrices, making it difficult to obtain analyte-free blank matrices for method validation. This poses challenges in the preparation of calibration standards, leading to uncertainty and reduced consistency in the results. This study aimed to develop a rapid hydrophilic interaction liquid chromatographic method coupled with tandem mass spectrometry. It could analyze 47 amino acids and related compounds, present in various biological matrices (human plasma, urine, and cyst fluids). The calibration curves were constructed by stripping each matrix to obtain a surrogate matrix. The slopes of the different calibration curves were systematically evaluated and the analytical results were compared with results obtained through the method of standard addition. The calibration curves established using both the standard addition method and the matrix stripping method were parallel, hence matrix stripping effectively mitigates matrix effects under a variety of matrix conditions, thereby assuring the accuracy and reliability of quantitative analysis results. To further investigate the applicability of this method to large-scale sample analysis, the analysis of plasma samples and urine samples obtained from patients with two types of kidney disease was performed.

Conventional inductively coupled plasma mass spectrometry (ICP-MS) merely measures total phosphorus within nucleic acid matrices and cannot resolve intact target DNA from degraded oligonucleotide fragments or matrix-originated phosphorus contaminants. In this work, we report a hyphenated size-exclusion chromatography-ICP-MS (SEC-ICP-MS) workflow for direct mass quantification of double-stranded DNA fragments spanning hundreds to thousands of base pairs. Purified DNA fragments are introduced directly via HPLC injection for size-based separation. [31]P[16]O signals and [115]In signals are simultaneously monitored by ICP-MS, with indium serving as the internal standard. External calibration using certified inorganic phosphorus solution enables phosphorus quantification, and DNA mass concentrations are subsequently derived from the fixed stoichiometric phosphorus fraction of each defined DNA sequence. The instrumental limit of detection for phosphorus reaches 28 ppb, corresponding to an absolute mass limit of 112 pg. Method trueness was validated with two independent nucleic acid certified reference materials, with relative deviations falling between 1.2 and 5.0%, verifying favorable quantitative accuracy. The workflow was applied to two synthetic DNA fragments (300 bp and 2000 bp), giving average mass concentrations of 141.8 ± 3.7 mg·kg[-][1] and 110.5 ± 3.1 mg·kg[-][1], respectively. Orthogonal cross-validation via the digital polymerase chain reaction (dPCR) produced consistent measurements of 140.5 ± 5.1 mg·kg[-][1] (300 bp) and 113.3 ± 4.3 mg·kg[-][1] (2000 bp), jointly corroborating the robust reliability of this fragment-resolved SEC-ICP-MS quantification strategy. The developed SEC-ICP-MS workflow delivers robust, fragment-resolved absolute quantification for short-to-long DNA constructs and exhibits compatibility with functionalized modified nucleic acids.

Mass spectrometry imaging (MSI) is a robust analytical tool for resolving the spatial localization of diverse metabolites in a tissue sample simultaneously. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is particularly advantageous for analyzing leaf tissue, since it bypasses the cuticle and uses endogenous water in the sample as a matrix, avoiding delocalization and reducing costs associated with sample preparation. While direct analysis of fresh tissue offers substantial benefits, it also presents a less-discussed challenge: plants initiate a cascade of metabolic responses to various forms of stress (e.g., mechanical wounding, heat stress), so freshly-harvested tissue can undergo major metabolic changes before and during analysis. Herein, a method is introduced which retains the benefits of IR-MALDESI MSI but increases spatial resolution and quenches metabolic processes by quickly freezing leaf tissue within the source enclosure immediately after harvest. Leaf samples were mounted on a glass slide with carboxymethylcellulose gel immediately after harvest and moved into the source enclosure, humidity was purged, and the sample was frozen using the Peltier-cooled stage. Camera images demonstrated minimal ice deposition on the sample surface, and evaluation of the phenylpropanoid pathway suggested that freezing the sample inhibited post-harvest stress responses during and prior to analysis. Frozen tissue analysis significantly decreased ablation spot diameter from 65.4 to 43.9 µm, enabling approximately a 1.5-fold increase in spatial resolution with a conventional 2.97-µm IR laser.

Residual inorganic ions in automated online solid-phase extraction (SPE) workflows can alter electrospray ionization and bias LC-MS/MS quantification of perfluoroalkyl substances (PFAS) in environmental waters. We evaluated the influence of modest salt concentration (10-30 mg/L NaCl or CaCl2) on the PFAS analytical response using an online SPE-LC-MS/MS system in negative ESI-MRM (Electrospray Ionization-Multiple Reaction Monitoring) mode. PFBA (50 ng/L) was non-detectable under salt-free conditions but produced well-defined peaks after salt addition, with detection limits improving to 0.17-0.22 ng/L (LOD) and 0.57-0.74 ng/L (LOQ) at 20-30 mg/L of NaCl and CaCl2. The isotopically labeled internal standard remained stable across conditions, but the PFBA/[13]C4-PFBA area ratio increased from 1.67 to 1.89, demonstrating incomplete compensation for salt-dependent response changes by isotope dilution. A matrix effect assessment across the full PFAS target list revealed a wide range from - 74% to + 1005%, with short-chain compounds exhibiting the strongest enhancements (up to tenfold for PFPeA) and longer-chain PFAS showing weaker or negative effects. NaCl amplified chain length-dependent contrast more strongly than CaCl2 did, producing divergent responses, particularly for C8-C9 compounds. These findings demonstrate that the environmentally relevant salt concentrations substantially alter PFAS response patterns in automated online SPE-LC-MS/MS systems, with implications for method validation and data interpretation. The ionic composition should be explicitly considered when quantifying PFAS in water matrices with varying ionic composition, particularly for short-chain compounds where internal standardization provides incomplete correction.

DNA is widely employed in biological science and materials science, driving a growing demand for its effective preservation. The conventional cryopreservation method is highly resource-intensive, requiring substantial storage space and continuous energy input. These limitations highlight the urgent need to develop cost-effective and efficient alternative strategies. In this study, we creatively extended the application of chitosan derivatives to the preservation of extracellular DNA, based on our previous work on chitosan-mediated nucleic acid enrichment and detection. Among different types of chitosan derivatives, chitosan oligosaccharide lactate (COL) was identified for its outstanding DNA-binding and re-release capabilities. Upon complexation with COL, DNA is effectively protected against major environmental stressors including enzymatic hydrolysis, oxidative damage, and ultraviolet irradiation, which were the major environmental stressors. This protective interaction significantly enhances the stability of DNA under non-cryogenic conditions, which in turn minimizes the losses in DNA concentration and integrity while preserving critical sequence information. Furthermore, the binding and release of COL with DNA could be achieved handily by pH adjustment. Moreover, COL could be readily available and amenable to large-scale production. Collectively, our findings provide a great potential method for realizing convenient and cost-effective DNA preservation at room temperature.

Marine macroalgae are increasingly recognized as sustainable and chemically diverse sources of valuable biomolecules. However, the short-chain peptide fraction (2-4 amino acids) of edible and invasive seaweeds remains poorly explored, mainly because conventional proteomics workflows are generally optimized for longer peptide sequences. In this study, an untargeted LC-HRMS suspect screening workflow was developed to characterize short-chain peptides generated by hydrolysis with Alcalase®, an alkaline protease from Bacillus licheniformis hydrolysis in 14 marine macroalgae species belonging to green, brown, and red phyla collected along the Atlantic coast of southern Spain. Overall, 333 short-chain peptides, predominantly dipeptides, were putatively annotated through accurate-mass filtering and manual MS/MS interpretation. Multivariate and univariate analyses revealed distinct peptide distribution patterns among algal groups, with brown algae showing a higher abundance of hydrophobic dipeptides associated with predicted bioactive properties according to in silico screening tools. Notably, invasive brown algae displayed peptide profiles largely comparable to edible species, supporting their potential valorization as sustainable sources of short-chain peptides. Exploratory in silico analyses further suggested the occurrence of peptide sequences potentially associated with antioxidant, antihypertensive, antidiabetic, sensory, and antifungal properties. Several peptides were also predicted to contribute to umami-related flavor attributes, highlighting the relevance of these matrices for future investigation of food-related peptide functionality. Although the proposed annotations and predicted bioactivities require orthogonal confirmation and experimental validation, the results provide a comprehensive exploratory overview of marine macroalgal short peptidomes. Overall, this study expands current knowledge on algae-derived short-chain peptides and demonstrates the applicability of untargeted LC-HRMS workflows for the characterization of underexplored peptide fractions in complex marine matrices.

Neurotransmitters (NTs) play crucial roles in brain function by mediating communication between neurons at synaptic junctions and are considered an indicator for determining neurological diseases. Therefore, early and sensitive detection and monitoring are critical for maximizing the effectiveness of treatment. For this purpose, a highly active nanoflower (NF)-based colorimetric sensor system was developed for the determination of neurotransmitters. Herein, laccase@manganese dioxide NFs (lac@MnO2 NFs) were successfully synthesized via an in situ approach by the incorporation of manganese dioxide nanoparticles (MnO2 NPs) into laccase nanoflowers (lac NFs). The lac@MnO2 NFs retained 100% relative activity at pH 7, while lac NFs showed negligible activity (below 1%) under identical conditions, which is attributed to the synergistic integration of MnO2 NPs that exhibit laccase-like activity and facilitate rapid electron transport. Lac@MnO2 NFs were applied for the colorimetric detection of various neurotransmitters (NTs) including epinephrine (EP), dopamine (DA), and levodopa (L-DOPA). The analytical performance of the developed colorimetric sensor was evaluated, demonstrating a limit of detection (LOD) of 1.376 μM for EP, 0.891 μM for DA, and 0.825 μM for L-DOPA, with relative standard deviation values less than 7%. In addition, to broaden the application of NFs in colorimetric sensors, a lac@MnO2 NF-based portable agarose gel kit was designed. The developed sensing platform can be utilized for on-site detection of DA, demonstrating a low LOD of 3.33 μM and a wide detection range of 5-400 μM. These findings suggest that lac@MnO2 NF-based colorimetric sensors hold substantial potential for future clinical and biomedical applications.

This study presents a novel hybrid biosensor integrating chicken IgY antibodies and quantum dot-labeled DNA aptamers for rapid, sensitive detection of staphylococcal enterotoxin A (SEA) in food samples. The system leverages the unique advantages of avian IgY, which avoids false positives from staphylococcal protein A interference, combined with high-affinity aptamers (K d= 84 nM) selected through magnetic bead-SELEX for specific detection. The QD-conjugated aptamer reporter system enables visual fluorescence readout with a detection limit of 1 ng/mL within 45 min, offering a faster workflow, operational simplicity, and an estimated consumable cost of <$1 per test under bulk-production conditions. Validation studies using spiked and field-collected food matrices demonstrated strong agreement with PCR-based analysis, including complete concordance in the 50 tested food samples, supporting the platform's reliability for food safety monitoring. This biosensor represents a significant advancement in point-of-need toxin detection, offering laboratory-grade accuracy in a field-deployable format that requires no specialized equipment, making it particularly valuable for resource-limited settings and routine food safety screening programs.

Consumption of food containing residues of nitrofuran metabolites may pose potential risks to human health. Although many countries have prohibited the use of nitrofurans as veterinary drugs, their illegal application persists. The detection of nitrofuran (NF) metabolites is essential for ensuring food safety. In this study, we designed and synthesized a pair of novel isotope-coded derivatization (ICD) reagents, 2-(4-formylphenyl)-1-phenyl-1H-naphtho[1,2-d]imidazole (d0-PPIB) and 2-(4-formylphenyl)-1-(pentadeuteriophenyl)-1H-naphtho[1,2-d]imidazole (d5-PPIB), using phenyl as the isotope-coded group. With the aid of the ICD reagents, a sensitive and accurate method for determination of NFs using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established. The instrumental analysis time is remarkably short, with the four target analytes separated within just 4 min. The derivatization process was efficiently completed in only 15 min using microwave-assisted techniques, substantially reducing the overall experimental duration. The method achieved excellent sensitivity, with detection limits ranging from 0.011 to 0.019 μg/L, and the measurement error ranged from -2.2 to 3.5%. The intra- and inter-day precision was consistently high, with relative standard deviations (RSDs) below 5%. This ICD-based strategy effectively mitigates matrix effects while reducing costs and improving accuracy. Using one pair of ICD reagents (d0/d5-PPIB), multiple analytes are concurrently derivatized to generate their isotope-labeled internal standards, markedly decreasing commercial stable isotope-labeled (SIL) internal standard consumption. The method was successfully applied to quantify NF metabolites in three food samples.

Originally developed for small molecules in the late 1970s and early 1980s, isotope dilution mass spectrometry (IDMS) also has become the "go to" method of quantification for those wishing to characterize the mass fraction or amount of substance content of proteins in matrix-based certified reference materials (CRMs). While instrumental factors can impact the accuracy of IDMS methods, the use of double IDMS, higher order approaches, and "exact matching" have done a lot to improve the accuracy of the results. This review shows the development of IDMS for the quantification of proteins from being a method only used for value assignment of primary calibrators using isotope-labelled peptides as internal standards to a method for quantification of proteins in complex biological matrices such as serum and blood using isotope-labelled recombinant intact proteins as internal standards. It will discuss the challenges and limitations of currently used organic IDMS peptide-based approaches regarding the definition of the measurands, equilibration of sample and internal standard, digestion efficiency, and stability and suitability of the internal standards. This paper reviews the steps taken to assess the accuracy of the peptide- and protein-based IDMS methods used for the characterization of CRMs and the requirement to ensure comparability in laboratory diagnostics down to the patient level. Furthermore, it will give an outlook on future challenges, such as including structural and protein activity information in the results.

Accurate in-line monitoring of particle size and solid concentration in turbid, multiphase systems remains a significant challenge in pharmaceutical manufacturing. Most particle measurement techniques are developed for off-line analysis, while recent in-line methods mainly rely on imaging and chord length distribution (CLD) measurement. Complementing these approaches, the spatially and angularly resolved diffuse reflectance measurement (SAR-DRM) system serves as a process analytical technology analyzer, capturing detailed, configuration-dependent spectral responses of particle suspensions. This study systematically evaluates SAR-DRM's effectiveness in analyzing a wide range of particle diameters (≤90-800 µm) and concentrations (1-10 wt%) for polystyrene suspensions. The visible-NIR and NIR measurements from its multiple spatial-angular fiber configurations are further analyzed to assess their potential in complementing other in-line techniques, improving interpretability and analytical performance. The results show that combining specific SAR-DRM configurations through data augmentation markedly enhances model performance compared to individual configurations or co-adding methods, reducing error by over 50% in some cases. Moreover, data fusion of SAR-DRM with CLD measurements yields the most accurate models, particularly for mid-sized particles, decreasing the prediction error to 10-12 µm. The study highlights SAR-DRM as a promising process analytical technology analyzer for particulate process monitoring, paving the way for advanced hybrid modelling in pharmaceutical and chemical manufacturing. It also underscores the complementary sensing capabilities of SAR-DRM and CLD measurements, providing a robust multisensory platform for real-time, quantitative analysis in complex, high-turbidity systems.

Direct analysis of raw matrices by Direct Analysis in Real Time mass spectrometry (DART-MS) remains limited by logistical and operational challenges, including complex handling and positioning, risks of sample degradation, and contamination of the analytical instrument. The Permeably Enclosed Raw Matrices (PERM) approach, introduced here, provides a standardized and instrument-safe strategy in which samples are confined within a semipermeable enclosure, enabling practical and reproducible analysis while preventing particulate release and moderating thermal exposure. The method was evaluated using six chemically and physically diverse matrices of forensic, toxicological, and food relevance: seized tablets, seized powders, roasted coffee, human hair, plant material, and spiked whole human blood. Across all samples, PERM-DART-HRMS delivered S/N ratios spanning four orders of magnitude (4-44 k). The approach enabled the identification of both major and low-abundance constituents - including MDMA, MDA, diazepam, lorazepam, 3-MMC, 25I-NBOMe, N-MEC, caffeine, endogenous lipids such as cholesterol, and sesquiterpenes such as dehydrocostus lactone - without extraction, solubilization, or chromatographic separation. Comparative analysis against conventional DART-HRMS workflows demonstrated effective prevention of instrumental contamination and markedly improved operational reliability and efficiency. Altogether, PERM-DART-MS provides a unified, preparation-free platform for rapid chemical screening, offering a practical and reliable alternative for multidisciplinary applications in forensic, toxicological, and food science contexts.