Gold nanobipyramid-based colorimetric sensor for rapid on-site detection of microcystin-LR in drinking water.

Photo-thermo-refractive (PTR) glass is a key material for optical devices, yet the synergistic mechanism between its raw material precursors remains unclear. This study systematically investigates the individual and combined effects of silver precursors (Ag2O and AgNO3) and sodium salts (Na2CO3 and NaNO3) on the structural evolution and crystallization behavior of Si-Na-Al-Zn-based PTR glasses. Through a combination of spectroscopic (UV-Vis, FTIR, Raman), thermal (DSC), and microscopic (SEM) characterizations, we demonstrate that the precursor combination profoundly influences the glass network homogeneity, ion mobility, and phase separation behavior. The results reveal that the AgNO3 and NaNO3 combination fosters a highly homogeneous and thermally stable network, facilitating the formation of a uniform distribution of silver nanoparticles and, subsequently, a dense nanoscale precipitation of NaF crystals. This work elucidates the critical synergistic mechanism between precursors, providing a fundamental basis for the precise composition design of high-performance PTR glasses for advanced optical applications.

Progress in sperm cell separation and analytical techniques for semen-epithelial mixed stains: a forensic perspective.

A simple colorimetric immunosensor using a paper-based analytical device (PAD) and a sandwich immunoassay was developed for the detection of high-sensitivity C-reactive protein (hs-CRP), a crucial risk predictor for cardiovascular diseases (CVDs). The capture antibody was immobilized onto magnetic beads to facilitate immunomagnetic separation (IMS) of CRP from the sample matrix, while the detection antibody was labelled with biotin. This biotinylated antibody subsequently linked to streptavidin conjugated with β-galactosidase (βgal), allowing a colorimetric assay due to the enzymatic reaction between βgal and chorophenol red-β-D-galactopyranoside (CRPG). The use of the highly sensitive CPRG substrate significantly enhanced the performance of the colorimetric hs-CRP detection with an LOD of 0.10 mg L-1 and a detection range of 0.30-3.0 mg L-1, which is clinically sufficient for the assessment of CVD risk. Furthermore, both intraday and interday measurements confirmed satisfied precisions with % RSD of less than 5%. Finally, the developed method was applied to determine CRP in a certified human serum reference material. The obtained result was found to be insignificantly different from the true value, confirming the reliability of the sensor. This simple, portable and instrument-free method, therefore, holds great promise for development as a screening test kit for CVD risk assessment in resource-limited settings.

Isomeric post-translational modifications (PTMs) on proteins challenge proteomic analyses due to their identical mass and fragmentation patterns. We evaluate high-resolution ion mobility spectrometry (IMS) for separating three naturally occurring acyl-lysine isomer pairs on histones: crotonyl/methacryl, butyryl/isobutyryl, and l-/d-lactyl. These PTMs were chemically installed on lysine residues 9 and 18 (K9 and K18) of synthetic histone H3 peptides (residues 3-15 and 3-25). Using trapped IMS (TIMS), we observe half-height separation of H3[3-15] peptides possessing crotonyl/methacryl and l/d-lactyl marks, and the lactyl isomers of H3[3-25] can be distinguished. In contrast, multipass cyclic IMS (cIM) achieves baseline or near-baseline resolution for every pair, except the longest butyryl/isobutyryl peptide isomers, despite their collision cross-section differences of ≈1%. We show that the resolution increased with the square root of the cIM pass number, allowing baseline separation within 300 ms. Beyond separation, structure-mobility relationships emerge: branched modifications (isobutyryl, methacryl) yield more compact gas-phase conformations than their linear analogs (butyryl, crotonyl). For the doubly crotonylated/methacrylated peptides studied, both PTM identity and site determine the mobility. These results demonstrate IMS as a sensitive method for the elucidation of the acyl-modified histone peptide fine structure by resolving isomeric PTM ambiguity. This addresses a persistent analytical bottleneck and should be included in routine proteomics MS-based workflows.

We present an advanced analytical strategy that exploits the unique capabilities of tandem-trapped ion mobility spectrometry (Tandem-TIMS) to combine high-resolution ion mobility separation with targeted collisional activation and controlled gas-phase trapping. This approach enables the insitu generation of subcomplexes from native-like protein complexes of a selected charge state and allows direct evaluation of their kinetic stability and structural integrity in the gas phase. Using this workflow, we investigated the gas-phase stability of insitu generated streptavidin subunits. Streptavidin tetramers 15+ were mobility-selected and subjected to CID at 160 V between TIMS-1 and TIMS-2, producing monomers, dimers, and trimers. The resulting subcomplexes were then stored in TIMS-2 for up to 10.3 s, during which their collision cross-section distributions remained unchanged, indicating high kinetic stability in the gas phase. Overall, this study highlights the versatility of Tandem-TIMS as an analytical platform for advanced, mobility-resolved measurements that provide new structural and kinetic insights into biological systems.

Ossiculoplasty for Trauma.

Heterogeneous expression of a single surface protein within one cell population can drive major functional differences, yet low-expression subtypes remain difficult to isolate. Conventional tube-based immunomagnetic separation collapses all labelled cells into one positive fraction and thus cannot resolve small differences in marker abundance. Here, we present MagSculptor, a microfluidic platform for high-resolution magnetic fractionation of low-expression EpCAM-defined subtypes within one immunomagnetically labelled population at a time. Arrays of soft-magnetic strips create localized high-gradient zones that map modest differences in bead loading onto distinct capture positions, yielding High (H), Medium (M), Low (L), and Negative (N) fractions. Finite element method simulations of coupled magnetic and hydrodynamic fields quantify the field gradients and define an operating window. Experimentally, epithelial cancer cell lines processed sequentially under identical settings show reproducible subtype partitioning. In a low-EpCAM model (MDA-MB-231), conventional flow cytometry, under standard EpCAM staining conditions, did not yield a robust EpCAM-positive gate, whereas MagSculptor still revealed graded subpopulations. Western blotting confirms a monotonic decrease in EpCAM abundance from H to N, and doxorubicin assays show distinct in vitro drug sensitivities, while viability remains above 95%. MagSculptor thus helps extend immunomagnetic separation from binary enrichment to multi-level isolation of low-expression subtypes and provides a convenient front-end for downstream functional and molecular analyses.

Accurate enumeration of CD4+ and CD8+ T lymphocytes is essential for HIV management, yet conventional flow cytometry remains largely inaccessible in resource-limited settings. Current point-of-care testing (POCT) approaches, including lateral flow assays and fluorescence-based imaging methods, offer improved accessibility but typically compromise accuracy and yield semi-quantitative results. Here, we present a magnetic-activated smartphone microflow cytometry (MACC) platform that enables rapid, highly accessible, and fully quantitative T lymphocyte counting at the POCT. MACC integrates microfluidic immunomagnetic cell separation with smartphone-based bright-field imaging, providing high-sensitivity, highly accessible analysis without requiring sophisticated laboratory equipment or fluorescent labels. A degassing-driven microfluidic pumping mechanism ensures stable microflow generation for reliable continuous analysis, while smartphone imaging enables clear differentiation of targeted lymphocytes from non-lymphocytes. The complete assay, including magnetic bead labeling, chip operation, hands-on procedures, and automated cell-counting analysis, is completed within 24 min. Validation with HIV-infected patient samples demonstrated strong concordance between MACC and conventional flow cytometry for CD4+ and CD8+ counts as well as CD4/CD8 ratio measurements, with minimal bias. By combining high accessibility, cost-effectiveness, and ease of operation, MACC represents a promising alternative to traditional methods, facilitating decentralized HIV monitoring and expanding diagnostic accessibility in resource-limited settings.

Characterization of Asian lacquers by atmospheric solids analysis probe high resolution tandem mass spectrometry coupled with cyclic ion mobility separation

Rapid detection of intact pathogens on a microfluidic device by combining immunomagnetic separation and viability dyes

High-resolution mobility-based ion separations in Structures for Lossless Ion Manipulations (SLIM) have been useful for ion mobility separations for a variety of molecular classes in the gas phase. Here, we present multipass SLIM separations for gas-phase proteins in their near-native state exhibiting charge-state-dependent arrival time distributions using carbonic anhydrase (29 kDa), alcohol dehydrogenase (148 kDa), and apo-transferrin (79 kDa). The experimental CCS values were obtained from calibration curves for the arrival times of Agilent Tune Mix ions. For multipass separations, the ATDs were converted to CCS values by deconvoluting the multipass arrival times into accurate single-pass values amenable to the single-pass calibration curves. Mass spectra of carbonic anhydrase (CA) showed three different charge states (z = 9+ to 11+). Their corresponding mobility peaks were baseline-separated by using 8-m single-pass separations. When compared to the corresponding drift tube ion mobility (DTIMS) measurements, the CCS values obtained from DTIMS and SLIM were in agreement within experimental error. Single-pass analysis of alcohol dehydrogenase (ADH) exhibits three predominant charge states (z = 23+ to 25+) with mobility overlap between adjacent charge states. The mobility peak resolution for ADH improved with multipass separations (up to 24-m path length). In addition, CCS distributions obtained for charge states z = 16+ to 18+ of apo-transferrin reveal a transition from a compact unimodal form (z = 18+ and 19+) to broader multimodal CCS distributions for z = 16+. For apo-transferrin, 40-m multipass separations were performed allowing for complete isolation of the selected mobility range corresponding to z = 17+, leading to selective isolation of a narrow arrival time window. The extended mobility separations provided minimal alterations to the structure of the proteins, and the experimentally derived CCS values showed minimal change as a function of the separation time or number of passes. Mobility-based ion separations for native-like proteins, using SLIM, open opportunities for native-IMS applications as well as other manipulations enabled by SLIM-like mobility-selective isolation and collection.

Immunomagnetic separation is an invaluable tool in biomedical and food safety applications due to their high specificity and efficiency in capturing target cells. This study is a comprehensive investigation of the Ag-Ab reaction and the effects of several factors such as IgG concentration, incubation temperature, and incubation time on the binding efficiency of target cells to the immunomagnetic beads aiming to maximize captured E. coli O157 cells from ground beef samples. Our findings reveal a concentration-dependent relationship between IgG dilution and binding efficiency, with lower dilutions (1:100) yielding higher binding percentages. Additionally, the optimal incubation temperature for bead-cell interactions was identified as 32 °C, with deviations above or below this temperature impairing binding efficiency. Longer incubation times (up to 120 min) significantly improved antibody coating on the beads, enhancing their ability to capture target cells. The study also demonstrated a strong correlation between bacterial load and binding efficiency, highlighting the sensitivity of the immunomagnetic separation method in detecting E. coli O157 even at low concentrations. These findings underscore the importance of precisely controlling experimental parameters to maximize the efficiency and specificity of immunomagnetic separation techniques, providing a valuable framework for researchers aiming to refine bead-based cell capture protocols for diagnostic or research applications.

Complex carbohydrates, such as N-linked glycans, are highly important biomolecules with roles ranging from signaling to recognition and immune response. Ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as a rapid and orthogonal analytical technique to condensed-phase separations for studying carbohydrates, but many challenges exist in their analyses with IMS-MS due to their isomeric and conformational heterogeneity. Specifically, glycan IMS-MS separations often display more peaks than what can be predicted based on structure and/or much broader than expected peaks presumably from their metal-adducted conformers. This has precluded IMS-MS from being routinely used to analyze complex glycans largely because of the reduction in overall peak capacity and thus difficulty in deconvolving mixtures. In this work, we present a traveling wave-based ion heating strategy that uses activating traveling wave conditions. We demonstrated that this ion heating approach can improve peak capacity for individual glycan species as well as for those in mixtures. Importantly, we did not observe any significant loss in sensitivity and comparable resolution to glycans analyzed at gentle traveling wave conditions. Additionally, we demonstrated that isomeric glycans could be repeatedly cycled resulting in scalable resolution without significant ion losses. Overall, our approach can be broadly implemented on any traveling wave-based IMS-MS platform, and we envision utility toward other molecular classes desiring improved IMS-MS peak capacities.

Development and evaluation of CD45-conjugated magnetic particles-based host cell depletion for enhanced metagenomic next-generation sequencing in bloodstream infection.

Integrated immunomagnetic separation and catalase-inhibited colorimetry for rapid visual detection of Staphylococcus aureus

Advancements in molecular biology have facilitated the ability to detect microbes of interest in low abundance within complex samples. Although these technologies are extremely powerful, they typically accommodate only very small volumes of liquid samples as inputs; making sample volume a critical constraint for many molecular methodologies. Because testing volumes are often restricted to the microliter range, methods that concentrate target microbes can broaden the applicability of these detection devices. Immunomagnetic separation (IMS) is an example of a sample preparation method capable of selectively concentrating targets; utilizing magnetically-sensitive materials coated with biorecognition elements to isolate targets of interest. While the commercial availability of micron-sized, conjugation-ready, superparamagnetic particles has amplified the success of IMS for interrogating samples <10 mL, querying of large sample volumes with these particles is often financially restrictive if performed routinely. Therefore, a cost-effective alternative that can be employed for large-volume samples is presented. Here, a low-cost coating allows the conjugation of antibodies to the surface of inexpensive permanent magnets; ultimately creating an economical solid support for the selective capture of microorganisms in both buffer and ground beef homogenate (a complex food matrix). The broad utility of this method was further demonstrated by capturing either E. coli O157:H7 or Salmonella enterica through a simple antibody substitution. Novel techniques aimed at releasing target cells from the magnet via UV-light were also investigated, although the results were not definitive. Overall, expansion of IMS to large-volume food samples using this simple and economical solution could transform downstream detection capabilities in diagnostic applications.

Multiomics approaches enable a comprehensive characterization of complex biological systems by simultaneously investigating multiple molecular layers. Generating multiple omics datasets from a single sample is crucial to minimize biological variability and ensure cross-layer consistency, which is critical for robust downstream data analysis. However, existing workflows often require adaptation to the specific experimental context and instrumental setup. This study systematically compared two established protocols for the simultaneous extraction of metabolites, lipids, and proteins from HepG2 cells: (i) a biphasic extraction with subsequent overnight protein digestion from the interphase pellet, and (ii) a monophasic extraction involving on-bead protein digestion. For the monophasic approach, we further investigated the effects of bead size and digestion conditions. Metabolomics samples were analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry; lipidomics and proteomics samples were analyzed by nano-scale liquid chromatography coupled with ion mobility separation and high-resolution tandem mass spectrometry. Each method was evaluated in terms of total feature count, selectivity, reproducibility, handling complexity, and overall performance. While neither protocol was optimal across all criteria, the monophasic extraction using paramagnetic beads with shortened incubation time proved to be the most reproducible, efficient, and cost-effective solution for in-house multiomics workflows in HepG2 cells.

IntroductionPolymorphonuclear-myeloid-derived suppressor cells (PMN-MDSC) are elevated in COVID-19 patients, playing a crucial role in suppressing the SARS-CoV-2 specific T-cell response and serving as an early marker for disease progression. In this study, we investigated the involvement of PMN-MDSC from COVID-19 patients in the formation of extracellular traps (ET).MethodsFifty RT-PCR–confirmed severe COVID-19 patients admitted to the ICU and ten healthy donors were enrolled. PBMC were isolated from peripheral blood by density gradient centrifugation, and PMN-MDSC frequency was evaluated by flow cytometry. PMN-MDSC were isolated by immunomagnetic separation. ET extrusion was analyzed by immunofluorescence imaging. Apoptosis of pulmonary microvascular endothelial cells cultured with PMN-MDSC was measured by flow cytometry.ResultsWe found that platelet-rich plasma (PRP) from COVID-19 patients, unlike that from healthy donors, induced ET formation by PMN-MDSC. Furthermore, the PRP-induced ET was found to be independent of Toll-like receptor 4 (TLR4) signaling. Interestingly, the SARS-CoV-2 Spike protein itself can trigger ET formation via a TLR4-dependent pathway. Additionally, PMN-MDSC induced endothelial cell apoptosis through an ET-independent mechanism.DiscussionThese findings highlight a previously unrecognized contribution of PMN-MDSCs to the thrombotic complications in severe COVID-19 cases, underscoring their detrimental impact on disease progression.

The first results of using a new type of ultrahigh-resolution mass analyzer based on a planar multipass time-of-flight mass spectrometer with periodic reflecting lenses (Y-MRT MS) for bottom-up whole-proteome analysis are presented. The instrument achieves a resolving power in a range of 600,000-800,000 for peptide ions across the whole m/z range, with a high repetition rate of 300 Hz (averaged to 0.5-4 Hz for enhanced dynamic range). In preliminary experiments for human cell lines, MCF-7 and HeLa, single-shot 30 min gradient HPLC separations of 1 μg proteolytic digests yielded, on average, over 4000 protein groups in MS/MS-free proteome analyses using the DirectMS1 method. Combining three technical runs increased these numbers to 4500 protein groups at 1% FDR. Peptide ion mass measurements demonstrated an accuracy of 70-130 ppb across the whole m/z range, with a dynamic range exceeding 104. In DIA mode (SWATH-DIA, 20 Th window, 30 min gradient), 4350 protein IDs were obtained at 1% FDR on average in single-shot LC-MS/MS runs. These results highlight the Y-MRT mass analyzer's potential for bottom-up proteomics. Further improvements in proteome coverage and analysis time are anticipated with optimized HPLC configurations and the integration of gas-phase ion mobility separation.