The Analyst

The estimation of the biological sex of archeological remains is crucial information in bioarchaeology and forensic anthropology. In recent years, proteomics based on molecular sexual dimorphism have emerged as a preferred method, particularly because of its minimally-invasive approach to extracting amelogenin X and Y proteins from tooth enamel. However, there is an increasing demand to accelerate this process while facilitating the analysis of large archaeological assemblages. This study presents a novel high-throughput targeted paleoproteomics method for biological sex estimation using MALDI-CASI-FTICR mass spectrometry. This approach combines the strengths of existing methods, including ultra-high resolution, significantly reduced processing times, targeted analysis, and scalability to large archaeological sample sets. The method was initially validated on modern individuals with known sex and subsequently applied to 130 adult and juvenile individuals from medieval Great Moravia (present-day Czech Republic). Biological sex was successfully estimated for all but one of the individuals. The results not only provide a more efficient biological sex estimation but also help to resolve a few errors in sex assessment previously encountered with osteomorphological and tooth morphometric techniques. The implementation of this method significantly improves the accuracy and efficiency of biological sex estimation, offering a powerful tool for anthropological research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/706309v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1ede7e6org.highwire.dtl.DTLVardef@13d2f5org.highwire.dtl.DTLVardef@17ee44dorg.highwire.dtl.DTLVardef@1be9dd9_HPS_FORMAT_FIGEXP M_FIG C_FIG

1.BackgroundHuman infertility affects approximately 17.5% of the global population, with male factors accounting for nearly half of all cases. The identification of reliable molecular biomarkers is crucial for improving the diagnosis and assessment of male fertility. In this study, we developed and optimized an untargeted high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) workflow for comprehensive lipidomic and metabolomic profiling of human spermatozoa using only 1.25 million cells per sample. ResultsCompared to previous reports, our optimized method achieved unprecedented analytical depth, identifying 473 lipid species and 955 structurally annotated metabolites, corresponding to nearly 7.600-fold improvements in detection efficiency per cell over published approaches. Lipidomic analysis revealed cholesterol, fatty acids, phosphatidylcholines, and phosphatidylethanolamine plasmalogens as the most abundant lipid classes, consistent with the structural complexity of the sperm plasma membrane. Metabolomic profiling showed strong enrichment of lipid-related and steroidogenic pathways, including phospholipid biosynthesis, glycerolipid metabolism and androgen and estrogen metabolism. The integration of lipidomic and metabolomic data highlighted functionally interconnected pathways related to membrane dynamics, energy metabolism, and hormone biosynthesis. ConclusionsOverall, this work establishes a robust, sensitive, and scalable analytical framework enabling high-coverage molecular characterization of spermatozoa from limited sample material, laying the groundwork for future biomarker discovery and clinical applications in male infertility research. One Sentence SummaryDevelopment of a highly sensitive untargeted HPLC-ESI-MS/MS lipidomic and metabolomic workflow that achieves unprecedented molecular coverage from only 1.25 million human spermatozoa, revealing interconnected lipid and metabolic pathways and providing a robust foundation for biomarker discovery in male infertility. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/703749v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@10b3132org.highwire.dtl.DTLVardef@1caf850org.highwire.dtl.DTLVardef@746adborg.highwire.dtl.DTLVardef@1135539_HPS_FORMAT_FIGEXP M_FIG C_FIG

Carbapenemases are major drivers of carbapenem resistance in Gram-negative bacteria and pose a critical threat to last-line antibiotic therapy. Rapid identification of carbapenemase classes is essential for appropriate treatment and epidemiological surveillance; however, current functional methods lack class-level resolution and may yield false-negative results for OXA-48-like enzymes. In this study, we developed and validated an assay based on liquid chromatography-mass spectrometry with trapped ion mobility spectrometry-time-of-flight [LC-MS (timsTOF)] for simultaneous detection and class-level differentiation of five clinically relevant carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like). The method employs three carbapenem substrates (meropenem, imipenem, and ertapenem). A total of 55 clinical isolates were analyzed using a standardized 2-hour incubation protocol, with a total analysis time of 7 min per sample. Ion mobility enabled unambiguous identification of the OXA-48-specific meropenem-derived {beta}-lactone based on its distinct collisional cross-section (185 [A]{superscript 2} vs. 191 [A]{superscript 2} for intact meropenem), despite identical mass and nearly identical retention time. This marker was detected in all OXA-48-like producers and was absent in all other groups. In contrast, imipenem and ertapenem did not provide comparable discrimination, highlighting the central role of meropenem. Distinct hydrolysis profiles enabled class-level differentiation supported by multivariate analysis. LC-MS (timsTOF) thus enables rapid, sensitive, and specific functional detection of carbapenemases within a single workflow. The ion mobility dimension is critical for accurate identification of OXA-48-like enzymes and supports the potential implementation of this approach in routine clinical microbiology laboratories. ImportanceThis study introduces an ion mobility-enabled LC-MS (timsTOF) approach for functional detection and class-level differentiation of clinically relevant carbapenemases within a single analytical workflow. By leveraging collisional cross-section measurements, the method enables reliable identification of OXA-48-like carbapenemase through detection of a meropenem-derived {beta}-lactone that is indistinguishable by mass alone. This directly addresses a major diagnostic limitation of conventional activity-based assays, which may yield false-negative results for OXA-48-like enzymes. The approach further demonstrates the potential of integrating ion mobility into routine clinical mass spectrometry to enhance specificity beyond traditional mass and retention time measurements. These findings support the development of next-generation diagnostic strategies capable of detecting both known and emerging resistance mechanisms without reliance on predefined targets.

BackgroundThe clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding specificity, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. ResultsWe have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R2 = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 minutes, a significant improvement over traditional methods. ConclusionsSE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The methods compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.

ObjectiveA catheter-free, non-radiolabeled method that permits in vivo measurement of tissue-specific glucose uptake does not exist. To address this gap, we sought to develop and validate a new, higher throughput mass spectrometry (MS)-based method that combines an injection of insulin with a non-radiolabeled glucose tracer, 2-fluoro-2-deoxyglucose (2FDG), to determine insulin-stimulated tissue-specific glucose clearance in conscious, unrestrained mice. MethodsInjections of saline or insulin with 2FDG were coupled with LC-Q Exactive Hybrid Quadrupole-Orbitrap (LC) MS-based measures of plasma 2FDG and tissue (2-fluoro-2-deoxyglucose-6-phosphate) 2FDGP to determine glucose clearance in mice under several different conditions. ResultsThe newly developed method was first applied to a dose response experiment in mice. Next, the ability of this method to quantify changes in glucose clearance in response to an insulin stimulus was assessed, and glucose clearance was compared between chow and high fat fed mice. Results from these studies showed that insulin-stimulated skeletal muscle and heart glucose clearance can be estimated following a bolus injection of tracer, and these fluxes are blunted in diet-induced obese mice. The broad applicability of this approach was then demonstrated by assessing glucose clearance in a mouse model with anticipated changes in insulin-stimulated skeletal muscle glucose metabolism. ConclusionsThe results validated a new LC-MS method to quantify insulin-stimulated tissue-specific glucose clearance in vivo without the use of catheters or radiolabeled tracers. The method offers great potential because it is designed for application to pre-clinical studies seeking high throughput tests and/or assays that can be coupled with discovery technologies such as genomics, proteomics and metabolomics. HIGHLIGHTSO_LIIn vivo glucose clearance can be estimated by a new non-radiolabeled method. C_LIO_LIThe plasma tracer to tracee ratio is required to determine tissue tracer phosphorylation. C_LIO_LIMeasures of plasma glucose and tracer kinetics are critical for data interpretation. C_LIO_LIThe new method can be combined with omics technologies such as metabolomics. C_LI

Colorimetric loop-mediated isothermal amplification (LAMP) on microfluidic paper-based analytical devices (PADs) offers a low-cost, disposable, and equipment-free alternative to liquid LAMP assays. However, amplification on PADs is consistently slower, by 5-46%, than reactions in tubes. To identify the origin of this delay, we evaluated heat transfer, diffusion in porous cellulose, and nonspecific adsorption of LAMP components across both high- and low-copy input regimes. Our results show that once thermal equilibrium is reached, reduced effective diffusion is the dominant contributor to the kinetic lag at low copy numbers, whereas nonspecific adsorption becomes the primary barrier at higher template concentrations. Pre-coating the paper with bovine serum albumin (BSA) mitigates adsorption. It narrows the tube-to-paper gap, thereby accelerating amplification of the SARS-CoV-2 ORF7ab synthetic gene by an average of 6 minutes, from 1E3 to 1E5 copies per reaction. These findings provide a mechanistic basis for the copy-number-dependent behavior of PAD LAMP and offer simple, low-cost strategies to improve the speed and reliability of PAD nucleic acid assays.

Digital immunoassays provide exceptional analytical sensitivity for detecting low-abundance biomarkers, but their broad adoption is limited by practical barriers. Commercial platforms are prohibitively expensive for routine use by individual laboratories, and laboratory-scale concepts typically describe specialized biosensors and sophisticated workflows. Here, we introduce a nanomembrane-based Catch-and-Display Immunoassay (CAD-IA) as an accessible digital immunoassay for common laboratory settings. In CAD-IA, fluorescent nanoparticles are "captured" by the nanoscale pores of ultrathin silicon nitride membranes through a pipette powered filtration. The captured nanoparticles serve as optically isolated hotspots for fluorescent immunocomplex formation when target antigen is present. Co-localization of the fluorescent particles and fluorescent immunocomplexes are then "displayed" and quantified by standard confocal microscopy to generate digital signals. CAD-IA is implemented using the {micro}SiM-DX (microfluidic device featuring an ultrathin silicon membrane for diagnostics) platform, which is manually assembled from mass produced, cost-effective components. Using the traumatic brain injury (TBI) biomarker S100B as a model, we demonstrate that CAD-IA provides consistent digital outputs and linear quantification with a dynamic range of at least two orders of magnitude when digital and analog analysis are combined on the same image sets. We further demonstrate that the assay maintains linearity in serum matrices and achieves suitable sensitivity (LoD = 0.02 g/mL) for clinically relevant diagnostic with the addition of tyramide signal amplification (TSA). While further optimization of CAD-IA is possible, these results constitute a proof-of-concept demonstration of a novel digital immunoassay that is accessible to most laboratory environments.

Maintaining precise isothermal conditions in portable nucleic acid amplification tests (NAATs) is critical for reproducible results but remains challenging with conventional single-sided thin-film heaters, which exhibit temperature gradients and strong dependence on ambient conditions. To close this gap, we engineered ThermiQuant VitroMini, a dual-sided heater design that achieves volumetric-level temperature uniformity using thin-film heaters while preserving optical transparency for real-time colorimetric loop-mediated isothermal amplification (LAMP) analysis on microfluidic paper-based analytical devices ({micro}PADs). The device integrates two independently regulated indium tin oxide (ITO) heaters (8 {Omega} each) controlled by independent proportional-integral-derivative (PID) algorithms. Heaters were evaluated under controlled ambient environments of 4 {degrees}C (refrigerated), 23 {degrees}C (room temperature), and 50 {degrees}C (oven). Analytical tests were performed using a colorimetric LAMP assay targeting the SARS-CoV-2 orf7ab gene on {micro}PADs preloaded with dried LAMP reagents, with time-lapse images (30 seconds interval) analyzed via Amplimetrics software. VitroMini maintained 65 {+/-} 0.5 {degrees}C across 4 to 50 {degrees}C ambient conditions and achieved a limit of detection of 50 copies/reaction (6.7 copies/{micro}L), with quantification times (Tq) linearly correlated with log10 DNA concentration. Dual-sided heating eliminated temperature bias, condensation artifacts, and ambient-dependent variability while preserving optical transparency for real-time LAMP quantification. ThermiQuant VitroMini bridges the gap between benchtop volumetric heaters and portable diagnostic devices, offering a compact, low-power, and field-deployable platform for decentralized molecular diagnostics and One Health applications.

Cell viability is a critical parameter in bioproduction, yet most facilities still rely on manual, offline assays. This work introduces a new label-free Digital Holographic Microscopy (DHM)-based viability prediction pipeline using a simple optical design compatible with both on-line and in-line probe implementation. Unlike previous approaches validated under controled laboratory conditions, the proposed pipeline was designed to operate across diverse CHO bioprocess conditions without calibration or parameter tuning. It was validated on a large, heterogeneous dataset comprising 40 cell cultures collected from industrial and academic sites, spanning multiple cell lines, culture media, process modes and cell densities up to 100 million cells/mL. Beyond viability estimation, exploratory analyses suggest that DHM-based monitoring can provide additional process-relevant insights, including early detection of viability decline and correlation with recombinant protein titer. Together, these results indicate that DHM has the potential to enable a new generation of non-invasive, multiparametric monitoring tools for advanced bioproduction control.

Gel electrophoresis has been a cornerstone laboratory technique for decades, yet it is often viewed as cumbersome, costly, and has remained confined to laboratory settings. Recent advances in DNA nanotechnology have repurposed electrophoresis as a primary readout for some biosensing applications such as DNA nanoswitches, where a conformational change in a DNA structure indicates the presence of a target molecule. Conventional gel electrophoresis setups not ideal for such targeted applications, with moderate equipment cost, excessive reagent use, and time-consuming processes. Here, we adopt a reductionist, application-driven approach to redesign gel electrophoresis specifically for DNA nanoswitch-based detection. We present a fully 3D-printable mini gel electrophoresis system that incorporates conductive plastic electrodes, demonstrating performance comparable to conventional systems using platinum electrodes. By optimizing the inter-electrode distance and running parameters, our system resolves the on/off states of DNA nanoswitches in as little as one minute. We further show that the device operates reliably at low voltages, including when powered by a USB power bank, and even enables instrument-free nanoswitch readout using an LED with a cell-phone camera. Our design substantially reduces the cost, voltage requirements, material usage, operational complexity, and experiment time. These improvements make gel-based biosensing more practical outside traditional laboratory environments, paving the way for broader adoption of gel electrophoresis in point-of-care and resource-limited settings.

Untargeted proteomics enables quantitative determination of host cell proteins (HCPs) in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. We report a prospective validation of label-free untargeted proteomics for HCP quantification using a total-error (TE) approach. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20-80 ng). Four independent assays (198 injections) supported hierarchical replication and one-way random-effects ANOVA variance decomposition with Welch-Satterthwaite adjustment. Dual entrapment analysis demonstrated empirical peptide-level false discovery proportions below 1% at q = 0.01. Deterministic parsimony inference ensured invariant protein-group definition. Weighted least-squares regression (R{superscript 2} = 0.993) identified stable proportional compression with recoveries of 81-85%. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision total SD ranged from 0.69% to 3.81% over the validated range. Accuracy profiles integrating empirical bias with a log- log variance model showed 95% {beta}-expectation and 95/95 content tolerance intervals fully contained within {+/-}30%, with a lower limit of quantification (LLOQ) of 20 ng. Abundance-stratified TE analysis revealed concentration-dependent calibration heterogeneity masked by aggregate-level estimation; stratum-specific {beta}-expectation intervals within {+/-}35% defined an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe, CCC = 0.998, LoA {+/-}9%) and cross-platform acquisition (Astral, CCC = 0.980, LoA {+/-}18%) remained within predefined {+/-}30% agreement limits. System suitability criteria were derived empirically from validation performance. This is the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a statistical framework applicable to other high-dimensional analytical methods requiring regulatory qualification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/710150v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f5331aorg.highwire.dtl.DTLVardef@ee2234org.highwire.dtl.DTLVardef@798eaorg.highwire.dtl.DTLVardef@c84034_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

Ambient metabolomics techniques such as laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS) enable fast, preparation-free fingerprinting of biological samples but are inherently limited by spectral congestion in the absence of chromatographic separation. While ion mobility spectrometry provides additional gas-phase separation, maintaining ion transmission under the transient signals characteristic of laser desorption, remains analytically challenging. Here, we define operating conditions for cyclic traveling-wave ion mobility spectrometry (cIMS) that preserve transmission under LA-REIMS duty-cycle constraints and systematically evaluate how cIMS integration reshapes biofluid fingerprints and enhances chemical specificity in chromatography-free metabolomics analysis. Under optimized single-pass conditions, cIMS separation reorganized LA-REIMS spectra into structured mass/mobility feature domains, enabling selective mobility-based filtering of matrix-derived salt cluster ions. This reduced non-biological background contributions by up to 35% of total spectral intensity while preserving over 90% of detected untargeted features. Although cIMS operation introduced a sensitivity penalty relative to time-of-flight-only acquisition, approximately 80% of the total ion current was recovered under optimized conditions. Mobility-resolved data revealed coherent homologous series and class-specific structural trends, particularly for lipids, supporting class-level annotation. Analysis of 101 metabolite and lipid standards covering a broad physicochemical range (logP -5.30 to 19.40) demonstrated comprehensive molecular coverage, high mass accuracy (mean 2.4 ppm), and good agreement with reference CCS values (mean deviation 4.0%), with isomer separation observed for biologically important secondary bile acids in extended separation cycles. Collectively, these results establish LA-REIMS-cIMS as a practical analytical strategy for enhancing chemical specificity and spectral interpretability in support of high-throughput large-scale metabolic fingerprinting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/709786v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@18a2dfdorg.highwire.dtl.DTLVardef@d165d6org.highwire.dtl.DTLVardef@1750291org.highwire.dtl.DTLVardef@fbbce9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Ion mobility spectrometry adds an orthogonal gas-phase separation to LA-REIMS, reorganizing complex biofluid spectra into distinct mass-mobility feature bands and improving molecular resolution in rapid ambient ionization metabolomics. C_FIG

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

Molecular recognition is a central component that confers detection specificity to all biosensors. The design and use of such molecules require consideration of properties including their affinity and selectivity, plus their ease of production and engineering, for downstream commercial purposes. Progesterone (P4), is a biomarker that is extensively for various diagnostic purposes. Examples include detection of P4 as an indicator of oestrus in cattle breeding, and ovulation in human IVF programs. P4 is also thought to promote strains of breast cancer, resulting in it being an environmental pollutant of interest. The present study focusses on in-silico molecular docking trials of P4 molecules with proteins such as antibodies and receptors. We describe the geometry of novel P4-binding pockets and predict key residues that favour high affinity and selectivity for P4. The in-silico molecular docking trials were performed on various mutants of an anti-P4 antibody that had lost their P4 specificity but retained selective recognition of steroids with structures closely related to cholesterol. Reverse-docking trials permitted the identification of novel scaffolds with favourable P4 binding properties. Future reports will validate the predictions of these studies through wet lab experiments. A further opportunity for this approach is to incorporate a scaffold functionality to permit binding of the protein or receptor to other molecules or sites within a biosensor electrode. These findings, and future studies, will assist in development of enhanced biosensing platforms with custom-designed P4 binders, aiding commercialisation using in-house developed reagents to meet IP requirements and minimise scaling costs. The steroid biotechnology market, valued at over $10 billion, also benefits from novel steroid binder designs, facilitating real-time steroid biomonitoring platforms for optimising steroid bioprocesses.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

The protein sample preparation methods for shotgun proteomics are nowadays well-established unlike the ones for whole protein analysis. The goal of my work has been to create a simple methodology which provides a single uncomplicated sample preparation tool for these two fields. Nowadays the bulk of proteomics work is done using detergents for protein solubilization. The presented concept, which is based on unspecific adsorption of protein molecules on wide pore materials, allows for protein capture and clean-up from solutions of the most commonly used sodium dodecyl sulfate detergent. It could also be applied to proteins in detergent-free solutions. After the capture and clean-up, proteins could be either cleaved for the downstream peptide analysis or eluted for the whole protein analysis. If required, the eluted whole proteins could be recaptured and cleaved into peptides. Depending on the experimental goals, the sample preparation device could be fitted with embedded proteolytic enzymes to simplify routine sample processing and/or reversed phase media for the downstream peptide or protein separation.

Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram-scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid-resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics-style sample preparation. Using human HeLa cells cultured in 13C-glucose-enriched and control media, we show that reliable relative {delta}13C measurements can be obtained from as few as 50 cells, corresponding to <10 ng of total protein, with a precision of approximately {+/-}9{per thousand}. The observed amino acid-specific labeling patterns are metabolically coherent and consistent with bulk measurements, while smaller cell numbers ([&le;]10 cells) do not yield statistically robust results. These findings establish the practical sensitivity threshold of FT IsoR MS at the low-nanogram level and demonstrate its suitability for isotope-resolved analyses of small cell populations, micro-organoids, and other low-input biological samples, thereby extending stable isotope analysis toward single-cell-scale applications.

Proximity ligation assay (PLA), in which the ligation of two DNA probes is greatly accelerated by the associating target molecules, has emerged as a highly sensitive technique for protein detection. The detection of the ligated DNA typically relies on PCR, which requires temperature cycling. In this study, we report on a novel discontinuous (DISCO)-LAMP assay that enables the wash-free detection of PLA products via loop-mediated isothermal amplification (LAMP). Due to the exponential amplification nature of LAMP, a careful balance between efficient amplification of the ligated full-length DNA and minimal background amplification from the individual constituent probes is essential but often challenging to achieve. After extensive template/primer design and assay optimization, DISCO-LAMP assay achieved a detection limit of 1 fM for the ligated DNA probe while maintaining undetectable background amplification at 1 nM of each individual probe. DISCO-LAMP detected Shiga toxin 2 (Stx2) with a limit of detection (LoD) of 100 fM when functionalized with Stx2-binders, as well as both Wuhan-1 and Omicron spike protein when functionalized with DS16, a newly engineered DARPin targeting a conserved epitope on the SARS-CoV-2 Spike protein. We believe DISCO-LAMP represents a versatile and efficient LAMP-based PLA technology that is readily adaptable for sensing diverse targets.

MicroRNAs (miRNAs) are ultra-short RNA molecules characterized by high sequence homology, frequent post-transcriptional modifications, and typically low abundance, particularly in circulating biofluids. These inherent biological features present substantial technical challenges for RT-qPCR- based quantification. Consequently, the development of miRNA RT-qPCR assays has required architectural adaptations at the reverse transcription (RT) stage to generate extended cDNA templates, thereby enabling effective downstream quantitative PCR amplification. One widely adopted approach involves the enzymatic addition of a poly(A) tail to the 3' end of miRNAs, followed by poly(T)-primed universal reverse transcription, which has gained broad acceptance due to its perceived sensitivity and simplified workflow. However, independent experimental evidence indicates that this architecture does not consistently provide the level of specificity required for reliable single-nucleotide (SN) discrimination, particularly when quantifying low-abundance circulating miRNA targets, as demonstrated in our previous study. An alternative strategy relies on miRNA-specific reverse transcription using stem-loop priming has been equally well accepted. When generically generated, this approach offers certain improved specificity, but its performance in resolving single-nucleotide differences remains limited. In this article, we employed precision engineering to maximize specificity for both reverse transcription and qPCR steps. By tailoring both primer design and reaction architecture to the specific sequence features of each miRNA, we enable robust single nucleotide discrimination among these ultra-short targets. Prototype of ten different miRNova assays quantifying miRNAs whose sequences are differed in various configurations were tested on synthetic miRNA targets. For miRNova assay validation, saliva samples were elite rugby players submitted to small RNA extraction, then RT-qPCR. Spike-in of synthetic targets was applied for each quantification point to characterized the sensitivity, specificity and accuracy of the assays. Comparative analysis was performed between miRNova and two commercially available kits on the same sample set. The obtained results show a superior performance of miRNova assays allowing for sensitive and accurate quantification of miRNAs in saliva samples. Altogether, this results in modular, reproducible assays optimized for low-abundance miRNA detection in challenging biofluids, including saliva, positioning the platform beyond existing sensitivity-focused solutions toward true diagnostic-grade specificity.