SLAS Technology

Extracellular adenosine triphosphate (eATP) is a mediator of purinergic signalling in the airways, implicated in mucociliary function, inflammation, and cough via activation of P2X3 receptors. Elevated airway eATP has been associated with multiple respiratory diseases, yet reliable measurement of eATP remains challenging due to its rapid enzymatic degradation and confounding contributions from intracellular ATP. Here, we describe an optimized, microwell plate-based luminescence method for quantifying eATP from human airway epithelial cell cultures and bronchoalveolar lavage (BAL) fluid with enhanced signal stability. Using a commercially available ATP detection assay with a prolonged luminescence half-life, we introduced a simple 0.45 {micro}m syringe filtration step to remove cells and thereby isolate extracellular ATP. This approach demonstrated ATP specificity via apyrase degradation, and provided a linear detection range from 5 nM to 5 {micro}M. Addition of ATP stabilization buffer preserved ATP levels in cell culture media for at least 4 hours at 4 {degrees}C and in human BAL samples for at least 6 weeks at -80{degrees}C. Applying this method to primary human bronchial epithelial cells revealed detectable eATP release, with preferential secretion at the apical surface under air-liquid interface conditions. Collectively, this optimized assay enables robust, high-throughput, and time-flexible quantification of eATP in both experimental and clinical airway samples. These methods support improved investigation of purinergic signalling in airway health and disease and may facilitate biomarker development relevant to eATP in the airways.

Microfluidic devices with surface-bound biomolecular patterns enable localised detection arrays, enzymatic catalysis, and gene expression. Photolithography is a contactless patterning method with high spatial control. However, while patterning open surfaces by photolithography is well-established, patterning enclosed microfluidic channels remains technically challenging. Such capability would enable in situ surface modification and precise pattern alignment to channel geometries. Here, we present a photolithographic method using commercially available reagents to pattern sealed microfluidic devices. We first coat surfaces with (3-Aminopropyl)triethoxysilane (APTES) to bond microfluidic chips and provide surface amine groups onto which photocleavable polyethylene glycol (PC PEG) compounds are bound. UV exposure using standard photolithography equipment selectively deprotects the amine groups, which can subsequently bind amine-reactive cargos. We demonstrate this methods versatility by patterning both glass and poly(dimethylsiloxane) (PDMS) surfaces with diverse cargoes: DNA, proteins and gold nanoparticles. We also compare covalent versus noncovalent DNA patterning. Covalently bound DNA patterns were denser and could be used for sequence-specific target DNA capture. However, noncovalently bound DNA yielded higher cell-free gene expression from surface-bound GFP templates.

The generation of clonal CHO cell lines is foundational to biologics manufacturing; however, labor-intensive cell culture workflows predominate in the field. We created the CLAIRE (Cell Line AI Recognition and Evaluation) tool to streamline end-to-end cell line development by integrating deep-learning image analysis with automated liquid handling. We benchmarked three object detection models for monoclonality verification and found DETR provides superior accuracy (>0.90 F1-score) in identifying single cells. To quantify the outgrowth of cell lines, we evaluated multiple zero-shot SAM2 segmentation models against a feature-based estimation method. Feature-based detection successfully identified diverse cell colony types while less robust performance was observed for SAM2 models, particularly for sparse density colonies. The pre-trained DETR and feature-based detection models were wrapped in a task-focused user interface that outputs cell line hitpick lists compatible with a Lynx LM1800 liquid handler in addition to custom scripts automating cell passaging and sampling. This approach yielded an end-to-end 36 day CLD workflow capable of generating high-titer cell lines for multiple complex antibody structures. Here, we open-access our trained models, user interface, and Lynx automation scripts to provide a modular toolkit useful for clonal cell line engineering projects. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/703387v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f72e70org.highwire.dtl.DTLVardef@109c54dorg.highwire.dtl.DTLVardef@7867b1org.highwire.dtl.DTLVardef@dfa61e_HPS_FORMAT_FIGEXP M_FIG C_FIG

1.Adipocyte size is an independent predictor of several metabolic disorders, including type 2 diabetes, liver and cardiovascular diseases. However, technical limitations due to the fragile nature of mature adipocytes have restricted the functional analyses of size-separated adipocytes using conventional methods. Therefore, we have developed a microfluidic device, based on deterministic lateral displacement, for sorting intact, mature adipocytes. Cell-size distribution was determined from time-lapse recordings inside the device, in separate outlets, and by Coulter counter analysis of the collected cell fractions. This approach allowed size-separation with minimal size-overlap with mean diameters of (small fraction) 47 {micro}m and (large fraction) 82 {micro}m based on Coulter counter measurements. Viability of the separated cells was verified by insulin stimulation and western blotting of key insulin signaling proteins. The sample recovery, comparing input versus output material, was relatively high, 42% for the large fraction with a purity of 93%. We demonstrate that microfluidics is a suitable approach to overcome the limitations of sorting mature adipocytes according to size. Together, the high recovery rate, high throughput capacity, accurate separation and the fact that the cells maintained hormonal response after sorting provides compelling evidence of the strength and usability of the microfluidic approach for exploring adipocyte function in relation to size.

High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, namely the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 114 and 155 nm) by using light-scatter based detection thresholds (SSC, FSC, VSSC) across a concentration range from 106 to 109 particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding for their qualitative and quantitative analysis. We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.

Accurate quantification of fungi is important for a myriad of applications but remains challenging. Previously, we demonstrated that an approach called the adenine-HPLC method can quantify bacteria, including those with aggregating properties that are difficult to quantify using conventional methods, by measuring cellular adenine derived from DNA and converting the adenine amount to genome copy number, without being influenced by cell morphology. However, in this study, when this adenine-HPLC method was applied to the quantification of budding yeast as a model fungus, accurate measurement proved impossible. This limitation was attributed to adenine release from other adenine-containing biomolecules, such as RNA and ATP, and we therefore developed a method that suppresses adenine release from these molecules. This method involves reducing the temperature of the acid treatment and prewashing the cells before acid treatment. In addition, we incorporated a process that corrects for the naturally occurring free adenine level as background during total adenine measurement. The improved adenine-HPLC method based on these modifications enables accurate quantification of budding yeast using genomic DNA content in whole cells as the quantification unit.

Frequent blood testing is a routine but burdensome reality for many children, particularly those with chronic, rare, or medically complex conditions. Repeated clinic, hospital, and laboratory visits can disrupt family life, increase stress for children and caregivers, and limit access to timely monitoring and research participation. Despite advances in pediatric care, blood collection has remained largely tethered to in-person clinical settings. This study validates a new model: safe, effective, parent-administered pediatric blood collection performed at-home. We evaluated the RedDrop ONE capillary blood collection device in a real-world, parent-administered home setting to determine whether non-clinical caregivers can reliably collect clinically meaningful blood samples from children without venipuncture, specialized training, or in-clinic support. Conducted under Institutional Review Board (IRB) oversight, this observational usability study enrolled 50 children aged 3-17 years across a geographically diverse U.S.-based pediatric population, including healthy and medically fragile children with chronic autoimmune and rare diseases. All study activities, including enrollment, consent, instruction, collection, and sample return, were completed remotely, reflecting real-world adoption conditions rather than controlled clinical environments. Parents successfully collected blood samples from their children at home with high consistency, low perceived pain, and strong overall acceptance. Across collections, blood and serum volumes were sufficient and reproducible, and laboratory analysis confirmed strong analytical concordance between samples collected from two different anatomical sites, arm and leg. Parents reported high confidence using the device, short collection times, and a high likelihood of completing collections on the first attempt. Importantly, both parents and children rated the overall experience as better than expected, and parents consistently reported that the RedDrop ONE experience was superior to traditional finger-prick and needle-based venous blood draws. Parents reported minimal child discomfort and greater flexibility by avoiding in-clinic phlebotomy visits. These benefits are especially meaningful for families managing chronic or rare pediatric conditions that require repeated blood monitoring. By enabling blood collection at-home, this model reduces travel burden, scheduling constraints, and procedural anxiety while maintaining analytical reliability. This study also demonstrated that parent-administered pediatric blood collection can support real-world clinical workflows beyond research. All samples were successfully shipped overnight at ambient temperature and processed by a CLIA-certified laboratory, supporting feasibility for remote pediatric patient monitoring and decentralized clinical trials. While lipid testing served as the representative clinical use case, the volumes and consistency achieved exceeded volume thresholds commonly required for advanced downstream applications, including proteomics, metabolomics, transcriptomics, and genomic analyses. Taken together, these findings validate parent-administered pediatric blood collection as a practical, scalable alternative to in-clinic phlebotomy for many use cases. By shifting blood collection from the clinic to the home, this approach has the potential to reduce reliance on in-person phlebotomy, integrate seamlessly into routine pediatric care, and expand access to monitoring and research for families who face geographic, logistical, or medical barriers. For health systems, researchers, and parents alike, this study supports a future in which clinically meaningful pediatric blood collection is no longer limited by healthcare facility location but instead centered on the child and family.

Collecting cells from zebrafish embryos for genotyping is critical to rapid research with these model organisms. The standard collection process is manual, labor-intensive, time-consuming, and requires a skilled person to perform it. To overcome this challenge, researchers are exploring the development of automated genotyping tools for live animals, which would significantly enhance the efficiency and accuracy of genetic screening in zebrafish and other species. The focus of this research was to optimize the Zebrafish Embryo Genotyper (ZEG), an automated system used for the rapid extraction of cellular material from zebrafish embryos. This system rapidly vibrates a roughened chip containing a zebrafish embryo to collect genetic material safely and efficiently. The aim was to improve the efficiency of DNA collection from the chips used with the ZEG by identifying the key factors that contribute to the process. First, the chips were modified to resolve issues associated with loss of sample volume from the chip wells due to evaporation during processing. Second, we experimented with three critical parameters - sample volume in the wells, the vibrational frequency of the system, and the operation time - on the quantity of DNA collected. The performance was evaluated by measuring embryo survival and quantifying the DNA collected. The sensitivity (previously 90%) of the DNA collection and embryo survival (previously 95%) of the were both found to be greater than 95% after optimization. The optimized design parameters (15 {micro}L solution volume, 2.4 V, and a 5-minute run with 5 s alternating on/off) provided a >50% increase in DNA collection compared to the previous designs and parameters. The proposed chip design and operation do not appear to cause any apparent adverse effects on the development or survival of the embryos.

Batch effects are recognized as major sources of technical confounding in high-throughput assays. However, their impact on organoid studies receives little attention in the literature. As organoids gain prominence as a class of emerging new approach methodologies (NAMs), consideration of batch variation will become increasingly important to ensure data reproducibility and accurate interpretation in pre-clinical and clinical studies. In this manuscript, we provide a practical description of our work in detecting, characterizing, and correcting batch effects in a prior published retrospective clinical colorectal cancer organoid drug-response study. We outline the workflow we employed, including exploratory diagnostics, experimental drift detection, and statistical adjustment. We detail the methods employed to evaluate batch effects, monitor longitudinal drift, and select approaches to remove technical artifacts, preserve biological signal and test for robustness. Our experience demonstrates that in even modestly sized studies, results can be adversely affected by insufficient consideration and attempts at ameliorating batch effects. By documenting the challenges we encountered and the solutions implemented within our study, we hope that we can provide a seminal practical reference for organoid researchers and enable increased discussion and adoption of robust batch-compensation practices in the organoid field, ensuring that the topic is more routinely addressed, improved, and eventually standardized.

Live-cell imaging (LCI) provides researchers the opportunity to understand biological phenomena at a temporal resolution and is achieved using dedicated imaging systems. These studies enable insight into dynamic phenotypic changes occurring in cells, which may otherwise be missed when studying fixed samples. Access to advanced microscopy is disproportionately available to researchers in high-income countries, whereas researchers in low-to middle-income countries (LMICs) are severely underrepresented in the adoption of such technologies. A major barrier to the dissemination of advanced microscopy centres around economic inequalities, with the cost of high-end imaging systems often being prohibitively expensive. Recognition of such disparities has motivated the wider microscopy community to manufacture frugal microscopes that are accessible to researchers in resource-constrained settings. The OpenFlexure Microscope (OFM) is an open source, customisable, 3D-printed microscope suitable for medical research and field-diagnostics. We have made adaptations to the OFM to enable its use for live-cell imaging in humid tissue culture incubators. By moving major electronic components outside of the microscope, we remove the risk of corrosion of the Raspberry Pi and Sangaboard used to operate the instrument. We tested four common 3D-printing polymer materials for increased thermal robustness and found ASA is the best plastic to print the main body of the microscope, offering both durability and image stability in 24- to 48-hour time course experiments. We have also created an optional 3D-printable weighted-hammock system to reduce external vibration artefacts during image acquisition. Critically, electronic modifications included custom extension cables from the motors and camera to the Raspberry Pi and Sangaboard, and the inclusion of 22 ohm ({Omega}) resistors to reduce the current to the stepper motors, preventing detrimental temperature increases inside sealed incubators during prolonged powering of the instrument. To remove dependence on WiFi connections for setting up timelapse experiments, we generated a simple application with a graphical user interface (GUI) that can be installed locally on a Raspberry Pi and is specifically designed for setting up timelapse experiments without extensive computational knowledge or experience. We validated our LCI-OFM adaptations with a 48-hour treatment of MDA-MB-231 breast cancer cells with the chemotherapeutic drug docetaxel, showcasing how the modified microscope can seamlessly feed into established bioimaging pipelines and generate biologically meaningful results. For researchers in LMICs, this adapted LCI-OFM provides new opportunities to study locally-relevant health challenges with timelapse microscopy, enabling deeper insight into biological dynamics and supporting the generation of preliminary data critical for securing grant funding and access to more advanced imaging systems in purpose-built regional imaging hubs.

Parenteral drugs must meet strict release criteria to ensure patient safety upon administration. Sterility is a critical requirement, and is typically assessed using the compendial U.S. Pharmacopoeia (USP) <71> sterility test. However, with the growing demand to reduce batch release times, the tests 14-day incubation period is becoming a concern, emphasizing the need for more rapid alternatives. Here, we compared the performance of the calScreener+ isothermal microcalorimetry (IMC) device (Symcel) to that of the compendial USP <71> sterility test, using a panel of sixteen microorganisms (six USP <71> reference strains and ten field isolates) in two inoculum sizes (100 and 5 CFU). The IMC-based method detected a higher number of positive samples compared to the compendial method (95.8% vs 87.5%; p < 0.05). Furthermore, IMC was consistently faster, reducing mean detection times from 43 hours to 19 hours at 100 CFU and from 46 hours to 28 hours at 5 CFU (p < 0.001). In conclusion, the calScreener+ IMC device shows promise as a rapid and sensitive alternative to the compendial sterility test, with the potential to speed up batch releases without compromising patient safety.

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.

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.

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.

Cultivated meat production requires robust and validated analytical methods for comprehensive characterization. While transcriptomics-based approaches establish the foundational profile of molecular analysis, proteomics provides additional resolution that further enhances scientific certainty in both product development and safety characterization. However, the industry adoption of proteomics is currently hindered by technical complexity and a critical lack of analytical standardization, which leads to significant workflow-dependent variations in proteome coverage. To address this gap, we investigated the influence of key workflow steps (digestion, cleanup, LC-MS conditions) on the proteome profile of cultivated duck biomass. We compared five bottom-up sample preparation protocols - two traditional in-solution options (urea and SDC-based protocols), two device-based approaches (PreOmics iST and EasyPep kits), and an innovative protocol (SPEED), and demonstrated that device-based protocols offered the highest peptide yield and proteome coverage. However, optimization allowed cost-effective in-solution methods to achieve comparable performance. Specifically, an optimal digestion time of 3 hours at 37{degrees}C and the use of polymer-based desalting columns significantly enhanced protein identification ([~]4500 - 5000 IDs). Moreover, data independent acquisition (DIA) provided deeper proteome coverage than data dependent acquisition (DDA) with higher precision ([~]6500 vs 5000 IDs). The validated Standard Operating Procedures presented here establish a standardized framework for bulk bottom-up proteomics in cultivated meat, facilitating the generation of reliable and comparable data required for robust multi-omics characterization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/713501v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@5b61b8org.highwire.dtl.DTLVardef@16c7e65org.highwire.dtl.DTLVardef@1de21d2org.highwire.dtl.DTLVardef@7e984a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIComplexity and non-standardization limit MS-proteomics use in cultivated meat (CM). C_LIO_LICM protein profile varies with sample prep, LC-MS, and data processing pipeline. C_LIO_LIDevice-based and optimized cost-effective protocols offer a high proteome coverage. C_LIO_LIProteomics can complement transcriptomics for a comprehensive CM characterization. C_LIO_LIProposed standardized methods ensure reliable data for future regulatory submissions. C_LI

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.

The fallopian tube serves as a sperm reservoir, and it is the site where the oocytes become fertilized. Here, we describe development of an organ-on-a-chip microfluidic model of the fallopian tube (FT Chip) lined by primary human epithelial cells and stromal fibroblasts derived from the FT ampulla. Abundant tissue folds lined by hormone-responsive, epithelial cells resembling those seen in vivo formed on-chip, but not in epithelial organoids cultured in gel cultures. Comparative time-resolved analysis of human sperm versus oocyte-sized microparticles introduced into the epithelial channel in the presence of estradiol revealed that sperm movement was significantly reduced, while the oocyte-sized particles increased, relative to movements in acellular chips. When the non-hormonal contraceptive TDI-11861 was administered to the chip, dose-dependent inhibition of human sperm motility was detected. Thus, this FT Chip may offer a human preclinical tool to study FT physiology and assess the efficacy and mechanism of action of contraceptives.

Capillary zone electrophoresis (CZE)-mass spectrometry (MS) has been proposed as a powerful analytical tool for bottom-up, top-down, and native proteomics (multi-level proteomics) decades ago to analyze complex biological samples at the levels of peptides (bottom-up), proteoforms (top-down), and complexoforms (native). However, its broad adoption has been impeded by the limited robustness and reproducibility. Here, we present multi-level proteomics data from nearly 170 CZE-MS runs ([~]170 hours of instrument time), demonstrating qualitatively (i.e., the number of identified peptides and proteoforms, the number of detected complexoforms, and their migration time) and quantitatively (i.e., peptide, proteoform, and complexoform intensity) reproducible measurement of complex samples with varying levels of complexity, i.e., Escherichia coli cells, HeLa cells, and human plasma. CZE-MS-based native proteomics enabled the detection of hundreds of complexoforms up to 800 kDa from the complex systems via consuming only nanograms of protein material. The results indicate that CZE-MS is sensitive and reproducible enough for broad adoption for multi-level proteomics-based biomedical research.

The development of chemiluminescent immunoassays (CLIAs) is a complex and iterative process that relies on costly laboratory infrastructure, limiting its accessibility and application across healthcare settings and disease areas. Here, we detail the CLIA Mobile Development Kit (CLIAMDK) a modular, mobile, and inexpensive platform to assess image sensors, smartphones and data processing workflows for CLIA development. For its demonstration, we developed two CLIAs targeting renin and aldosterone, key biomarkers for diagnosing primary aldosteronism. The results from our performance study, including 50 patient samples, demonstrate the potential of our platform in a real-world scenario. We found that the performance of our mobile reader platform is comparable to that of a state-of-the-art plate reader, with a Lower Limit-of-Detection (LLoD) approaching 41 femtomolar. We envision that our platform will help accelerate CLIA development, make it more accessible, and lay the foundations for novel, distributed, yet highly sensitive diagnostic tests.

Subtyping of ketoacidosis, a metabolic state characterized by blood acidification due to various causes, remains challenging in forensic casework. Postmortem omics samples paired with machine learning offers an independent tool to address this challenge. However, such data, especially related to real forensic cases, are rare. In Sweden, high-resolution mass spectrometry data routinely collected in forensic toxicology, can be leveraged for metabolomic analysis. Here, we integrate postmortem metabolomics and machine learning models to detect and subtype ketoacidosis-related deaths using real forensic cases in Sweden. From femoral blood samples of 109 alcoholic ketoacidosis cases, 220 diabetic ketoacidosis cases, 140 hypothermia cases, and 1,229 controls (hanging cases), we developed and tested three machine learning models, which achieved over 90% accuracy in ketoacidosis detection and over 80% in subtyping. Validation with independent cohorts (21 starvation cases, 29 alcoholic controls, and 40 diabetic controls) confirmed robustness with over 80% of starvation cases classified as ketoacidosis-related. Feature clustering highlighted metabolites such as cortisol to be important for subtyping. In summary, our findings demonstrate that combining machine learning with postmortem metabolomics enables accurate detection and subtyping of ketoacidosis-related deaths, which is useful for forensic casework.