Biosensors and Bioelectronics

African swine fever virus (ASFV) poses a major transboundary threat to global swine production, underscoring the need for rapid and field-deployable diagnostic tools. Although quantitative polymerase chain reaction (qPCR)-based assays are the standard molecular assay for ASFV detection, their reliance on centralized laboratory infrastructure, multi-step sample preparation, and trained personnel limit their utility for timely decision-making at the point of need (PON). Here, we report a portable molecular diagnostic platform that enables colorimetric quantitative loop-mediated isothermal amplification (qLAMP) directly from diluted whole blood on microfluidic paper-based analytical devices ({micro}PADs). The assay targets the conserved ASFV viral protein 72 (VP72) and topoisomerase II (TOPII) genes and incorporates objective image-based colorimetric signal analysis to reduce user-dependent interpretation. Using plasmid DNA spiked into whole blood diluted to 5% (v/v) in 5% D-mannitol, the {micro}PAD-LAMP assay achieved a limit of detection (LOD) of 25 copies per reaction (67 copies/{micro}L of whole blood sample) for VP72 targets with no observed cross-reactivity against nine common swine pathogens, demonstrating 100% analytical sensitivity and specificity during in-house testing and 90% and 92% analytical sensitivity and specificity respectively in an external laboratory evaluation. The complete assay was performed within 60 minutes using a portable heating and imaging platform. Together, these results demonstrate a simple, DNA extraction-free molecular diagnostic approach that enables rapid and reliable ASFV detection from whole blood applicable to field-relevant conditions.

Interleukin-6 (IL-6) is a pleiotropic cytokine whose aberrant elevation drives life-threatening conditions, including sepsis, cytokine storm, and autoimmune disorders, yet existing clinical detection methods demand centralized laboratory infrastructure and multi-hour assay times incompatible with rapid point-of-care decision-making. Here, we report an impedimetric aptasensor built on a programmable tetrahedral DNA nanostructure (TDN) interface anchored to a disposable gold screen-printed electrode (Au-SPE) for the ultrasensitive, label-free detection of IL-6. By systematically varying the number of thiolated base vertices from zero to three, we establish a clear and previously unreported structure-function relationship between multipodal anchoring geometry and charge-transfer resistance modulation: tripodal thiolation yields the most rigid, upright, and electrochemically responsive interface, producing the steepest analytical signal gain upon IL-6 binding at the apex-localised aptamer. Under optimised conditions (pH 7.0, 0.05 uM TDN, MCH passivation), the aptasensor exhibits a linear dynamic range of 0.0001-0.001 pg/mL, a limit of detection of 55 ag/mL, and a sensitivity of 1.55x107 ohm (pg mL-1)-1. Selectivity evaluation against seven physiologically relevant interferents such as TNF-, BSA, glucose, urea, ascorbic acid, glycine, and cysteine confirms negligible cross-reactivity, with relative responses ranging from 0.57% to 14.35% of the IL-6 signal. Spike-recovery experiments in human serum yield recoveries of 74.0-87.6% (%RSD < 4.5%), and the sensor retains functional activity for at least 21 days under refrigerated storage. This work demonstrates that thiolated vertex number is a critical and tunable design parameter for TDN-based biosensors, offering a modular, disposable platform for sub-femtogram cytokine detection with direct applicability to early sepsis diagnosis and inflammatory disease monitoring.

Continuous monitoring of proteins in complex biological fluids is essential for advancing personalized medicine, yet existing biosensors are often limited by instability, single-use designs, and insufficient sensitivity. Here, we describe a detailed protocol for the fabrication and operation of molecular pendulum (MP) electrochemical sensors integrated with an active-reset mechanism to enable real-time, reversible, and ultrasensitive protein detection. The protocol is described in two parts. First, we describe the microfabrication of gold microelectrodes and their nanostructured modification, followed by assembly of DNA-based pendulum probes with redox reporters and affinity receptors that translate binding into kinetic electron-transfer shifts. Second, we detail the sensing and active-reset approach, which detects the target analyte and applies tunable oscillatory potentials to accelerate its dissociation, regenerate sensor surfaces, and extend operational lifetime. The protocol includes detailed guidance on device fabrication, surface functionalization, sensing and reset cycles, and data analysis. When implemented, MP sensors with active-reset achieve pg/ml sensitivity, rapid equilibration, and robust performance across biofluids and in situ models, enabling continuous protein monitoring over extended periods. This combined technology represents a biosensing platform with significant potential, opening new avenues for wearable and implantable molecular monitoring, early disease detection, and personalized therapeutic guidance.

Despite the significant technical advancement in spatial transcriptomics, its clinical usage is largely untapped. Here, we develop an integrated system, ENDO-Genome, for minimally invasive in-body transcript sampling to facilitate live spatial transcriptomic analysis of human internal organs. This is achieved by integrating a nanoarrayed biochip with existing endoscope to perform pressure-sensor-calibrated "Touch & Go" RNA extraction directly from human internal organs, including the highly vascularized liver or kidney, without the need for tissue biopsy, voiding any bleeding risks. By a demonstration using gastrointestinal endoscopy, multiplexed landscape of 55 mRNA transcripts was obtained from multiple locations of human intestinal tract via a 5-minute operation in routine examinations. Benefiting from a sequencing-free approach, each assay costs less than 10 US dollars. For the clinical study involving 15 Crohn' s disease (CD) patients, no complication case was reported out of 47 ENDO-Genome operations, showcasing the gentle deposition and excellent safety of the technique. The live spatial transcriptomics provides direct in vivo pictures of the heterogenous spatial transcriptional programs underlying CD pathological response at different intestinal locations, revealing distinct ileal phenotypes. This is manifested by unique microscale scattering of inflammation gene clusters, along with the discovery of a tissue-specific cooperative mechanisms between inflammation and RNA methylation regulations at single- or multi-cell scales.

Accurate and decentralized quantification of serotonin, also known as 5-hydroxytryptamine (5-HT), in biological fluids is critically important for the diagnosis, prognosis, and therapeutic monitoring of neurological and psychiatric disorders. However, conventional analytical methods generally rely on centralized laboratory infrastructure, skilled personnel, and labor-intensive sample processing, which restrict their applicability in rapid near-patient and point-of-care settings. Herein, we report a portable molecularly imprinted polymer (MIP)-based electrochemical sensing platform for selective and on-site detection of serotonin using screen-printed carbon electrodes (SPCEs). The biomimetic recognition interface was fabricated through direct electropolymerization of a polydopamine recognition layer in the presence of serotonin as the template molecule, followed by template extraction to generate complementary recognition cavities for selective rebinding. The sensor fabrication parameters, including monomer concentration, electropolymerization cycles, template-to-monomer stoichiometry, and electrolyte pH, were systematically optimized to achieve improved sensitivity, selectivity, and signal stability. Under optimized conditions, the MIP/SPCE sensor exhibited a broad linear response from 10 pM -10 uM in phosphate buffer, with a correlation coefficient of R2 = 0.974 and an ultralow limit of detection of 0.16 pM. The analytical applicability of the platform was further validated in spiked artificial serum, where the sensor achieved an LOD of 0.12 pM, satisfactory recovery values of 88.66-96.02%, and acceptable precision with RSD values [&le;] 8.43% (n=3), confirming its reliability in a complex biological matrix. The developed sensor demonstrated excellent selectivity toward serotonin against physiologically relevant interferents, maintaining signal retention between 99% and 101%. In addition, the platform showed high operational repeatability with an RSD of 0.45%, good inter-electrode reproducibility with an RSD of 6.3%, and long-term storage stability, retaining 90-110% of its initial response over 28 days. Importantly, cross-platform validation using a smartphone-coupled potentiostat demonstrated strong analytical agreement with laboratory-grade instrumentation, as evidenced by R2 = 0.9967 and a slope of 1.023. These findings establish the proposed MIP/SPCE platform as a simple, low-cost, portable, and smartphone-compatible electrochemical device for field-deployable serotonin monitoring in clinically relevant samples.

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.

Differentiating overlapping dementia pathologies, such as frontotemporal dementia and Alzheimers disease, calls for the simultaneous measurement of several blood biomarkers, yet electrochemical sensors remain predominantly single-target and dependent on labels and sample preparation. Here we report SMART-NeuroDx, a reagent-free electrochemical array that quantifies pTau217, GFAP, pTau181, and NfL directly from unprocessed plasma and serum in under 35 minutes. Four surface-confined redox-active molecularly imprinted polymer and aptamer-MIP recognition matrices are electropolymerized onto a four-working-electrode porous-gold printed-circuit array, using potential-assisted electrostatic gating keyed to each targets isoelectric point to prevent cross-channel template contamination during synthesis. Label-free Faradaic responses from the redox-active polymer backbone are acquired on a custom battery-powered STM32G4 handheld potentiostat and converted to concentration from five voltammetric features using cross-validated Random Forest and XGBoost regressors. The handheld unit reproduced the baseline fidelity of a commercial benchtop workstation and resolved sub-picogram pTau217 (limit of detection 0.087 pg mL-{superscript 1}) across marker-appropriate dynamic ranges, with inter-chip relative standard deviation at or below 5.34% and coefficients of determination of 0.94 to 0.99 against reference concentrations. Each channel retained selectivity in plasma and serum against competing neurological and inflammatory proteins, with non-specific signal deviation held below 10% by the hydrated PyPEG interfacial shell. The platform establishes reagent-free, simultaneous four-analyte neurodegeneration sensing on a single point-of-care device.

Rapid, selective detection of bacterial pathogens remains a central challenge. Here we report a label-free electrochemical biosensing approach that leverages protein-protein interaction (PPI)-derived peptides as recognition elements for rapid detection of Listeria monocytogenes (LM). The sensor design is inspired by the interaction between the LM virulence factor Internalin A (InlA) and the human host receptor E-cadherin (E-Cad1). Peptides derived from the InlA-binding domain of E-Cad1 were engineered as molecular recognition elements, with the E-Cad1(15-24) peptide displaying micromolar affinity and selective binding towards LM. Immobilization of these peptides on gold electrodes enabled bacterial detection by electrochemical impedance spectroscopy within 10 minutes, without labels or external signal amplification. A low peptide surface density was associated with enhanced binding-site accessibility and may facilitate multivalent interactions between the bacterial surface and the immobilized peptides. The platform produced a detectable response at experimentally tested concentrations as low as 1 CFU mL {superscript 1} and exhibited excellent selectivity under the conditions examined. This work introduces a chemically programmable, PPI-inspired biosensing paradigm that uses a reductionist approach and could potentially be extended to other pathogen targets.

Square-wave voltammetry (SWV) is widely used for electrochemical biosensing because it enables sensitive, temporally resolved measurement of redox reporter signals. However, automated quantification of SWV signal remains challenging for long duration and in vivo measurements, where voltammograms can exhibit changing baselines, heterogeneous noise, peak drift, outliers, and interfering faradaic processes. Here, we introduce the Adaptive Square-Wave Voltammetry Iterative Fitting Toolkit (ASWIFT), an automated method for robust SWV signal extraction based on iteratively reweighted regularized smoothing. ASWIFT is available as both an open-source Python package and a downloadable desktop application. The method adaptively estimates the baseline, selects regularization strengths, fits the redox peak, and reports peak height without trace-specific parameter tuning. Across simulated datasets spanning diverse baseline, peak, noise, and concentration-response conditions, ASWIFT produced less systematic bias and more consistent signal estimates than existing methods. We further evaluated ASWIFT using in vitro doxorubicin measurements and in vivo DNA-based kanamycin sensor measurements collected in rat blood and interstitial fluid, demonstrating agreement with established methods. These results support ASWIFT as a robust framework for automated SWV analysis in real-time electrochemical biosensing.

Background: Rapid and accurate measurement of procalcitonin (PCT) is useful for diagnosing bacterial infections and guiding antibiotic therapy, yet current laboratory-based immunoassays require centralised infrastructure, delaying clinical decision-making and limiting access in low-resource settings. We developed a battery-free, smartphone-connected electrochemical lateral flow assay with linked analytics for the detection of PCT (ELLA-PCT) enabling quantitative testing at the point of care without conventional laboratory instrumentation. Methods: We designed a competitive electrochemical lateral flow assay using gold nanoparticles co-functionalised with a PCT-specific DNA aptamer and ferrocene hexanethiol as a redox reporter. A miniaturised near-field communication (NFC) potentiostat embedded within a disposable cassette enabled wireless electroanalytical measurements using a smartphone. Analytical performance was assessed in buffer and serum, including limit of detection (LOD), linearity, specificity, and stability. Clinical evaluation was performed on 27 serum samples from nine adults undergoing antibiotic treatment for suspected bacterial infection, with results compared against the reference Time-Resolved Amplified Cryptate Emission (TRACE) assay. Findings: ELLA-PCT achieved an LOD of 46 pg/mL and a linear detection range of 0.5-100 ng/mL. The ELLA-PCT assays remained stable for three months under ambient storage conditions, and cross-reactivity with calcitonin, C-reactive protein, and interleukin-6 remained below clinically relevant thresholds. Clinical results showed strong correlation with TRACE (R2 = 0.979, p < 0.0001), with a mean bias of 0.05 ng/mL and narrow 95% limits of agreement (-0.69 to 0.89 ng/mL). Importantly, ELLA-PCT delivered results in only 30 minutes without requiring external power or clinical laboratory instrumentation. In comparison to TRACE, the total turnaround time was reduced by at least 50%, which typically requires at least one hour from sample collection to results at a hospital setting. Interpretation: ELLA-PCT is an antibody-free, smartphone-connected test that provides laboratory-grade quantitative PCT measurement using only an NFC-enabled electroanalytical sensor and a mobile phone. The platform has a strong potential to decentralise diagnosis of infectious diseases, support antibiotic stewardship, and enable remote, real-time monitoring in outpatient and resource-limited settings. Larger studies are needed to evaluate the use of whole-blood samples and the integration of the platform into digital clinical workflows.

Monitoring pH and extracellular acidification rate (ECA) in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins,OEP and OEPK, embedded in a plasticized PVC matrix containing a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime-based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of the protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.

The outbreak frequency and geographic distribution of viral pathogens are continuously expanding, making enhanced genomic surveillance an urgent global public health need. Parallel library preparation combining next-generation sequencing (NGS) and third-generation sequencing (TGS) can substantially improve the coverage and resolution of genomic surveillance, representing a key strategy for strengthening surveillance. Here we developed a complete sample-to-result system integrating a programmable active-matrix digital microfluidic (AM-DMF) chip with a bioinformatics analysis pipeline. Compared with conventional manual protocols used in public health laboratories, our system reduces reagent consumption by 72%, shortens library preparation time by 45% and decreases the inter-batch coefficient of variation (CV) by 20%. In 20 RT-qPCR-confirmed clinical samples, the system achieved complete concordance for viral identification and assigned serotypes/genotypes consistent with sequencing-based phylogenetic analysis. This system is field-deployable and enables rapid virus serotyping as well as in-depth genomic surveillance. TeaserA digital microfluidic platform integrating short- and long-read sequencing enables rapid comprehensive viral genome analysis.

Rapid tests with visual readout can quickly help identify and triage at-risk patients. However, multiplexed visual tests (MVTs) for targets with clinical thresholds above the limit of detection -- e.g., rogue autoantibodies (raAbs) -- are lacking because test interdependency and cross-reactivity make optimization intractable. Here, we introduce a conceptual and experimental framework to synchronize visual readout and clinical thresholds for multiple, cross-reacting targets simultaneously, and illustrate it with a 3D-printed, structurally-preprogrammed capillaric MVT for anti-interferon (IFN)- and -{omega} raAbs and anti-SARS-CoV-2 spike protein (anti-SCoV2) antibodies. Using design of experiments, we sought and identified parameters that collectively govern the background of all tests (buffer composition, ionic strength), and ones that individually govern assay signal and sensitivity (capture probe density, sample volume), thus enabling both collective background reduction and independent tuning of test line visual threshold. The instrument-free MVT is highly sensitive (pg-ng mL-1), reproducible (CV<10% in plasma), and completed in <1 h. We benchmarked the threshold-calibrated MVTs to microplate ELISA with 41 COVID-19 patient plasma samples yielding ROC-AUCs of 0.97, 1.00, and 0.98 for anti-IFN-, -IFN-{omega} and -SCoV2 tests, respectively. The proposed framework for synchronizing multiple visual readouts with respective clinical thresholds, combined with capillarics, opens the door to instrumentation-free MVTs for point-of-care use.

Electrical stimulation (ES) and transepithelial/transendothelial electrical resistance (TEER) measurements are essential techniques in cell biology and tissue engineering, yet commercial devices for these applications cost between USD 2,500-9,000 and typically offer only one functionality. We present LATEER (Low-cost Arduino-based TEER and Electrical stimulation device), an open-source hardware platform that combines both ES and TEER measurement capabilities at a total cost below USD 100. The device features four independent channels, configurable pulsatile signals (amplitude up to 8.2 V, frequency 0.1-500 Hz, pulse width [&ge;]0.1 ms), and a resistance measurement range of 300 {Omega} to 1 M{Omega}, with <5% error for R {gtrsim} 4.7 k{Omega}. LATEER uses commercially available graphite pencil leads as electrodes ([~]USD 2 vs. USD 350 for commercial Ag/AgCl electrodes), which demonstrated excellent biocompatibility in cell culture. The system includes 3D-printed electrode holders compatible with standard 12-well and 24-well plates, allowing microscope visualization without electrode removal, and a Python-based graphical user interface for parameter configuration and real-time data acquisition. Because the electrodes remain fixed in the plate lid and only a single cable enters the incubator, both stimulation and resistance measurement can run continuously under standard culture conditions (37 {degrees}C, 5% CO2) without removing the plate or repositioning the electrodes, avoiding the temperature excursions and placement variability inherent to manual chopstick measurements. Validation with human pluripotent stem cell-derived cardiomyocytes demonstrated reliable frequency capture (electrical pacing) of the contracting monolayer, with a capture threshold between 250 and 400 mV/mm and controlled pacing across the 0.5-5 Hz range. TEER functionality was verified with mesenchymal stem cells, where the device resolved cell-density-dependent differences in electrical resistance in real time. All design files, firmware, and software are freely available under the CERN-OHL-S v2 license, enabling replication and customization by research laboratories worldwide. HighlightsO_LIAn open-source device combines electrical stimulation and TEER measurement under $100 C_LIO_LIGraphite electrodes offer biocompatibility at 0.6% cost of commercial alternatives C_LIO_LIFour independent channels with configurable parameters and real-time data logging. C_LIO_LIContinuous run setup in-incubator; no electrode repositioning needed C_LIO_LIValidated with stem cell-derived cardiomyocytes, achieving frequency capture (threshold 250-400 mV/mm) C_LIO_LI3D-printed holders enable microscope visualization without electrode removal C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/743263v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@98f9deorg.highwire.dtl.DTLVardef@13c73aborg.highwire.dtl.DTLVardef@1cdf099org.highwire.dtl.DTLVardef@16ed0cf_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications Table O_TBL View this table: org.highwire.dtl.DTLVardef@4ef802org.highwire.dtl.DTLVardef@7c651borg.highwire.dtl.DTLVardef@d20013org.highwire.dtl.DTLVardef@102fc89org.highwire.dtl.DTLVardef@111b927_HPS_FORMAT_FIGEXP M_TBL C_TBL

Gold electrodes are attractive substrates for bioelectronic and cell-free synthetic biology platforms because they are conductive, chemically stable, biocompatible, and readily functionalized through thiol-gold chemistry. Here, gene-length DNA monolayers assembled on planar gold electrodes as reusable templates for cell-free protein expression are investigated. Using thiol-modified sfGFP genes, DNA surface density is shown to be tunable by changing the DNA concentration during incubation, with the immobilized genes able to support cell-free sfGFP expression directly from the electrode surface. Further, the effects of applied voltage, storage, repeated reactions, reducing agents, and protein fouling on monolayer stability and expression output are examined. While some conditions lead to loss of reusable expression activity, dense chemisorbed monolayers can retain partial function under neutral, non-reducing conditions and are relatively robust to protein exposure. In contrast, low-density physisorbed monolayers show a stronger relationship between DNA loss and expression output. Finally, when using gold-mediated fluorescence quenching to monitor changes in DNA conformation, surface-bound DNA demonstrates electrophoretic addressability. Together, these results establish DNA- functionalized planar electrodes as a promising foundation for modular, addressable cell-free expression platforms.

Digital bioassays enable precise single-molecule quantification but are difficult to adapt to point-of-care testing (POCT) because conventional protocols include off-chip complex processes for sample treatment and sealing, requiring hardware and workflow complexity. We present OASSIS (Open Aqueous two-phase Separation System for Integrated Single- molecule digital bioassay platform), an oil-free and open ATPS platform that localizes both targets and signals in femtoliter-scale dextran (DEX) droplets beneath a polyethylene glycol (PEG) phase. We integrated a CRISPR-Cas13a assay system with a novel, branched fluorescent reporter conjugated to a dextran-binding domain (DBD), which ensures signal retention within the DEX droplets after cleavage. Fluorescence recovery after photobleaching experiments confirmed this robust signal confinement. OASSIS not only performs amplification-free digital RNA detection but also enables serial sample introductions through its open-format architecture that progressively improve sensitivity: the limit of detection (LOD) improved from 1.08 fM (first introduction) to 0.34 fM (third introduction). Furthermore, OASSIS demonstrated specific detection and [~]10-fold enrichment from a complex, denaturant-treated nasopharyngeal swab matrix. Together, these results demonstrate that the open-format architecture of OASSIS provides a practical route toward sensitive, low-complexity POCT and clinical diagnostic applications.

Urothelial carcinoma (UC) is among the most common malignancies worldwide and is known to exhibit a high recurrence rate. The relative lack of validated, non-invasive biomarkers for the disease challenges early detection and negatively impacts patient outcomes. The linear polysaccharide hyaluronan (HA) has been recognized as a potential source of diagnostic information for UC, with its urinary concentration shown to be predictive of disease severity. Here, we use solid-state nanopore (SSNP) sensing to investigate the value of urinary HA size distribution as an independent and complementary predictor of UC. We show that, when combined with urinary concentration, HA size distribution provides a significant improvement to the differentiation of healthy individuals from those with urinary tract diseases in general (AUC = 0.91, p < 0.05), as well as differentiation of individuals with UC from those without (AUC = 0.87, p < 0.05). These results establish the potential of SSNP-based HA profiling for non-invasive diagnostics of UC.

Different in vitro models are widely used as experimental platforms to assess neuronal responses to metabolic stress and test potential treatments for patients with ischemic stroke. Results of those studies depend on the stress models used, and the link between cell viability-based readouts and electrophysiological activity remains poorly explored. We investigated the neuronal network activity of human-derived neuronal networks generated from human induced pluripotent stem cells (hiPSCs) under three commonly used metabolic stress models: hypoxia alone, oxygen and glucose deprivation (OGD), and hypoxia combined with different concentrations of glutamate. We aim to clarify the differences between three commonly used in vitro models, including the relation between microscopic and electrophysiological readouts. These conditions produced distinct effects on neuronal network activity. Hypoxia alone induced a progressive decline in activity over time. In contrast, OGD triggered a biphasic response, characterized by an early increase in activity followed by a decline. High concentration glutamate exposure under hypoxia also altered network dynamics, inducing a triphasic pattern consisting of a rapid activity decrease, a transient increase, and a subsequent decline. Across all these pathological conditions, neuronal activity progressively declined and converged toward network failure after prolonged hypoxia. Following reoxygenation, recovery was limited and condition-dependent: hypoxia alone, OGD, and high glutamate conditions showed limited recovery. On the other hand, low glutamate concentration was associated with good recovery. Microscopic assessment revealed that cellular viability was differentially affected across conditions. OGD was associated with the highest levels of cell death, whereas glutamate exposure, particularly at high concentrations, led to a marked reduction in synaptic puncta despite partial preservation of cell viability. These findings highlight that commonly used in vitro ischemia models induce distinct neuronal responses and highlight the importance of integrating electrophysiological and structural analyses to better characterize metabolic stress in human neuronal networks better.

Extracellular Vesicles (EVs) are small membrane-bound particles secreted by cells that play key roles in intercellular communication, gene regulation and modulation of cell function. They are involved in both physiological and pathological processes and, due to their ability to transport biomolecules across biological barriers, have emerged as promising tools for use as drug delivery vehicles and biomarkers with diagnostic and prognostic applications. Various methodologies are currently employed for the isolation, characterization, and analysis of EVs, including Ultracentrifugation (UC), Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and Flow Cytometry. Flow Cytometry has emerged as a powerful technique capable of providing a multiparametric analysis of individual EVs. Recent advancements have led to the development of cytometers with higher sensitivity and increased limit of detection, enabling the detection and sorting of nanoscale particles--a technique known as Nano-Flow Cytometry. In this study, we show the optimization of small particle sorting, termed nanoFACS, via the CytoFLEX SRT. This method enables sorting based on size or fluorescence, enhancing reproducibility and broadening the potential for application in biological and clinical assays. Furthermore, we demonstrate the utility of nanoFACS in isolating nanoparticles from complex biofluids and in detecting miRNA using molecular beacons (MBs) highlighting its potential in both basic research and translational applications.

BackgroundContinuous glucose monitoring (CGM) systems are used not only for retrospective assessment of the glycemic profile but also for real-time decision-making, including automated insulin delivery. Accordingly, CGM performance characterization must capture not only the agreement of individual paired values but also the systems ability to reproduce the direction, rate, amplitude, and shape of glucose concentration change. Summary metrics, most notably MARD, cannot establish whether an observed deviation reflects an error in the formation of the test profile itself, a constant sensor offset, amplitude compression, a change in response rate, temporal misalignment, or hysteresis. ObjectiveTo adapt a programmable flow-based in vitro platform for the separate assessment of the experimentally delivered glucose profile and the dynamic response of CGM systems, and to propose a set of metrics that decomposes dynamic error into its components. MethodsGLU profiles were generated by programmable mixing of solutions at a constant total flow rate of 2 mL/min. Actual GLU concentration was independently measured with a SUPER GL2 glucose analyzer. Four static levels, three repeats of a 5.5[-&gt;]12.0[-&gt;]5.5 mmol/L profile, three repeats of a 6.0[-&gt;]3.0[-&gt;]6.0 mmol/L hypoglycemic profile, three 5.0[-&gt;]15.0[-&gt;]5.0 mmol/L profiles at different rates, one complex 4[-&gt;]18[-&gt;]3[-&gt;]12[-&gt;]5.5 mmol/L profile, and two proof-of-concept sensor experiments at 100- and 200-min transitions were investigated. Dynamic response was characterized by bias, MAE, RMSE, MARD, amplitude transfer coefficient K_A, rate transfer coefficients K_up and K_down, normalized shape RMSE, residual shift, and hysteresis loop area. ResultsAt the static levels, measured GLU exceeded the programmed value by 0.234-0.780 mmol/L. In the repeated 5.5[-&gt;]12.0[-&gt;]5.5 profiles, the ratio of actual to programmed rate was 0.978-1.083 on the rising phase and 0.987-1.157 on the falling phase, while the amplitude transfer coefficient was 0.967-1.066. In the hypoglycemic profile, minimum GLU was 2.55- 2.96 mmol/L, and time below 3.0 mmol/L was 15.2-72.6 min. The measured rates of 0.0519, 0.1045, and 0.2027 mmol/L/min preserved the intended ratio of approximately 1:2:4. In the complex profile, the programmed plateau of 18 mmol/L was not reached: mean measured GLU was 16.20 mmol/L. For CGM-A, K_A was 0.682 and 0.650, and K_up/K_down were 0.666/0.730 and 0.634/0.626; the corresponding values for CGM-B were 1.228 and 1.128, and 1.564/1.328 and 1.276/1.145. Hysteresis loop area differed 5- to 10-fold between the two sensor responses, exceeding an order of magnitude at the 100-min transition. ConclusionThe programmed concentration should be treated as a control setpoint, rather than as a reference measurement. The "programmed trajectory -- measured glucose -- CGM output" cascade first allows quantitative assessment of the agreement between the programmed and actually realized profile and only then separate characterization of sensor response. Decomposition of dynamic error into amplitude, rate, shape, and hysteresis components reveals differences that a single MARD value or correlation coefficient cannot capture.