High-throughput Testing Research Articles

CRISPR/Cas9-Mediated Genome Editing of T4 Bacteriophage for High-Throughput Antimicrobial Susceptibility Testing.

The accurate and effective determination of antimicrobial resistance is essential to limiting the spread of infectious diseases and ensuring human health. Herein, a simple, accurate, and high-throughput phage-based colorimetric sensing strategy was developed for antimicrobial susceptibility testing (AST). Taking advantage of the CRISPR/Cas9 system, the genome of the T4 phage was modularly engineered to carry lacZ-α (lacZa), a marker gene encoding the α-fragment of β-galactosidase (β-gal). T4lacZa phages were identified by blue-white selection and then used for a biosensing application. In this strategy, the bacterial solution is exposed to the T4lacZa phage, causing target bacteria to overexpress β-gal. Upon the addition of a colorimetric substrate, the β-gal initiates an enzymatic reaction, resulting in a solution color change from yellow to red. This sensing strategy offers a visual way to monitor bacterial growth in the presence of antibiotics, enabling the determination of bacterial antimicrobial susceptibility. As a proof of concept, our developed sensing strategy was successfully applied to identify 9 different multidrug-resistant Escherichia coli (E. coli) in urine samples with 100% specificity. Compared with conventional disk diffusion susceptibility tests, the engineered phage-based sensing strategy can shorten the detection time by at least half without losing detection sensitivity, providing an alternative high-throughput method for AST in clinical diagnosis.

On-Chip NADH Detection in Multicellular Models Using an AlGaN/GaN Photodetector Array with Enhanced Sensitivity.

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme, existing in its oxidized form (NAD+) and reduced form (NADH). Both are essential in cellular redox reactions and are implicated in energy production and cancer. Current NADH detection methods often involve complex optical measurements. We propose a miniaturized, on-chip photoelectric sensor array using AlGaN/GaN two-dimensional electron gas (2DEG) photodetectors for NADH quantification. The device exhibits an ultralow dark current and ultrahigh UV light responsivity, enabling sensitive NADH detection. By exploiting the absorbance disparity between NADH and NAD+, our sensor achieves rapid, sensitive detection, surpassing commercial assays. It effectively detects NADH levels in 3D multicellular models, promising cancer screening and monitoring. This sensor platform offers a significant advancement in NADH quantification, with the potential for high-throughput testing and point-of-care diagnostics. Our study presents an efficient approach for NADH sensing, addressing the need for rapid and sensitive detection methods in biomedical research and clinical practice.

Zebrafish arterial valve development occurs through direct differentiation of second heart field progenitors.

Bicuspid Aortic Valve (BAV) is the most common congenital heart defect, affecting at least 2% of the population. The embryonic origins of BAV remain poorly understood, with few assays for validating patient variants, limiting the identification of causative genes for BAV. In both human and mouse, the left and right leaflets of the arterial valves arise from the outflow tract cushions, with interstitial cells originating from neural crest cells and the overlying endocardium through endothelial-to-mesenchymal transition (EndoMT). In contrast, an EndoMT-independent mechanism of direct differentiation of cardiac progenitors from the second heart field (SHF) is responsible for the formation of the anterior and posterior leaflets. Defects in either of these developmental mechanisms can result in BAV. Although zebrafish have been suggested as a model for human variant testing, their naturally bicuspid arterial valve has not been considered suitable for understanding human arterial valve development. Here, we have set out to investigate to what extent the processes involved in arterial valve development are conserved in zebrafish and ultimately, whether functional testing of BAV variants could be carried out. Using a combination of live imaging, immunohistochemistry and Cre-mediated lineage tracing, we show that the zebrafish arterial valve primordia develop directly from SHF progenitors with no contribution from EndoMT or neural crest, in keeping with the human and mouse anterior and posterior leaflets. Moreover, once formed, these primordia share common subsequent developmental events with all three aortic valve leaflets. Our work highlights a conserved ancestral mechanism of arterial valve leaflet formation from the SHF and identifies that development of the arterial valve is distinct from that of the atrioventricular valve in zebrafish. Crucially, this confirms the utility of zebrafish for understanding the development of specific BAV subtypes and arterial valve dysplasia, offering potential for high-throughput variant testing.

Full-Thickness Perfused Skin-on-a-Chip with In Vivo-Like Drug Response for Drug and Cosmetics Testing

In this study, we present a novel 3D perfused skin-on-a-chip model fabricated using micro-precision 3D printing, which offers a streamlined and reproducible approach for incorporating perfusion. Perfused skin models are well-regarded for their advantages, such as improved nutrient supply, enhanced barrier function, and prolonged tissue viability. However, current models often require complex setups, such as self-assembled endothelial cells or sacrificial rods, which are prone to variability and time-consuming. Our model uses projection micro-stereolithography 3D printing to create precise microcapillary-like channels using a biocompatible resin, overcoming the drug-absorbing properties of PDMS. A customized chip holder allows for the simultaneous culture of six perfused chips, enabling high-throughput testing. The engineered skin-on-a-chip features distinct dermis and epidermis layers, confirmed via H&E staining and immunostaining. To evaluate drug screening capabilities, inflammation was induced using TNF-α and treated with dexamethasone, with cytokine levels compared to 2D cultures and human skin biopsies. Our 3D model exhibited drug response trends similar to human skin, while showing reduced cytotoxicity over time compared to biopsies. This perfused skin-on-a-chip provides a reliable, physiologically relevant alternative for drug and cosmetics screening, simplifying perfusion setup while preserving key benefits.

Application of high-throughput drug sensitivity testing in children with relapsed and refractory acute leukemia

To explore the current application of high-throughput drug sensitivity (HDS) testing in children with relapsed and refractory acute leukemia (RR-AL) and analyze the feasibility of salvage treatment plans. A retrospective collection of clinical data from children with RR-AL who underwent HDS testing at the Department of Children's Hematology and Oncology of the First Affiliated Hospital of Zhengzhou University from November 2021 to October 2023 was conducted, followed by an analysis of drug sensitivity results and treatment outcomes. A total of 17 children with RR-AL underwent HDS testing, including 7 cases of relapsed refractory acute myeloid leukemia and 10 cases of relapsed refractory acute lymphoblastic leukemia. The detection rate of highly sensitive chemotherapy drugs/regimens was 53% (9/17), while the detection rate of moderately sensitive chemotherapy drugs/regimens was 100% (17/17). Among the 17 RR-AL patients with highly and moderately sensitive chemotherapy drugs and regimens, the MOACD regimen (mitoxantrone + vincristine + cytarabine + cyclophosphamide + dexamethasone) accounted for 100%, with the highest inhibition rate for single-agent mitoxantrone (94%, 16/17), and the highest inhibition rate for targeted therapy being bortezomib (94%, 16/17). Nine patients adjusted their chemotherapy based on HDS testing results, with 4 undergoing hematopoietic stem cell transplantation. Four patients achieved disease-free survival, while 5 died. Eight patients received empirical chemotherapy, with 2 undergoing hematopoietic stem cell transplantation; 4 achieved disease-free survival, while 4 died. HDS testing can identify highly sensitive drugs/regimens for children with RR-AL, improving the rate of re-remission and creating conditions for subsequent hematopoietic stem cell transplantation.

Valved Microwell Array Platforms for Stepwise Liquid Dispensing.

The fabrication of microarray chips and the precise dispensing of nanoliter to microliter liquids are fundamental for high-throughput parallel biochemical testing. Conventional microwells, typically featuring a uniform cross section, fill completely in a single operation, complicating the introduction of multiple reagents for stepwise and combinatorial analyses. To overcome this limitation, we developed an innovative valved microwell array. Using ultraviolet (UV)-curing resin three-dimensional (3D) printing, these multilayer configurations can be rapidly fabricated through direct template printing and polydimethylsiloxane (PDMS) casting. Each microwell incorporates a microvalve structure, truncating fluids within the upper metering well and allowing transfer to the bottom reservoir well under centrifugal force. Sequential operations enable the introduction of multiple reagents, facilitating orthogonal combinations for complex assays. We explored four types of valving methods: DeepWell, Expansion, Bottleneck, and Membrane valve, each offering varying degrees of design complexity, operational efficiency, robustness, and precision. These methods constitute a versatile toolkit to accommodate a broad spectrum of analytical requirements. Our innovative approach redefines microwell architecture, direct manufacturing techniques, and stepwise fluid dispensation in microarrays.

B-157 High Resolution Liquid Chromatography - Mass Spectrometry (LC-MS) For Rapid, High-Throughput Clinical Testing of β2-Transferrin in Human Nasal Secretions

Abstract Background Leakage of Cerebrospinal Fluid (CSF) can result from trauma, lacerations, and surgery, and if untreated can result in potentially life-threatening meningitis. CSF leaks can be diagnosed through the detection of β2-Transferrin (β2-Tf) from nasal secretions via gel electrophoresis. β2-Tf is a uniquely glycosylated proteoform of Transferrin found primarily in CSF, which can be distinguished from the most abundant Transferrin proteoform (β1-Tf) by its altered electrophoretic mobility. However, gel-based detection of β2-Tf requires hours of labor and has low specificity due to the numerous alternative glycoforms of Transferrin. Higher-throughput assays are thus desirable for improved patient care. Recently, our group applied High-Resolution Mass Spectrometry (HR-MS) coupled to Microprobe In-Emitter Elution (MPIE) to discriminate Transferrin isoforms. This method successfully resolved β2- from β1-Tf in nasal secretions derived from patient samples with suspected CSF leakage, but has relatively low sample-to-sample throughput. Herein, we describe a novel approach utilizing Liquid Chromatography Mass Spectrometry (LC-MS) to rapidly profile Transferrin glycoforms from patient nasal secretion samples. Furthermore, we explore solutions to address significant blood contamination in nasal secretions, which may otherwise interfere with this approach. Furthermore, we explore solutions to address significant blood contamination in some nasal secretions, which may otherwise interfere with this approach. Methods Our approach consists of two primary steps: 1) Sample processing and high abundance protein depletion via commercial filtration and depletion columns. 2) LC-MS analysis of the resulting extract to resolve Transferrin glycoforms by retention time and m/z. This approach retains the high specificity of the previously demonstrated MPIE-MS method, but takes advantage of existing high-throughput liquid chromatography platforms to reduce the sample-to-sample analysis time. Secretion samples were analyzed using a Vanquish HPLC system coupled to a Q-Exactive Plus Mass Spectrometer (ThermoFisher). Broadband mass spectra were collected (FWHM resolution of 17,500 at 200 m/z), and the deconvoluted average masses were used to identify Transferrin glycoforms. Results Our approach was benchmarked using remnant nasal secretion samples from patients with known CSF leakage. Examining 9 samples, the resulting ESI spectra revealed peaks at 79,554 Da and 78,008 Da, consistent with β1-Tf (Pred. MW (Average): 79,554.71 Da) and β2-Tf (Pred. MW (Average): 78,008.35 Da) within ∼10 ppm mass accuracy. The 79.5 kD peak was observed in all samples, while the 78 kD peak was observed in 7 of 9 samples. Samples with missing β2-Tf were marked by significant blood contamination. Genetic variants (2/9 samples) resulted in mass shifts for both β1- and β2-Tf variants, but Transferrin glycoforms identifiable due to the preserved mass shift between the β1- and β2-Tf peaks (∼1546.4 Da). An optimized LC-MS gradient enables rapid (∼10 minute) sample-to-sample injection times. Samples with blood contamination represent an additional challenge for identification of β2-Tf, owing to ion suppression from blood-derived β1-Tf. We have developed enrichment and depletion methods to increase the sensitivity for β2-Tf in blood contaminated samples, and anticipate that these approaches will reduce or eliminate this challenge. Conclusions Our approach enables high-throughput, MS-based measurement of Transferrin glycoforms (i.e. β1- and β2-Tf) from clinical nasal secretion samples for the first time.

Performance of single PN/A reactor under wide fluctuation of nitrogen load.

Partial nitritation-anammox (PN/A) is a cost-effective technology in high ammonia-nitrogen wastewater treatment. However, PN/A is prone to instability as the ammonia-nitrogen sharply fluctuates. In this study, a packed bed reactor is employed to construct a single-stage PN/A system to investigate the operational characteristics and explore the denitrification mechanism. The effluent NH4+-N concentration, ammonia nitrogen removal rate (ARE), and total nitrogen removal rate (TNR) could be sustained at about 60mg/L, 80%, and over 70%, respectively, when the influent nitrogen load rate (NLR) is changed from 0.733 to 0.879kg-N/m3/day. Both ARE and TNR are decreased when NLR continues increasing to 1.026kg-N/m3/day. The influent NLR decreases from 0.879 to 0.147kg-N/m3/day, and ARE and TNR reached 98% and 85.4%, respectively. Therefore, the denitrification effect of the reactor could be recovered, and the excellent nitrogen removal capacity could be obtained within a wide range of influent NLR. Moreover, the high-throughput sequencing and metagenomic testing indicate that the Proteobacteria and Planctomycetes that the PN/A functional strains (i.e., ammonia-oxidizing bacteria (AOB) and anammox bacteria (AnAOB)) account for 38.8% in the sludge. The relative abundance of Nitrospira containing the nitrite-oxidizing bacteria (NOB) has dropped to 0.01%, and the functional gene nxr of the nitrite oxidation process is also inhibited. The relative expression of the functional gene is dominated by the short-range nitritation and anammox oxidation, which demonstrates that the nitrogen removal is mainly dominated by nitritation-anammox.

Customizable OpenGUS immunoassay: A homogeneous detection system using β-glucuronidase switch and label-free antibody

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a β-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction. To enhance the signal-to-background ratio of the immunoassay, a GUS switch mutant that exhibits reduced background activation was developed by screening several additional mutations at the diagonal interface residue H514. Moreover, we optimized the composition of the reaction buffer, including organic solvents, salt, and surfactant. Under optimal conditions, we customized OpenGUS immunoassays for Cry j 1, human C-reactive protein, and human lactoferrin, achieving around 10–20-fold maximum fluorescence (15 min) or colorimetric (2 h) responses with picomolar to low nanomolar level detection limit, simply by using commercially available IgGs. Additionally, the three analytes were successfully detected in complex matrices similar to those used in practical applications. We believe that this customizable OpenGUS immunoassay will pave the way for the prompt development of rapid and sensitive homogeneous immunoassays for point-of-care diagnostics, high-throughput testing, and onsite environmental assessments.

Genetic dissection of stem and leaf rachis prickles in diploid rose using a pedigree-based QTL analysis.

Prickles are often deemed undesirable traits in many crops, including roses (Rosa sp.), and there is demand for rose cultivars with no or very few prickles. This study aims to identify new and/or validate reported quantitative trait loci (QTLs) associated with stem and leaf rachis prickle density, characterize the effects of functional haplotypes for major QTLs, and identify the sources of QTL-alleles associated with increased/decreased prickle density in roses. QTL mapping using pedigree-based analysis (PBA), and haplotype analysis were conducted on two multi-parental diploid rose populations (TX2WOB and TX2WSE). Twelve QTLs were identified on linkage groups (LGs) 2, 3, 4, and 6. The major QTLs for the stem prickle density were located between 42.25 and 45.66 Mbp on chromosome 3 of the Rosa chinensis genome assembly, with individual QTLs explaining 18 to 49% of phenotypic variance (PVE). The remaining mapped QTLs were minor. As for the rachis prickle density, several QTLs were detected on LG3, 4, and 6 with PVE 8 to 17%. Also, this study identified that ancestors R. wichurana 'Basye's Thornless', 'Old Blush', and the pollen parent of M4-4 were common sources of favorable alleles (q) associated with decreased prickle density, whereas 'Little Chief' and 'Srche Europy' were the source of unfavorable alleles (Q) in the TX2WOB and TX2WSE populations, respectively. The outcomes of this work complement other studies to locate factors that affect prickle density. These results can also be utilized to develop high-throughput DNA tests and apply parental selection to develop prickle-free rose cultivars.

Chicoric acid acts as an ALOX15 inhibitor to prevent ferroptosis in asthma

BackgroundChicoric acid (CA) is a crucial immunologically active compound found in chicory and echinacea, possessing a range of biological activities. Ferroptosis, a type of iron-dependent cell death induced by lipid peroxidation, plays a key role in the development and advancement of asthma. Targeting ferroptosis could be a potential therapeutic strategy for treating asthma. PurposeThe purpose of this study was to explore the screening of ALOX15, a pivotal target of ferroptosis in asthma, and potential therapeutic agents, as well as to investigate the promising potential of CA as an ALOX15 inhibitor for modulating ferroptosis in asthma. MethodsThrough high-throughput data processing of bronchial epithelial RNA from asthma patients using bioinformatics and machine learning, the key target of ferroptosis in asthma, ALOX15, was identified. An inhibitor of ALOX15 was then obtained through high-throughput molecular docking and molecular dynamics simulation tests. In vitro experiments were conducted using a 16HBE cell model induced by house dust mite (HDM) and lipopolysaccharide (LPS), which were treated with the ALOX15 inhibitor (PD146176), CA treatment, or ALOX15 knockdown. In vivo experiments were also carried out using a mouse model induced by HDM and LPS. ResultsThe composite model of ALOX15 and CA in molecular dynamics simulations shows good stability and flexibility. Network pharmacological analysis reveals that CA regulates ferroptosis through ALOX15 in treating asthma. In vitro studies show that ALOX15 is highly expressed in HDM and LPS treatments, while CA inhibits HDM and LPS-induced ferroptosis in 16HBE cells by reducing ALOX15 expression. Knockdown of ALOX15 has the opposite effect. Metabolomics analysis identifies key compounds associated with ferroptosis, including L-Targinine, eicosapentaenoic acid, 16-hydroxy hexadecanoic acid, and succinic acid. In vivo experiments demonstrate that CA suppresses ALOX15 expression, inhibits ferroptosis, and improves asthma symptoms in mice. ConclusionOur research initially identified CA as a promising asthma treatment that effectively blocks ferroptosis by specifically targeting ALOX15. This study not only highlights CA as a potential therapeutic agent for asthma but also introduces novel targets and treatment options for this condition, along with innovative approaches for utilizing natural compounds to target diseases associated with ferroptosis.

Faster ciguatoxin extraction methods for toxicity screening

Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.

Evaluating Nanoparticulate Vaccine Formulations for Effective Antigen Presentation and T-Cell Proliferation Using an In Vitro Overlay Assay.

Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates' ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay's effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting Neisseria gonorrhoeae (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel® (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay's potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations.

Looping Flexible Fluoropolymer Microcapillary Film Extends Analysis Times for Vertical Microfluidic Blood Testing.

The microfluidic measurement of capillary flow can be used to evaluate the response of biological samples to stimulation, where distance and velocity are altered. Melt-extruded multi-bored microfluidic capillaries allow for high-throughput testing with low device cost, but simple devices may limit control over sample flow when compared to the more complex "lab-on-a-chip" devices produced using advanced microfluidic fabrication methods. Previously, we measured the dynamics of global haemostasis stimulated by thrombin by dipping straight vertical microcapillaries into blood, but only the most rapid response could be monitored, as flow slowed significantly within 30 s. Here, we show an innovative method to extend both the stimulation process and flow measurement time without increasing the cost of the device by adding simple loops to the flexible extruded device. The loops enable longer time-scale measurements by increasing resistance to flow, thereby reducing the dependence on high stimulus concentrations for rapid reactions. The instantaneous velocity and equilibrium heights of straight and looped vertical microcapillary films were assessed with water, plasma and whole blood, showing that the loops create additional frictional resistances, reduce flow velocity and prolong residence times for increased time scales of the stimulation process. A modified pressure balance model was used to capture flow dynamics with the added loop. Looped devices loaded with thrombin and collagen showed an improved detection of blood stimulation responses even with lower stimulus concentrations, compared to straight vertical capillaries. Thrombin-activated blood samples in straight capillaries provided a maximum measurement zone of only 4 mm, while the looped design significantly increased this to 11 mm for much longer time scale measurements. Our results suggest that extending stimulation times can be achieved without complex microfluidic fabrication methods, potentially improving concentration-response blood stimulation assays, and may enhance the accuracy and reliability. We conclude adding a loop to low-cost extruded microfluidic devices may bring microfluidic devices closer to delivering on their promise of widespread, decentralized low-cost evaluation of blood response to stimulation in both research and clinical settings.

Multiwell-based G0-PCC assay for radiation biodosimetry

In major radiological events, rapid assays to detect ionizing radiation exposure are crucial for effective medical interventions. The purpose of these assays is twofold: to categorize affected individuals into groups for initial treatments, and to provide definitive dose estimates for continued care and epidemiology. However, existing high-throughput cytogenetic biodosimetry assays take about 3 days to yield results, which delays critical interventions. We have developed a multiwell-based variant of the chemical-induced G0-phase Premature Chromosome Condensation Assay that delivers same-day results. Our findings revealed that using a concentration of phosphatase inhibitor lower than recommended significantly increases the yield of cells with highly condensed chromosomes. These chromosomes exhibited increased fragmentation in a dose-dependent manner, enabling to quantify radiation damage using a custom Deep Learning algorithm. This algorithm demonstrated reasonable performance in categorizing doses into distinct treatment groups (84% and 80% accuracy for three and four iso-treatment dose bins, respectively) and showed reliability in determining the actual doses received (correlation coefficient of 0.879). This method is amendable to full automation and has the potential to address the need for same-day, high-throughput cytogenetic test for both dose categorization and dose reconstruction in large-scale radiation emergencies.

A smartphone-based tunable tyrosinase functional mimic modulated portable amperometric sensor for the rapid and real-time monitoring of catechol

A smartphone-based electrochemical sensor for rapid detection of catechol (CC) was designed. The system contained a smartphone, a hand-held detector, and tyrosinase enzyme mimicking material modified screen-printed carbon electrode (SPCE). Metal nanoparticles confined in porous carbon have been widely used as enzyme mimics owing to their enhanced electrochemical activity. However, the fabrication of enzyme-mimicking catalysts in a scalable and tunable manner is challenging. A two-step synthesis strategy was followed: carbonization of the silica template to form cubic-structured mesoporous carbon (CMC) and high-temperature calcination for copper confinement into CMC (Cu–CMC). The non-covalent interactions enable the Cu precursors to coordinate with the carbon atoms in the face-centered cubic lattice of CMC. The uncalcined material is the as-synthesized (As–Cu–CMC), while the 900 °C calcined are stage 1 (Cu–CMCS1) and stage 2 (Cu–CMCS2) materials with different Cu loadings. The Cu–CMCS2 possess a large pore volume (0.224 cm3g−1) and high surface area (104.2 m2/g). The Cu–CMCS2 modulated sensors enable rapid and real-time detection of CC. Under optimized conditions, the linear range of 1.0–1000 µM (0.0 V vs. integrated Ag/AgCl reference electrode) with the sensitivity and lower detection limit of 10.21 µA µM−1 cm−2 and 0.099 μM (S/N=3) was achieved. The excellent sensor performance is attributed to the increased number of Cu vacancies that mimic the native tyrosinase enzyme. The sensor connected to a portable potentiostat enable high-throughput real-time testing of the CC without sophisticated equipment. Furthermore, the sensor detected CC in green tea and water samples, and the results were validated using UV–visible spectrophotometry and high-performance liquid chromatography.Abbreviations: CMC, cubic-structured mesoporous carbon; Cu–CMCS2, copper loaded stage 2 material; CC, catechol; CMS, cubic mesoporous silica; SPCE, screen-printed carbon electrode; HF, hydrofluoric acid; P123, Pluronic 123; TEOS, Tetraethyl orthosilicate; AA, ascorbic acid; Glu, glucose; HQ, hydroquinone; 4-NP, 4-nitrophenol; CA, caffeic acid; RS, resorcinol; Cu–CMCS1, copper loaded stage 1 material; UV–Vis, UV–visible spectrophotometry; FE-SEM, field-–emission scanning electron microscopy; EDS, energy–dispersive X–-ray spectroscopy; Si, silica; TEM, transmission electron microscopy; NPs, nanoparticles; SAED, selected area electron diffraction; FT-IR, Fourier-Transform Infrared Spectroscopy; BET, Brunauer–Emmett–Teller; BJH, Barrett–Joyner–Halenda; TGA, Thermogravimetric analysis; CV, cyclic voltammetry; ipa, anodic peak current; ECSA, electrochemically active surface area; EIS, electrochemical impedance spectroscopy; Rct, charge-transfer resistance; CA, chronoamperometry; LOD, limit of detection.

(Invited) Targeted Research to Develop Next Generation PEM Water Electrolyzers

The utilization of low-carbon intensity hydrogen as an energy carrier promises a path to the decarbonization of sectors including electricity, heavy-duty transportation, and industry at scale. The United States Department of Energy is investing into this opportunity by heavily supporting research and development efforts for hydrogen production at scale. In recent years, the DOE has established the H2NEW consortium which consists of nine national labs and additional partners to focus on the advancement of water electrolysis. It also launched the Hydrogen Energy Earthshot in 2021, targeting the reduction of clean hydrogen production cost by 80% from $5 to $1 per 1 kg H2 produced in 1 decade. For these efforts to be successful, techno-economic analyses are used to identify the research and development areas with the greatest impact, supporting targeted studies on scalable manufacturing and materials that lower capital costs, produce hydrogen efficiently, and enable durable operation over the system lifetime.In this presentation the challenges and trade-offs of electrolyzer operation for renewable hydrogen generation will be reviewed, and results from targeted NREL studies on components, materials and interfaces will be discussed in detail, including for example PTL morphology, PTL coatings, and PTL/catalyst layer interactions and their effect on operation with thin membrane materials, hydrogen crossover and voltage loss contributions. The presentation also touches on any advanced in-situ diagnostic development that was required for the studies, such as high pressure operation, hydrogen crossover quantification, high throughput testing, and spatial diagnostics.

Stability of Bipolar Plates in Proton Exchange Membrane Water Electrolysis: Dissolution Studies of Titanium and Stainless Steel

Due to its high current densities and fast system response proton exchange membrane water electrolysis (PEMWE) is an ideal system for green hydrogen production from renewable energies. However, its widespread use is still hindered by the high costs rendering the produced green hydrogen not competitive. Therefore, a cost reduction is highly needed. The bipolar plates (BPPs) are causing a colossal factor in material costs. They are usually produced from titanium and coated with even more expensive materials such as gold or platinum. A possible, cheaper alternative is stainless steel. However, it is suspected to suffer from corrosion leading to the leaching of cations that can poison the catalyst or membrane.[1] Thus, we investigated the stability of BPP materials, the currently used titanium, and its cheaper alternative stainless steel (316L).[2] Therefore, we use a modified scanning flow cell setup coupled to an inductively coupled plasma mass spectrometer for investigating the stability of both materials on-line, as well as with scanning electron microscopy. To mimic the conditions at the BPP under operation, deionized (DI) water and highly diluted (0.5 mM) H2SO4 are chosen as electrolytes combined with a sample temperature of 60 °C.For Ti, we show that the dissolution is negligible, whereas 316L corrodes. For 316L, in DI water mainly Cr dissolves, while in the H2SO4 solution, the dissolution is more pronounced, and all alloy components are detected. Besides the pH value, the applied potentials play a crucial for its stability. However, our findings suggest that the contamination for a typical full cell architecture remains below 1 ppm, even for the highest measured dissolution rate of SS 316L.These capabilities of on-line high-throughput stability tests for BPP materials are, furthermore, used to investigate the influence of contaminations, such as F- or Cl-, and of the temperature-dependence on the stability of 316L. These results give further insights into 316L stability as BPP material and therefore contribute to the development towards cost-effective PEMWE.[1] a) N. Rojas, M. Sánchez-Molina, G. Sevilla, E. Amores, E. Almandoz, J. Esparza, M. R. Cruz Vivas, C. Colominas, Int. J. Hydrogen Energy 2021, 46, 25929-25943; b) H. Becker, J. Murawski, D. V. Shinde, I. E. L. Stephens, G. Hinds, G. Smith, Sustain. Energy Fuels 2023, 7, 1565-1603.[2] L. Fiedler, T. C. Ma, B. Fritsch, J. H. Risse, M. Lechner, D. Dworschak, M. Merklein, K. J. J. Mayrhofer, A. Hutzler, ChemElectroChem 2023, e202300373. Figure 1

Microdissected tumor cuboids: a microscale cancer model for large-scale testing that retains a complex tumor microenvironment.

Current cancer disease models fail to faithfully recapitulate key features of the human tumor microenvironment (TME), such as immune and vascular cells, while simultaneously enabling high-throughput drug tests. We have recently developed a precision slicing method that optimizes the yield of large numbers of cuboidal microtissues ("cuboids", ∼(400 µm) 3 ) from a single tumor biopsy. Here we demonstrate that cuboids from syngeneic mouse tumor models and human tumors retain a complex TME, making them amenable for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as cellular architecture, cytokine secretion, proteomics profiles, and response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong cytokine and drug responses, including to immunotherapy. Overall, our results suggest that cuboids could provide invaluable therapeutic information for personalized oncology applications, and could help the development of TME-dependent therapeutics and cancer disease models, including for clinical trials.

Plants Restoration Drives the Gobi Soil Microbial Diversity for Improving Soil Quality.

Desertification and salt stress are major causes of terrestrial ecosystem loss worldwide, and the Gobi, representing a salt-stressed area in inland China, has a major impact on the ecosystems and biodiversity of its surrounding environment. The restoration of the Gobi Desert is an important way to control its expansion, but there are few studies on the evaluation of restoration. In this study, soils under different restoration scenarios, namely, soils in restored areas (R1, R2), semi-restored areas (SR1, SR2), and unrestored control areas (C1, C2), were used to investigate differences in microbial diversity and physicochemical properties. The results showed that the soil was mainly dominated by particles of 4-63 μm (26.45-37.94%) and >63 μm (57.95-72.87%). Across the different restoration levels, the soil pH (7.96-8.43) remained basically unchanged, salinity decreased from 9.23-2.26 to 0.24-0.25, and water content remained constant (10.98-12.27%) except for one restored sample in which it was higher (22.32%). The effective Al, Cu, and Zn in the soil increased, but only slightly. Total organic matter (TOM) decreased from 3.86-5.20% to 1.31-1.47%, and total organic nitrogen (TON) decreased from 0.03-0.06% to 0.01-0.02%, but the difference in total organic carbon (TOC) was not significant. High-throughput testing revealed that the bacterial population of the restored area was dominated by A4b (6.33-9.18%), MND1 (4.94-7.39%), and Vicinamibacteraceae (7.04-7.39%). Regarding archaea, samples from the restored areas were dominated by Marine Group II (76.17-81.49%) and Candidatus Nitrososphaera (6.07-9.75%). PCoA showed that the different restoration levels were the main cause of the differences between the samples. Additionally, salinity was the dominant factor that induced this difference, but it was inhibited by the restoration and targeted enrichment of some of these functional genera. Desert restoration should therefore focus on conserving water rather than adding nutrients. Planting salt- and drought-tolerant vegetation will contribute to the initial restoration of the desert and the restoration of the microbiological content of the soil as it migrates over time, creating a cycle of elements. Restoration stimulates and enhances the microbial diversity of the soil via beneficial microorganisms.