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At least 19 recordsLinked to original sources

Microfluidics to Follow Spatiotemporal Dynamics at the Nucleo-Cytoplasmic Interface During Plant Root Growth.

Nuclear dynamics refers to global/local changes in the molecular and spatial organization of genomic DNA that can occur during development or in response to environmental stress signals and eventually impact genomic functions. In plants, nuclear dynamics relies notably on the connection of the nucleus with the cytoskeleton during development. It orchestrates genomic functions in response to developmental and environmental cues. This is particularly true in the plant root system, which is constantly exposed to a wide range of internal and external stimuli. Currently, studying nuclear dynamics in a growing root is challenging due to limitations regarding real-time imaging for quantitative analyses under controlled conditions. Microfluidic systems for plant cell studies are valuable analytical tools that provide precise control of culture conditions together with live-imaging capabilities at high temporal and spatial resolutions. Herein, we describe a microfluidic platform to unravel dynamically and noninvasively nuclear organization in the seedling root system exposed to various treatments. As exemplified here, our microfluidic platform can be conveniently used for real-time microscopy imaging and quantitative analysis of fine nuclear morphological changes upon modifying cytoskeleton dynamics. Importantly, our system can be applied to a wide variety of microscopic means including high-resolution microscopy to investigate diverse subcellular compartments or nuclear domains in Arabidopsis thaliana roots.

Plant Roots

Multiplexed microfluidic chip for cell co-culture.

Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually-addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1 h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.

Microfluidics

Unlocking Zeptomolar Single-Molecule Detection by Synergizing Digital Microfluidics and Digital CRISPR.

Accurate diagnosis relies on the highly sensitive and quantitative detection of multiple immune-related biomarkers. However, current detection methods still face significant limitations in sensitivity, specificity, and background signal control. Here, we introduce DDA (Dual-Digital immunoAssay), a fully automated, universal immunoassay platform that synergizes digital microfluidics with digital Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based amplification. This "dual-digital" strategy pushes the detection limit into the zeptomolar (zM) regime, enabling unprecedented sensitivity for single-molecule analysis. The DDA platform is built upon a digital microfluidic microwell array chip, integrating magnetic bead-based immunocapture with RNA-guided CRISPR/Cas13a signal amplification. This system enables a fully automated, "sample-in, answer-out" workflow. By systematically optimizing the entire process, DDA significantly reduces background noise and enhances detection sensitivity, achieving a limit of detection (LOD) down to 100 zM for key protein biomarkers. This represents a >100-fold improvement over leading commercial ultrasensitive assays. With single-molecule resolution and full automation, DDA provides a robust solution for the precise quantification of low-abundance immune biomarkers. As a proof-of-concept, we demonstrate its ability to accurately quantify key heart-failure-associated biomarkers, including NT-proBNP (LOD: 1 aM), IL-6 (LOD: 1.5 aM), and TNF-α (LOD: 2.5 aM), directly in complex serum samples. This platform holds great promise for automated multibiomarker screening and risk assessment, showcasing its powerful potential for the early diagnosis of major diseases such as cardiovascular diseases, cancers, neurodegenerative disorders, and infectious diseases.

Humans

Liquid biopsy-based detection of circulating and exfoliated cholangiocarcinoma tumor cells from blood and bile using heparan sulfate octasaccharides on integrated microfluidic systems.

Early diagnosis of cholangiocarcinoma (CCA) remains challenging because existing diagnostic approaches often lack sufficient sensitivity for reliable detection of early-stage disease. Circulating tumor cells (CTCs) in blood and exfoliated tumor cells (ETCs) in bile represent valuable targets for liquid biopsy-based detection; however, their low abundance and the complexity of clinical sample analysis pose substantial technical challenges for reliable enrichment and identification. Herein, we present a reproducible workflow for isolating and identifying CCA tumor cells from blood for CTCs and bile for ETCs using synthetic cell-surface heparan sulfate (HS) octasaccharide-functionalized magnetic beads (MBs) on integrated microfluidic systems. The method combined sample pre-processing, magnetic bead-based enrichment, controlled low-shear mixing and immunofluorescence-based identification into a unified workflow compatible with distinct clinical sample types. Key operational parameters, including MB concentration, mixing frequency, and pressure settings, were detailed to facilitate consistent performance. Using this workflow, tumor cell capture rates of approximately 70% in bile (for ETCs) and blood (for CTCs) were achieved, with a total processing time of 60-90 min per sample under clinically relevant low-abundance conditions. The platform enables reliable detection of as few as 1 tumor cell per mL of blood or bile. This method provides a practical and adaptable strategy for glycosaminoglycan-mediated liquid biopsy applications and may be extended to other tumor-cell enrichment workflows involving heterogeneous cell-surface interactions.

Humans

Measuring single-cell susceptibility to antibiotics within monoclonal bacterial populations.

The emergence of new resistant bacterial strains is a worldwide challenge. A resistant bacterial population can emerge from a single cell that acquires resistance or persistence. Hence, new ways of tackling the mechanism of antibiotic response, such as single cell studies are required. It is necessary to see what happens at the single cell level, in order to understand what happens at the population level. To date, linking the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics remains challenging due to the trade-offs between the resolution and the field of view. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.

Anti-Bacterial Agents

Iron-Deprivation Liposomes for Cancer Therapy.

Targeting iron homeostasis in tumor cells represents a promising anticancer strategy, as iron plays essential roles in tumor growth, invasion, and metastasis. Although deferoxamine can effectively chelate iron, its clinical application is limited by poor membrane permeability, short half-life, and lack of targeting capability. To overcome these challenges, we designed and synthesized a family of DFO-bearing lipids by modifying the molecule with dual fatty acid chains of varying lengths, and co-assembled them with auxiliary lipids via microfluidics to construct "iron-deprivation" liposomes. Among them, the medium-chain DFO-C12-liposomes exhibited the highest cellular uptake, iron-deprivation efficiency, and anticancer activity in vitro, markedly depleting mitochondrial iron, disrupting Fe-S cluster synthesis, suppressing mitochondrial respiration, and inducing autophagy. Furthermore, DFO-C12-liposomes efficiently coordinated Mn2 + via DFO-Mn2 + chelation, providing MRI capability while inducing iron deprivation-mediated ferroptosis. In addition, the iron-deprivation liposomes can encapsulate anticancer drugs such as doxorubicin, leading to an enhanced antitumor effect through the combination of iron deprivation and chemotherapy for osteosarcoma treatment. In summary, the "iron-deprivation" liposomes integrate iron chelation, imaging functionality, and chain-length-dependent cellular uptake into a versatile nanoplatform for regulating tumor iron homeostasis and achieving enhanced antitumor efficacy through multimodal therapeutic strategies.

alkyl chain‐length engineering

Phase Transition of Wax Enabling CRISPR Diagnostics for Automatic At-Home Testing of Multiple Sexually Transmitted Infection Pathogens.

Sexually transmitted infections (STIs) significantly impact women's reproductive health. Rapid, sensitive, and affordable detection of these pathogens is essential, especially for home-based self-testing, which is crucial for individuals who prioritize privacy or live in areas with limited access to healthcare services. Herein, an automated diagnostic system called Wax-CRISPR has been designed specifically for at-home testing of multiple STIs. This system employs a unique strategy by using the solid-to-liquid phase transition of wax to sequentially isolate and mix recombinase polymerase amplification (RPA) and CRISPR assays in a microfluidic chip. By incorporating a home-built controlling system, Wax-CRISPR achieves true one-pot multiplexed detection. The system can simultaneously detect six common critical gynecological pathogens (CT, MG, UU, NG, HPV 16, and HPV 18) within 30 min, with a detection limit reaching 10-18 M. Clinical evaluation demonstrates that the system achieves a sensitivity of 96.8% and a specificity of 97.3% across 100 clinical samples. Importantly, eight randomly recruited untrained operators performe a double-blinded test and successfully identified the STI targets in 33 clinical samples. This wax-transition-based one-pot CRISPR assay offers advantages such as low-cost, high-stability, and user-friendliness, making it a useful platform for at-home or field-based testing of multiple pathogen infections.

Sexually Transmitted Diseases

Identification of genetic determinants that promote biofilm growth under heterotrophic conditions in Cupriavidus necator using transposon enrichment.

Cupriavidus necator is a metabolically versatile β-proteobacterium of growing interest for auto- and heterotrophic bioprocesses, yet the genetic determinants governing its biofilm formation remain largely uncharacterized, particularly under process-relevant heterotrophic conditions. Here, we applied a forward-genetics transposon-enrichment approach to identify loci which promote surface-associated growth. A high-density mini-Tn5 mutant library (26,185 insertion clones, exceeding the >17,000 required for genome-wide coverage) was cultivated as a biofilm in a microfluidic flow-cell system on fructose for 168 h, and the surface-associated community was characterized by deep sequencing. Twelve genes showed significantly elevated insertion frequencies, several with documented links to biofilm formation in other bacteria, including the ferrous-iron uptake system (feoA/feoB), galU, and a GSDEF/EAL dual-domain protein. The gene B2043 (E6A55_RS29530), encoding this c-di-GMP-metabolizing protein, was selected for validation by markerless deletion. Under static conditions, the ΔB2043 mutant showed a 1.69 ± 0.06-fold increase in biofilm-associated biomass (p = 5.16 × 10-15). Under flow-through conditions, the mutant attached faster, entered exponential growth ∼10 h earlier, reached its biovolume plateau ∼16 h earlier than the wild-type, and formed distinct tower-like structures. These results identify B2043 as a negative regulator of biofilm formation acting predominantly during attachment, provide the first experimental evidence for c-di-GMP-dependent biofilm regulation in C. necator H16, and establish a functional-genomics framework - together with eleven further candidate loci - for engineering productive biofilms in this organism.

Biofilm formation

Recent advances in environmental antibiotic resistance genes detection and research focus: From genes to ecosystems.

Antibiotic resistance genes (ARGs) persistence and potential harm have become more widely recognized in the environment due to its fast-paced research. However, the bibliometric review on the detection, research hotspot, and development trend of environmental ARGs has not been widely conducted. It is essential to provide a comprehensive overview of the last 30 years of research on environmental ARGs to clarify the changes in the research landscape and ascertain future prospects. This study presents a visualized analysis of data from the Web of Science to enhance our understanding of ARGs. The findings indicate that solid-phase extraction provides a reliable method for extracting ARG. Technological advancements in commercial kits and microfluidics have facilitated the efficacy of ARGs extraction with significantly reducing processing times. PCR and its derivatives, DNA sequencing, and multi-omics technology are the prevalent methodologies for ARGs detection, enabling the expansion of ARG research from individual strains to more intricate microbial communities in the environment. Furthermore, due to the development of combination, hybridization and mass spectrometer technologies, considerable advancements have been achieved in terms of sensitivity and accuracy as well as lowering the cost of ARGs detection. Currently, high-frequency terms such as "Antibiotic Resistance, Antibiotics, and Metagenomics" are the center of attention for study in this area. Prominent topics include the investigation of anthropogenic impacts on environmental resistance, as well as the dynamics of migration, dissemination, and adaptation of environmental ARGs, etc. The research on environmental ARGs has made significant advancements in the fields of "Microbiology" and "Biotechnology Applied Microbiology". Over the past decade, there has been a notable increase in the fields of "Environmental Sciences Ecology" and "Engineering" with a similar growth trend observed in "Water Resources". These three domains are expected to continue driving extensive study within the realm of environmental ARGs.

Drug Resistance, Microbial

Decoding the spatiotemporal patterns of food spoilage microbial communities: Integrating multi-omics and artificial intelligence to enable precision preservation.

In the global food supply chain, food wastage caused by spoilage has resulted in significant economic losses, food shortages, and environmental pressure. This process is fundamentally driven by the spatiotemporal dynamics of microbial communities. However, traditional research methods struggle to elucidate the complex mechanisms of spatial heterogeneity, interspecies interactions, and functional succession. This limits the development of effective preservation strategies. This review systematically reviews the cutting-edge progress of integrating multi-omics technologies and artificial intelligence (AI) to study food spoilage microbial communities, breaking through this bottleneck. We propose an intelligent theoretical framework that could potentially analyze microbial metabolic activities and predict dynamic shelf life if implemented. The conceptual framework integrates multidimensional data, including spatial metabolomics, temporal metatranscriptomics, single-cell transcriptomics, and longitudinal metagenomics. It can also be combined with AI models, such as graph neural networks. The article elaborates on the principles and applications of spatio-temporal monitoring technologies, such as nano secondary ion mass spectrometry, hyperspectral imaging, and the Internet of Things sensing. Through illustrative cases of typical perishable foods, it also explores how such a multi-omics - AI system might be applied to spoilage warning and precise intervention. Additionally, the article addresses the current challenges in data coverage, model generalization, and federated learning implementation. Then the research further explores emerging areas such as engineered probiotics, edge AI, and microfluidic sensing. These areas are targeted at transforming food preservation from an empirical control approach to a data-driven, precise regulatory framework. This transformation provides theoretical support and technical approaches for developing a smart, sustainable food preservation system.

Multiomics

Biochemical and Structural Analyses of the Tardigrade DNA-Damage Suppressor Protein, Dsup.

Tardigrades are extremophiles that withstand harsh environments through unique molecular strategies. One such strategy involves Damage Suppressor (Dsup), a protein shown to protect cells from radiation-induced DNA damage. Little is known about the biochemical and structural characteristics of Dsup that lead to DNA protection. To gain insight into the mechanism of DNA protection by Dsup, we examined its fundamental biochemical and structural properties using mass photometry, biolayer interferometry, small-angle X-ray scattering, and microfluidic modulation spectroscopy. We found that Dsup is largely intrinsically disordered and binds DNA with high affinity via a multi-valent interface. This interaction induced conformational changes in both Dsup and the DNA, suggesting a potential structural mechanism of its DNA protection ability. We propose that Dsup alters DNA structure, possibly by partially unwinding it, to reduce its susceptibility to damage. These findings offer new insights into how a disordered protein such as Dsup functions as radioprotectants in extreme environments.

Tardigrada

Proteome Dynamics in iPSC-Derived Human Dopaminergic Neurons.

Dopaminergic neurons participate in fundamental physiological processes and are the cell type primarily affected in Parkinson's disease. Their analysis is challenging due to the intricate nature of their function, involvement in diverse neurological processes, and heterogeneity and localization in deep brain regions. Consequently, most of the research on the protein dynamics of dopaminergic neurons has been performed in animal cells ex vivo. Here we use iPSC-derived human mid-brain-specific dopaminergic neurons to study general features of their proteome biology and provide datasets for protein turnover and dynamics, including a human axonal translatome. We cover the proteome to a depth of 9409 proteins and use dynamic SILAC to measure the half-life of more than 4300 proteins. We report uniform turnover rates of conserved cytosolic protein complexes such as the proteasome and map the variable rates of turnover of the respiratory chain complexes in these cells. We use differential dynamic SILAC labeling in combination with microfluidic devices to analyze local protein synthesis and transport between axons and soma. We report 105 potentially novel axonal markers and detect translocation of 269 proteins between axons and the soma in the time frame of our analysis (120 h). Importantly, we provide evidence for local synthesis of 154 proteins in the axon and their retrograde transport to the soma, among them several proteins involved in RNA editing such as ADAR1 and the RNA helicase DHX30, involved in the assembly of mitochondrial ribosomes. Our study provides a workflow and resource for the future applications of quantitative proteomics in iPSC-derived human neurons.

Humans

Engineering extracellular vesicles for targeted siRNA delivery: Advances, therapeutic applications, and clinical translation.

Small interfering RNA (siRNA) therapeutics have emerged as a transformative approach for sequence-specific gene silencing, offering the potential to treat a broad spectrum of diseases by selectively suppressing disease-associated genes. However, the clinical translation of siRNA remains limited by rapid enzymatic degradation, poor cellular uptake, inadequate endosomal escape, and off-target effects, necessitating the development of efficient delivery systems. Extracellular vesicles (EVs) have gained considerable attention as natural nanocarriers owing to their excellent biocompatibility, low immunogenicity, intrinsic targeting capability, and ability to protect therapeutic cargo while traversing complex biological barriers. This review comprehensively discusses the biological characteristics of EVs, the molecular basis of RNA interference, and the major challenges associated with siRNA delivery [Fig. 1]. Recent advances in EV engineering, including cargo-loading strategies such as electroporation, sonication, extrusion, parent-cell engineering, and microfluidic approaches, together with surface functionalization using peptides, antibodies, aptamers, and hybrid nanoplatforms, are critically evaluated for improving targeting specificity and intracellular delivery. Furthermore, the therapeutic applications of engineered EV-mediated siRNA delivery in cancer, neurological disorders, liver diseases, cardiovascular diseases, inflammatory disorders, and infectious diseases are systematically summarized, highlighting their potential to enhance gene silencing while minimizing systemic toxicity. Current challenges related to large-scale manufacturing, cargo-loading efficiency, standardization, quality control, regulatory approval, and clinical translation are also discussed, together with emerging technologies involving synthetic biology, genome engineering, artificial intelligence, and multifunctional hybrid vesicles. Overall, engineered extracellular vesicles represent a highly versatile and biologically inspired platform for targeted siRNA delivery, providing a promising foundation for the development of next-generation precision RNA therapeutics and accelerating the clinical translation of gene-silencing strategies.

Extracellular vesicle engineering

Single-cell proteomics using mass spectrometry.

Over the past 2 to 3 years, mass-spectrometry-based single-cell proteomics (SCP) has experienced transformative improvements in microfluidic and robotic sample preparation, innovative MS1- and MS2-based multiplexing strategies, and specialized hardware (e.g., timsTOF Ultra 2, Astral), which have dramatically boosted sensitivity, throughput, and proteome coverage from picogram-level protein inputs. Concurrently, tailored computational workflows that encompass normalization, imputation, and no-code platforms have addressed pervasive missing data challenges and standardized analyses, collectively enabling high-throughput, reproducible profiling of cellular heterogeneity. This minireview summarizes the latest progress in SCP technology and software solutions, highlighting how the closer integration of analytical, computational, and experimental strategies will facilitate a deeper and broader coverage of single-cell proteomes.

Single-Cell Analysis

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals

Microdroplet-based high-throughput screening for antagonistic bacteria targeting penaeid shrimp pathogenic Vibrio harveyi.

Antagonistic bacteria that suppress the growth of specific bacteria have attracted attention as an antibiotics-independent strategy for infectious disease control in aquaculture. Microfluidics-based water-in-oil droplets (microdroplets) enable high-throughput screening of antagonistic bacteria in the field of medicine or agriculture. However, the use of this screening system in aquaculture has not yet been reported. In particular, penaeid shrimp aquaculture, one of the largest sectors of global aquaculture, has a strong demand for alternative disease control strategies because vaccination is ineffective. Here, we demonstrated a proof-of-concept study of microdroplet-based high-throughput screening system for antagonistic bacteria targeting penaeid shrimp pathogenic Vibrio harveyi. Using this screening system, we successfully isolated 18 bacterial candidates with potential growth-inhibitory activity, representing three genera (Pseudoalteromonas, Shewanella, and Tenacibaculum), from the bacterial community of kuruma shrimp Penaeus japonicus rearing water. 16S rRNA gene-based bacterial community analysis revealed that these isolates included several low-abundance, rare taxa. Although these isolates showed no inhibitory activity on agar plates, one out of four tested strains showed a trend toward improved survival during co-infection tests using kuruma shrimp. Overall, our study highlights both the potential and limitation of microdroplet-based antagonistic bacterial screening to accelerate the development of biological control strategies in shrimp aquaculture.

Animals

The Gyrolab platform for immunogenicity assessment and biotherapeutic and biomarker analysis: technical advances and bioanalytical applications.

Gyrolab is an automated ligand‑binding assay platform designed for the quantification of proteins with high sensitivity and broad dynamic range. Its low minimum required dilution and microfluidic disc format enable efficient sample processing while maintaining assay robustness, making it a valuable tool across drug‑development stages. This review summarizes published applications of Gyrolab for pharmacokinetic and toxicokinetic analysis and immunogenicity assessment through anti‑drug antibody detection. In addition, the platform's high-volume assay discs have facilitated its use in biomarker studies, allowing quantification of low‑abundance analytes. Representative examples from the literature are presented together with key assay elements, including analytes, matrices, dynamic ranges, and critical reagents. Because assay replicates remain an important consideration for Gyrolab workflows, recent publications addressing singlet versus duplicate strategies and their impact on data interpretation are also discussed. Finally, we provide real analytical data visualized using a three‑dimensional Gyrolab viewer to illustrate variability in duplicate measurements and to highlight future opportunities for improving assay reliability.

Humans

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article