Rapid and quantitative detection of low-abundance pathogens is critical for clinical diagnosis and disease surveillance. Digital recombinase polymerase amplification (dRPA) is a promising technique for rapid pathogen quantification. However, existing dRPA methods are limited by pre-amplification artifacts introduced during sample digitization. To address this, we developed a controllable initiation dRPA (ci-dRPA) method by incorporating photocleavable modifications into caged primers. Amplification is triggered only after complete sample digitization, when the caged primers are cleaved by UV light, thereby eliminating pre-amplification and ensuring synchronized reaction initiation. The feasibility, accuracy, and specificity of ci-dRPA were validated using a self-driven digital microfluidic (SDM) chip. This method achieves single-molecule digitalization and enables quantification across a wide dynamic range of 5-3 & times; 104 copies/mu L, with the entire process completed within 40 min. When tested on clinical oral pathogen samples, ci-dRPA showed high concordance with qPCR results. This work provides a generalizable solution to the challenges of dRPA initiation and offers a simple, rapid, and accurate platform for low-abundance nucleic acid detection.

Spinal cord injury (SCI) leads to significant locomotor, sensory, and autonomic deficits and remains refractory to current therapies. Extracellular vesicles from mesenchymal stromal cells (MSC-EVs) are potential acellular therapeutic strategy for tissue regeneration, but rapid clearance and poor lesion retention limit their efficacy; combining MSC-EVs with biomaterial depots can improve local retention and sustained release. Here, we engineered porous gelatin methacryloyl (GelMA) microspheres using a self-designed simple microfluidic device. MSC-EVs were physically loaded into porous GelMA microspheres, which were subsequently embedded into a GelMA scaffold to form GelMA microsphere-embedded GelMA scaffolds loaded with MSC-EVs (GMS-EVs scaffolds). Based on the satisfactory elastic modulus, suitable porous organized structure, and good biocompatibility, the GMS-EVs scaffolds could achieve local retention and sustained release of MSC-EVs both in vivoand in vitro. Implantation of GMS-EVs scaffolds showed significantly improved motor recovery, reduced glial scar formation, and enhanced neuronal regeneration compared to both SCI and GelMA-only controls in a rat spinal cord hemisection model. Mechanistically, these beneficial effects of GMS-EVs scaffolds were associated with the stimulation of the phosphoinositide 3-kinase/protein kinase B (PI3K-AKT) and extracellular signal-regulated kinase (ERK) signaling pathways in SCI rats. Overall, this study highlights the potential of scaffolds based on porous GelMA microspheres combined with MSC-EVs to enhance tissue repair in the injured spinal cord environment.

Recent studies on ionic diodes for ion transport regulation have shown promising functions with high rectification ratios. However, typical ionic diodes face challenges in attaining high output efficiency and ion flux due to their requirement of nanoscale dimensions for ion selectivity. In this article, we introduce a novel bipolar ionic diode constructed through the combined assembly of nanoparticles and a transformable polyelectrolyte network. Experimental and numerical studies were conducted to characterize and optimize the fabrication parameters, resulting in a remarkable rectification ratio exceeding 1000. The presented ionic diode possesses the unique capability to automatically transform from a dense to a loose structure in response to an applied potential bias. This feature enables high output efficiency and rapid ion accumulation. Leveraging these characteristics, we developed a fluorescence-based sensor with ultrahigh sensitivity for nickel ions, achieving a detection limit of 1 nM with a minimal sample volume of 50 μL─an improvement of 2 orders of magnitude over conventional bulk solutions with the same fluorescence indicator. This ionic diode demonstrates exceptional ability and flexibility in high-efficiency ion transport, positioning itself as a promising platform with broad applications for enhancing sensitivity in various fluorescence-based sensing applications within aqueous environments.

BACKGROUND:spinal cord injury (SCI) causes irreversible motor and sensory deficits with limited effective treatments. Mesenchymal stromal cells (MSCs) exert therapeutic effects largely through extracellular vesicles (EVs). Preconditioning MSCs with a hydrogen sulfide (H2S) donor enhance the therapeutic potential of EVs. OBJECTIVE:this study is aimed to develop a 3D-printed gelatin methacryloyl (GelMA) scaffold loaded with H2S-preconditioned MSC-derived EVs (H2S-EVs) to promote motor function recovery in SCI. METHODS:H2S-EVs were isolated from NaHS (an H2S donor)-preconditioned MSCs and incorporated into a 3D-printed GelMA scaffold (3D/GelMA/EVs). Scaffold mechanical properties and H2S-EVs. The scaffold's therapeutic efficacy was evaluated in a rat SCI model. RESULTS:MiRNA microarray revealed miR-7a-5p as the most upregulated miRNA in H2S-EVs. The 3D/GelMA/EVs scaffold exhibited an appropriate elastic modulus and porous structure, enabling sustained local EVs release. In vivo, the scaffold significantly improved motor function recovery in SCI rats. CONCLUSION:these results indicated that H2S-EVs provided an important therapeutic tool against SCI by miR-7a-5p and 3D/GelMA/EVs scaffolds were ideal biomaterials for the intervention of SCI.

Exosomes have the potential to be a noninvasive tumor biomarker for cancer diagnosis and classification. However, high‐efficiency capture and analysis of exosomes in complex biological samples remain challenging. Here, we propose a high‐throughput, rapid, ultrasensitive, and low‐detection approach based on a spatially patterned antibody barcodes for quantitative analysis of exosomes. The combination of carbon dots (CDs) self‐assembly substrate and microfluidic technology enables the patterning of antibody barcodes to capture exosomes. Then, the fluorescently labeled detection antibody CD63 is to react with the surface exosomal antigen CD63. The double‐positive detection approach not only recognizes the identification of exosomes but also demonstrates that the exosomes express the targeted membrane marker. Based on the serum exosome detection results, it achieves 100% accuracy to differentiate ovarian cancer patients from healthy donors, and 90% accuracy in patient subgroup distribution. At the same time, the detection approach has a low detection limit of 65 particles/μL. The technology of spatially patterned antibody barcodes is promising in biology studies, early disease diagnosis, and new biomarker screening.

Growing evidence implicates the brain-gut axis in depression pathogenesis, though the underlying mechanisms remain elusive. This study investigated the antidepressant potential of rifaximin, a non-absorbable antibiotic, and its mechanisms via the brain-gut axis in a rat chronic unpredictable mild stress (CUMS) model. We found that CUMS induced anxiety- and depression-like behaviors, impaired colonic endocrine cell function, and downregulated neuropeptide Y (NPY) expression in both the colon and medial prefrontal cortex (mPFC). CUMS also altered neuronal activation and disrupted key neurotransmitter (GABA, Glu, 5-HT) balance in the mPFC. Rifaximin treatment ameliorated these behavioral deficits, restored colonic endocrine function, and increased NPY levels in both the colon and mPFC. Furthermore, it normalized CUMS-induced alterations in neuronal activation and neurotransmitter balance. Crucially, functional knockdown of NPY in the mPFC not only reduced colonic NPY expression in control rats but also abolished the anxiolytic effects of rifaximin in CUMS-treated rats. In summary, this study suggests that in the CUMS model, rifaximin can play an anxiolytic and antidepressant effect, and its mechanism may be related to the rifaximin’s regulation on NPY mediated gut-brain axis between colon and mPFC.

Early and accurate detection of Alzheimer's disease (AD) is demanding for timely interventions to slow disease progression. Graphene field-effect transistors (GFETs) are promising in AD biosensors due to their surface sensitivity, nonlabeling, and quick signal detection. Here, we developed a dual-channel GFET integrated with Galinstan liquid-metal electrodes. These electrodes establish van der Waals contacts with graphene, simplifying the manufacturing process by eliminating the need for high temperatures or significant energy inputs. This liquid metal electrodes reduce channel damage and significantly boost charge carrier mobility, with electron mobility reaching 5320 cm2·V-1·s-1 and hole mobility at 5572 cm2·V-1·s-1, surpassing traditional gold electrodes. The enhanced electrical performance of the Liquid metal GFET (LM-GFET) facilitates the highly sensitive and specific detection of Alzheimer's disease (AD) biomarkers. Its dual-channel configuration allows for the simultaneous detection of amyloid-β (Aβ) peptides Aβ40 and Aβ42, and the calculation of the Aβ42/Aβ40 ratio, providing a reliable marker for early stage AD diagnosis. The demonstrated robustness, stability, and improved performance position the LM-GFET as an effective tool for noninvasive AD diagnostics, underscoring its potential in advancing graphene-based biosensors for clinical use.

Extracellular vesicles (EVs) is promising in predicting the efficacy of immune checkpoint inhibitor (ICI) therapies. But it is challenging to determine the level of circulating EVs due to their variations in spatial and temporal distribution. To address this, we developed an in situ EV detection platform integrating multiplex EV capture with microfluidic-generated immune-tumor spheroids. This platform enables in situ monitoring of EV secretion dynamics under ICI and chemotherapeutic treatments, capturing localized and temporal changes in EV release. Using predictive models, we identified EVs carrying programmed cell death ligand 1 (PD-L1) as the most robust predictors of spheroid viability during treatment. RNA sequencing further revealed that dynamic EV expression changes are driven by gene transcription, providing a temporal understanding of EV regulation. Our platform overcomes the limitations of traditional methods by offering a physiologically relevant system to study EV-mediated immune responses. By addressing the spatial and temporal heterogeneity of EVs, this work advances EV-based biomarker discovery and provides a foundation for optimizing personalized immunotherapies.

Single-cell multiomic and exosome analyses are potent tools in various fields, such as cancer research, immunology, neuroscience, microbiology, and drug development. They facilitate the in-depth exploration of biological systems, providing insights into disease mechanisms and aiding in treatment. Single-cell isolation, which is crucial for single-cell analysis, ensures reliable cell isolation and quality control for further downstream analyses. Microfluidic chips are small lightweight systems that facilitate efficient and high-throughput single-cell isolation and real-time single-cell analysis on- or off-chip. Therefore, most current single-cell isolation and analysis technologies are based on the single-cell microfluidic technology. This review offers comprehensive guidance to researchers across different fields on the selection of appropriate microfluidic chip technologies for single-cell isolation and analysis. This review describes the design principles, separation mechanisms, chip characteristics, and cellular effects of various microfluidic chips available for single-cell isolation. Moreover, this review highlights the implications of using this technology for subsequent analyses, including single-cell multiomic and exosome analyses. Finally, the current challenges and future prospects of microfluidic chip technology are outlined for multiplex single-cell isolation and multiomic and exosome analyses.

Exosomes, functional biomarkers involved in cancer progression, have gained widespread attention for promoting tumor formation, growth, and metastasis. Current bulk exosome detections in bodily fluids enable cancer functional analysis, but average secretion levels from cell populations, losing parent cell information and ignoring exosome heterogeneity from diverse cell subgroups, necessitating an effective platform for analyzing single-cell exosome functional heterogeneity. Here, a high-throughput platform is presented, capable of efficient single-cell isolation and multi-color exosome phenotype analysis, as well as quantifying trace exosomes secreted by single cells. Photothermal-driven single-cell chips achieve significant single-cell isolation efficiency (≈97%) within 5 min, facilitating the ultra-high throughput single-cell exosome analysis. By conducting mass spectrometry and protein interaction of breast cancer exosome phenotypic proteins, key exosome phenotypes are identified. Tens of thousands of single cells from breast cancer cell lines, and clinical tissues are analyzed, revealing various subgroup differences. The study finds more CD44 and EGFR co-expressing exosome subgroups in breast cancer cell lines, while immune-evasion PD-L1 high-phenotype exosome subgroups are primarily presented in complex tumor microenvironments, especially in HER2-positive tissues. This platform offers powerful single-cell isolation, exosome quantification, and phenotypic analysis capabilities, making it a powerful tool for advancing single-cell exosome analysis in cancer research.

Chimeric antigen receptor (CAR) T cells are widely used to treat hematological tumors due to their powerful ability to target and kill cancer cells, of which accurate function evaluation at the single-cell level is crucial to ensuring the efficacy of diagnosis and treatment. Currently, a universal platform to evaluate the efficacy of immune single cells rapidly, efficiently, and systematically is urgently needed. Here, the cytotoxicity, proliferative potential, and persistence of TIM3/CD28-modified CD19 CAR-T cells are evaluated in comparison with ordinary CD19 CAR-T cells through high-performance and throughput graphene oxide quantum dot (GOQD)-based single-cell microfluidic chips. Overall secretory factor expression, immune-therapy effect of different effector-target ratios, spatial immune-therapy effects, and subgroup type profiling are demonstrated to explicit the immunotherapy effect of TIM3/CD28-modified CD19 CAR-T cells. TIM3/CD28-modified CD19 CAR-T cells show stronger anti-tumor ability and maintain excellent immunotherapy effects even at low effector-target ratios and remote distances. TIM3/CD28 also strengthens the local targeting ability of TIM3/CD28-modified CD19 CAR-T. Importantly, TIM3/CD28-modified CD19 CAR-T exhibits more distinct Th1/Th2 long-term persistent and potent killer subgroups, which is very helpful for personalized therapy. Overall, this study provides a valuable approach that can be widely implemented to analyze current CAR-T combinations and evaluate the function of innovative CAR treatments in the future.

Angiogenesis is a key player in drug resistance to targeted therapies for breast cancer. The average expression of angiogenesis-related cytokines is widely associated with the treatments of target therapies for a population of cells or spheroids, overlooking the distinct responses for individuals. In this work, a highly integrated microfluidic platform is developed for the generation of monodisperse multicellular tumor spheroids (MTSs), drug treatments, and the measurement of cytokines for individual MTSs in a single chip. The platform allows the correlation evaluation between cytokine secretion and drug treatment at the level of individual spheroids. For validation, quantities of six representative proangiogenic cytokines are tested against treatments with four model drugs at varying times and concentrations. By applying a linear regression model, significant correlations are established between cytokine secretion and the treated drug concentration for individual spheroids. The proposed platform provides a high-throughput method for the investigation of the molecular mechanism of the cytokine response to targeted therapies and paves the way for future drug screening using predictive regression models at the single-spheroid level.

The illustration portrays the spatial interaction between individual CAR-T effector cells and single tumor target cells within microchambers, eliciting immune signals subsequently captured and detected via an antibody barcode resembling a rainbow pattern. This study addresses a critical gap by conducting a systematic and comprehensive assessment of CAR-T immunotherapy effects at a single-cell level using GOQD-based microfluidic chips. The findings reveal that the modification of TIM3/CD28 enhances the sustained anti-tumor efficacy of CD19 CAR-T cells, providing a robust platform for evaluating the functionality of emerging CAR therapy approaches in the future.

Nerve growth factor (NGF) is critical for peripheral ganglion cell survival. However, it cannot penetrate blood-brain barrier (BBB). Therefore, a carrier system is required to enhance its transport across BBB. First, the ultrastructure and permeability of BBB from mice modeled by NGF nanoparticle treatment were evaluated by electron microscopy. The influx of NGF into brain was then observed using laser spectroscopy and immunofluorescence. Finally, learning and memory abilities were assessed, and hippocampal cell apoptosis was assessed using fluorescent TUNEL staining. NGF nanoparticles (NPs) induced enhanced permeability of BBB, indicating that NGF NPs can enhance BBB permeability. In NGF nanoparticle group, FITC-NGF showed significant vascular infiltration, but not in control and model groups. NGF NPs stimulated Aβ scavenging ability to promote NGF passage through blood vessels. Compared with control group, NGF NPs group had a longer distance to find the platform on day 5 and a longer incubation period. And apoptosis in NGF nanoparticle group was lower than control group. NGF NPs can induce NGF to enter brain probably by increasing the permeability of BBB. NGF NPs can be used as a new strategy to deliver therapeutic drugs to the central nervous system.

Paeoniflorin (PAE) is an active ingredient extracted from peony. This study aimed to explore the mechanism by which liposome nanoparticles loaded with PAE protect neuronal damage in Parkinson’s disease. Model group, PAE group, PAE-Lips group, PAE-Lips+miR-135a agonist group, PAE-Lips+miR-135a inhibitor group, PAE-Lips+BAY11-7085 group, PAE-Lips+SC75741 group were designed. PCR, learning and memory ability testing, pole climbing test, etc. were used to determine the mechanism of PAE-Lips on Parkinson’s disease and whether it exerts effects through regulating miR-135a. PAE-Lips were successfully constructed. PAE-Lips improved Parkinson’s disease in rats and had a certain connection with miR-135a. Up-regulating miR-135a inhibited NF-κB pathway to a certain extent and improved Parkinson’s disease. It helped protect neurons. Further verification using PAE-Lips+miR-135a agonists, SC75741, BAY11-7085, etc. showed that PAE-Lips upregulated the expression of miR-135 and inhibited NF-κB pathway, which has a good protective effect on neurons in Parkinson’s disease. PAE-Lips can promote miR-135a to inhibit the NF-κB pathway, thereby protecting neuronal damage in Parkinson’s disease. This study will provide a new idea for the prevention and treatment of Parkinson’s disease, clarify the impact of PAE-Lips, miR-135a, NF-κB, BAY11-7085 and SC75741 on Parkinson’s disease, and provide a basis for the combined use of these interventions. The possibility of treating Parkinson’s disease more effectively deserves further exploration and research and provides a theoretical basis for the development of related therapeutic drugs.

Clinical semen quality assessment is critical to the treatment of infertility. Sperm DNA integrity testing provides critical information that can steer treatment and influence outcomes and offspring health. Flow cytometry is the gold standard approach to assess DNA integrity, but it is not commonly applied at the clinical level. The sperm chromatin dispersion (SCD) assay provides a simpler and cheaper alternative. However, SCD is low-throughput and non-quantitative - sperm assessment is serial, manual and suffers inter- and intra-observer variations. Here, an automated SCD analysis method is presented that enables quantitative sperm DNA quality assessment at the single-cell and population levels. Levering automated optical microscopy and a chromatin diffusion-based analysis, a sample of thousands of sperm that would otherwise require 5 hours is assessed in under 10 minutes - a clinically viable workflow. The sperm DNA diffusion coefficient (DDNA) measurement correlates (R2 = 0.96) with DNA fragmentation index (DFI) from the cytometry-based sperm chromatin structure assay (SCSA). The automated measurement of population-level sperm DNA fragmentation (% sDF) prevents inter-observer variations and shows a good agreement with the SCSA % DFI (R2 = 0.98). This automated approach standardizes and accelerates SCD-based sperm DNA analysis, enabling the clinical application of sperm DNA integrity assessment.

Droplet microfluidic techniques have long been known as a high-throughput approach for cell manipulation. The capacity to compartmentalize cells into picolitre droplets in microfluidic devices has opened up a range of new ways to extract information from cells. Spheroids and organoids are crucial in vitro three-dimensional cell culture models that physiologically mimic natural tissues and organs. With the aid of developments in cell biology and materials science, droplet microfluidics has been applied to construct spheroids and organoids in numerous formats. In this article, we divide droplet microfluidic approaches for managing spheroids and organoids into three categories based on the droplet module format: liquid droplet, microparticle, and microcapsule. We discuss current advances in the use of droplet microfluidics for the generation of tumour spheroids, stem cell spheroids, and organoids, as well as the downstream applications of these methods in high-throughput screening and tissue engineering.

The ongoing outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has spread globally and poses a threat to public health and National economic development. Rapid and high-throughput SARS-CoV-2 RNA detection without the need of RNA extraction and amplification remain a key challenge. In this study, a new SARS-CoV-2 RNA detection strategy using a microfluidic biochip for the rapid and ultrasensitive detection of SARS-CoV-2 without RNA extraction and amplification was developed. This new strategy takes advantage of the specific SARS-CoV-2 RNA and probe DNA reaction in the microfluidic channel, fluorescence signal regulation by nanomaterials, and accurate sample control by the microfluidic chip. It presents an ultralow limit of detection of 600 copies mL(-1) in a large linear detection regime from 1 aM to 100 fM. Fifteen samples were simultaneously detected in 40 min without the need for RNA purification and amplification. The detection accuracy of the strategy was validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR), with a recovery of 99-113%. Therefore, the SARS-CoV-2 RNA detection strategy proposed in this study can potentially be used for the quantitative diagnosis of viral infectious diseases.

One of the obstacles limiting progress in the development of effective cancer therapies is the shortage of preclinical models that capture the dynamic nature of tumor microenvironments. Interstitial flow strongly impacts tumor response to chemotherapy; however, conventional in vitro cancer models largely disregard this key feature. Here, a proof of principle microfluidic platform for the generation of large arrays of breast tumor spheroids that are grown under close-to-physiological flow in a biomimetic hydrogel is reported. This cancer spheroids-on-a-chip model is used for time- and labor-efficient studies of the effects of drug dose and supply rate on the chemosensitivity of breast tumor spheroids. The capability to grow large arrays of tumor spheroids from patient-derived cells of different breast cancer subtypes is shown, and the correlation between in vivo drug efficacy and on-chip spheroid drug response is demonstrated. The proposed platform can serve as an in vitro preclinical model for the development of personalized cancer therapies and effective screening of new anticancer drugs.

The progression of cardiovascular diseases is accompanied by myocardial injury and necrosis, heart failure, and inflammatory response. Accordingly, ultrasensitive and rapid detection of multiple biomarkers plays a vital role in clinical diagnosis and timely treatment. Here, we developed a novel Lys-AuNPs@MoS2 nanocomposite self-assembled microfluidic immunoassay biochip with digital signal output and applied it to the simultaneous detection of multiple serum biomarkers including inflammatory factors and cardiovascular biomarkers, PCT, CRP, IL6, cTnI, cTnT, and NT-BNP, with high throughput and sensitivity. The digital output signal was collected in the solid phase on the chip surface with two-dimensional distribution of targets. Lys-AuNPs@MoS2 nanocomposites self-assembled biochips could simultaneously detect all six biomarkers in 60 samples in 40 min with detection limit of a few to tens of pg/mL for all serum biomarkers. The microfluidic biochip based on Lys-AuNPs@MoS2 nanocomposites provides a promising method in applications for clinical diagnosis.