High-performance protein point-of-care testing (POCT) has become a critical clinical need, particularly for rapid decision-making in acute settings. However, current protein POCT technologies, such as lateral flow assays and electrochemical sensors, struggle to simultaneously achieve high sensitivity, operational simplicity, and robustness. In this work, an integrated digital microfluidic platform for multiplexed luminescence oxygen channeling immunoassay (iDMF-mLOCI) is developed, leveraging precise droplet manipulation on DMF to create a wash-free, multiplexed suspension assay for protein POCT. This system uniquely features: (1) Fast on-chip plasma separation (<30 s); (2) No washing cycles (100% bead retention, 23% faster processing versus conventional DMF immunoassays) and (3) High reproducibility (coefficient of variations (CVs) < 9.1%). These advancements enable sensitive multiplexed detection of protein biomarkers (Limits of detection (LODs): 2.2 pg/mL for interleukin-6 (IL-6), 0.05 ng/mL for procalcitonin (PCT), 0.2 ng/mL for heparin-binding protein (HBP)) using only 2.2 μL of trace sample per test. Utilizing the established iDMF-mLOCI platform, diagnostic capability for sepsis is assessed, which demonstrates excellent diagnostic accuracy (area under curve (AUC) = 0.934) and strong correlation with clinical widely used central laboratory testing methods (coefficient of determination (R2) > 0.94). This work defines and demonstrates a transformative sample-to-answer paradigm, seamlessly integrating simplicity, multiplexing, sensitivity, and precision, thereby advancing the frontier of high-performance POCT diagnostics for critical care.

Ultrasensitive detection of low-abundance protein biomarkers is crucial for early disease diagnosis but remains challenging for the conventional barcode beads-based suspension chip platform due to the limited detection sensitivity. Here, we report a conceptually novel reaction termed "radical-mediated in situ fluorescence dye deposition" (RIFD) as a simple interfacial signal amplification strategy to overcome the limitation. This first discovered RIFD follows a universal "three-element principle", where the coexistence of beads, free radicals, and dyes suffices for fluorescence labeling, eliminating the pre-conjugation of dyes to detection probes required in traditional methods. Mechanistic studies reveal that this radical-triggered RIFD possibly follows three distinct pathways, including amide condensation on lysine residues, covalent biphenyl formation on tyrosine residues, or radical-dye co-deposition. It facilitates local and ultrafast (within 5 min) dye-trapping specifically on target-positive barcode beads. Consequently, the established RIFD-based immunoassay achieves a limit of detection of 12 fg/mL for IL-10, a 100-fold improvement over the conventional suspension chip method, and also successfully differentiates Alzheimer's disease patients from healthy controls by quantifying low-abundance plasma p-Tau217. Multiplexed detection is further validated with a three-plex cytokines panel. Our reported RIFD represents a powerful in situ fluorescence labeling tool, advancing protein biomarker detection toward the sub-pg/mL level with broad implications for clinical diagnostics.

Therapeutic-coated vascular stents are one of the key directions in the clinical treatment of atherosclerosis. However, vascular injury and implant stimulation trigger a feedback loop between coagulation and inflammation, impairing endothelial cell (EC) function, which is critical for long-term success in atherosclerotic vessels. Here, we designed a self-adaptive covalent coating stent that integrates thrombin-responsive release of apixaban as a coagulation modulator and an antioxidant polyphenol as an anti-inflammatory. This pathologic microenvironment-responsive coating regulates coagulation-inflammation loop post-implantation. The nanogels within the coating dynamically response to thrombin, the upstream initiator of coagulation-inflammation, to release apixaban in high thrombotic risk conditions while retaining it in low-risk states. Epigallocatechin gallate (EGCG), functioning as a cross-linker and the ROS scavagener, detaches in response to oxidative stress. As such, the coating enables blockade of coagulation-inflammation loop, synergistically protects ECs from inflammatory damage, promotes endothelial regeneration, and inhibits endothelial-to-mesenchymal transition (EndMT), efficiently modulating the inflammatory microenvironment via localized and coordinated regulation of coagulation-inflammation loop. Furthermore, the implantation in an atherosclerotic rabbit model demonstrated that the coating inhibits plaque deterioration linked to inflammation and endothelial damage. This dual-responsive strategy offers a promising avenue for therapeutic stents by mitigating the coagulation-inflammation loop and supporting endothelial repair in atherosclerotic vessels.

Here we report PEI-mediated copper in situ deposition (CISD) as a general signal enhancement strategy for a commercial colloidal gold nanoparticles (AuNPs)-based lateral flow immunoassay (LFIA) platform. The copper shell could be coated on AuNPs which aggregate on the test line to form core-shell structured composites (AuNPs@Cu) to deepen the colorimetric signal of the strips. After critical elements that affect Cu deposition yields were optimized, a significant signal amplification has been acquired with limit of detection (LOD) improvements of 40 times by naked-eye identification and 100 times by quantitative instrumental analysis using human hemoglobin (hHb) as a target. This study demonstrates a common and simple strategy for upgrading the detection sensitivity of a commercial AuNPs-LFIA platform with a whole running time of 20 min.

The development of a detection methodology with high sensitivity, stability, and user-friendliness for quantification of proteins at subfemtogram levels is essential for clinical applications such as early screening, disease diagnosis, and monitoring disease progression. A traditional micropartition-based digital enzyme-linked immunosorbent assay (dELISA) results in significant bead loss due to intricate partitioning based on Poisson distribution, multistep reaction operations, nonglobal signal recognition, and reading modes, which have not yet achieved the ultimate detection sensitivity. This study introduces an ultrasensitive multiplexed digital immunoassay with extremely high bead analysis efficiency (HiBeA) through integrating the bead transfer strategy in multistep immunoreaction processing and flow cytometry detection mode. Typically, a bead analysis ratio over 95% was achieved, ensuring high sensitivity, efficiency, and stability of the established HiBeA using as few as only 5,000 beads. As a proof of concept, HiBeA was utilized for the multiplexed detection of IL-10 and IL-6, achieving detection limits of 5.9 and 8.8 fg/mL, respectively. This signifies a 3- to 4-fold enhancement in detection sensitivity under the same reaction time while using only 1% of the assay bead number compared to the commercial single-molecule array (SiMoA) system. HiBeA presents ultrasensitivity, robust detection stability based on tailored, multistep operation of immune-reaction, and the ability to perform multiplexed detection, thereby offering substantial prospects for the advancement of ultrasensitive clinical diagnostics.

The specific mechanisms underlying bacteria-triggered cell death and osteogenic dysfunction in host bone marrow mesenchymal stem cells (BMSCs) remain unclear, posing a significant challenge to the repair of infected bone defects. This study identifies ferroptosis as the predominant cause of BMSCs death in the infected bone microenvironment. Mechanistically, the bacteria-induced activation of the innate immune response in BMSCs leads to upregulation and phosphorylation of interferon regulatory factor 7 (IRF7), thus facilitating IRF7-dependent ferroptosis of BMSCs through the transcriptional upregulation of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4). Moreover, it is found that intervening in ferroptosis can partially rescue cell injuries and osteogenic dysfunction. Based on these findings, a hydrogel composite 3D-printed scaffold is designed with reactive oxygen species (ROS)-responsive release of antibacterial quaternized chitosan and sustained delivery of the ferroptosis inhibitor Ferrostatin-1 (Fer-1), capable of eradicating pathogens and promoting bone regeneration in a rat model of infected bone defects. Together, this study suggests that ferroptosis of BMSCs is a promising therapeutic target for infected bone defect repair.

As a natural cationic polymer material, the application of chitosan hydrogel for bone tissue engineering has been greatly limited due to its poor mechanical strength. Enzymatic mineralization has drawn increased attention to effectively improve the mechanical properties of hydrogels. In this study, carboxymethyl chitosan (CMCS) hydrogels cross-linked with different concentrations of genipin (2.5 %, 5 % and 10 %) were prepared and further mineralized through enzyme-induced biomimetic mineralization. The mechanical properties of the CMCS hydrogels were significantly increased as a result of mineralization, showing improvement of 1200-1500 % on storage moduli, and even exhibiting certain tensile behavior with the elongation rate of 30-35 %, likely due to the uniform formation and small size of mineralized products. Interestingly, the cationicity of chitosan also exerted an important modulation effect and the mineralization behavior and mechanical properties of mineralized hydrogels. In addition, the enzymatic mineralized hydrogels showed enhanced biocompatibility and osteogenic differentiation in-vitro, likely due to its superior mechanical properties and the introduction of calcium phosphate biominerals. In vivo experiments further suggest excellent bone-forming activity for the enzymatic mineralized hydrogels. Overall, tuning cationicity and enzymatic mineralization provide an effective approach for the preparation of chitosan hydrogels with superior mechanical and biological properties for bone tissue engineering application.

Abstract Objective To assess renal interstitial fibrosis (IF) using diffusion MRI approaches, and explore whether corticomedullary difference (CMD) of diffusion parameters, combination among MRI parameters, or combination with estimated glomerular filtration rate (eGFR) benefit IF evaluation. Methods Forty-two patients with chronic kidney disease were included, undergoing MRI examinations. MRI parameters from apparent diffusion coefficient (ADC), intra-voxel incoherent motion (IVIM), diffusion kurtosis imaging (DKI), and diffusion-relaxation correlated spectrum imaging (DR-CSI) were obtained both for renal cortex and medulla. CMD of these parameters was calculated. Pathological IF scores (1–3) were obtained by biopsy. Patients were divided into mild (IF = 1, n = 23) and moderate-severe fibrosis (IF = 2–3, n = 19) groups. Group comparisons for MRI parameters were performed. Diagnostic performances were assessed by the receiver operator’s curve analysis for discriminating mild from moderate-severe IF patients. Results Significant inter-group differences existed for cortical ADC, IVIM-D, IVIM-f, DKI-MD, DR-CSI V B, and DR-CSI V C. Significant inter-group differences existed in ΔADC, ΔMD, ΔV B, ΔV C, ΔQ B, and ΔQ C. Among the cortical MRI parameters, V B displayed the highest AUC = 0.849, while ADC, f, and MD also showed AUC > 0.8. After combining cortical value and CMD, the diagnostic performances of the MRI parameters were slightly improved except for IVIM-D. Combining V B with f brings the best performance (AUC = 0.903) among MRI bi-variant models. A combination of cortical V B, ΔADC, and eGFR brought obvious improvement in diagnostic performance (AUC 0.963 vs 0.879, specificity 0.826 vs 0.896, and sensitivity 1.000 vs 0.842) than eGFR alone. Conclusion Our study shows promising results for the assessment of renal IF using diffusion MRI approaches. Critical relevance statement Our study explores the non-invasive assessment of renal IF, an independent and effective predictor of renal outcomes, by comparing and combining diffusion MRI approaches including compartmental, non-compartmental, and model-free approaches. Key Points Significant difference exists for diffusion parameters between mild and moderate-severe IF. Generally, cortical parameters show better performance than corresponding CMD. Bi-variant model lifts the diagnostic performance for assessing IF. Graphical Abstract

Given the detrimental impact on non-target pollinators, there is a high demand for monitoring multiple neonicotinoid residues in the environment. In this study, we proposed a novel physical-biological coupled multiplexing system (PBCMS) to achieve a homogeneous and high-performance indirect competitive suspension array technology (ic-SAT) for multiple neonicotinoids monitoring. First, hapten-decorated microspheres (HDMs) were prepared by ligating the streptavidin-modified microspheres with the biotinylated hapten, according to a novel molecularly-defined single-stranded DNA (ssDNA)-grafted labeling approach. Taking thiamethoxam (THX) and imidacloprid (IMI) as models, the binding properties of two anti-neonicotinoid antibodies were profiled by establishing fluorescence monoplex ic-SAT assays. Subsequently, two HDMs (2.8 and 5.0 mu m) immobilized with THX and IMI haptens were synthesized and then implemented to fabricate a dualplex ic-SAT assay, while they were distinguished via a facile diameter-resolved decoding process through flow cytometry. Excitingly, the screening capacity of the dualplex ic-SAT assay was significantly expanded by employing antibodies with distinctive binding profiles. It enables the simultaneous monitoring of 7 representative neonicotinoid insecticides (THX, IMI, acetamiprid, clothianidin, thiacloprid, nitenpyram and imidaclothiz) and 4 major metabolites in a single test sample, with IC50 values ranging from 0.02 to 572.70 ng/mL. Furthermore, the dualplex ic-SAT assay was applied to honey, pollen and bee samples, demonstrating satisfactory recovery rates (74.1 %similar to 106.3 %) and nice agreement with LC-MS/MS analysis. In summary, the PBCMS offers an easy-to-implement, robust and cost-effective way for establishing user-defined ic-SAT, providing a powerful analytical tool to simultaneously monitor multiple neonicotinoid residues in environmental samples.

CaBPs-Na2FDP@CaCl2 exhibited dual functions of antitumor and osteogenesis. It achieved targeted therapy for tumors due to pH-response degradation and GLUT5-targeting, and promoted osteoblast differentiation in the form of Ca2+–CaM during degradation.

The degradation of oncoproteins mediated by proteolysis-targeting chimera (PROTAC) has emerged as a potent strategy in cancer therapy. However, the clinical application of PROTACs is hampered by challenges such as poor water solubility and off-target adverse effects. Herein, we present an ultrasound (US)-activatable PROTAC prodrug termed NPCe6+PRO for actuating efficient sono-immunotherapy in a spatiotemporally controllable manner. Specifically, US irradiation, which exhibits deep-tissue penetration capability, results in Ce6-mediated generation of ROS, facilitating sonodynamic therapy (SDT) and inducing immunogenic cell death (ICD). Simultaneously, the generated ROS cleaves the thioketal (TK) linker through a ROS-responsive mechanism, realizing the on-demand activation of the PROTAC prodrug in deep tissues. This prodrug activation results in the degradation of the target protein BRD4, while simultaneously reversing the upregulation of PD-L1 expression associated with the SDT process. In the orthotopic mouse model of pancreatic tumors, NPCe6+PRO effectively suppressed tumor growth in conjunction with US stimulation.

The emergence of digital immunoassays has advanced the sensitivity of protein analysis to ultrahigh sensitivity at the attomolar level. However, the background signal generated by the premixing of immunocomplexes and fluorogenic substrates can limit the precise quantification, especially in multiplexed assays. Herein, a bead-based SlipChip (bb-SlipChip) microfluidic device capable of massively parallel two-step sample loading is presented. The background signal can be suppressed through a two-step loading mechanism. Specifically, encapsulate the beads into the microwells first and then, through a slipping process, deliver the fluorogenic substrate in parallel into 281,200 microwells of 68 fL to perform the digital immunoassay. The quantification capability is demonstrated with a duplex assay of IL-6 and IL-10, achieving a limit of detection of 5.2 and 15.3 fg/mL, which is approximately 2-3 times improved compared to a commercial Simoa system. The bb-SlipChip provides a robust and universal method for digital immunoassay and can be extended to higher multiplexed detection as well as other biomedical applications involving microbeads.

The matrix viscosity plays a critical role in indirecting the cellular responses and apoptosis of tumor cells and exhibits somewhat different mechanotransduction mechanisms.

Osteoclasts are cells primarily involved in bone remodeling. Hyperactive osteoclastogenesis leads to pathological bone loss and microarchitectural deterioration. However, given the limitations of current osteoclast inhibitors, there is an urgent need for a novel antiresorptive agent with higher efficiency and fewer side effects. Cell-free fat extract (CEFFE) is the liquid fraction obtained from adipose tissues, which are enriched with a variety of adipokines. This study aims to explore its pharmacologic effect on hyperactive osteoclastogenesis. CEFFE exhibits excellent potentials to attenuate osteoclast-associated bone loss in ovariectomy mice and to inhibit osteoclastogenesis in primary monocytes. Furthermore, the cationic protein fraction of CEFFE (CEFFE-Cation) is identified as the main inhibitory component in osteoclast formation assay. According to liquid chromatography with tandem mass spectrometry analysis, the CEFFE-Cation fraction mainly consists of various antioxidant enzymes and cytokines, which endow it with a superior antioxidant capacity. Meanwhile, CEFFE-Cation is also capable of mitigating Ca2+ oscillation and subsequent nuclear factor of activated T-cells 1 nuclear translocation during osteoclastogenesis. Overall, this study elucidates the promising translational potential of CEFFE as a next-generation personalized antiresorptive agent for osteolytic bone disease treatment.

BackgroundEmergency psychological responding professionals are recruited to help deal with psychological issues as the Corona Virus Disease 2019 (COVID-19) continues. We aimed to study the neural correlates of psychological states in these emergency psychological responding professionals after exposure to COVID-19 related trauma at baseline and after 1-year self-adjustment. MethodsResting-state functional MRI (rs-fMRI) and multiscale network approaches were utilized to evaluate the functional brain activities in emergency psychological professionals after trauma. Temporal (baseline vs. follow-up) and cross-sectional (emergency psychological professionals vs. healthy controls) differences were studied using appropriate t-tests. The brain functional network correlates of psychological symptoms were explored. ResultsAt either time-point, significant changes in the ventral attention (VEN) and the default mode network (DMN) were associated with psychological symptoms in emergency psychological professionals. In addition, the emergency psychological professionals whose mental states improved after 1 year demonstrated altered intermodular connectivity strength between several modules in the functional network, mainly linking the DMN, VEN, limbic, and frontoparietal control modules. ConclusionBrain functional network alterations and their longitudinal changes varied across groups of EPRT with distinctive clinical features. Exposure to emergent trauma does cause psychological professionals to produce DMN and VEN network changes related to psychological symptoms. About 65% of them will gradually adjust mental states, and the network tends to be rebalanced after a year.

Here we report a brushed magnetic microbeads (BMBs) carrier with host-guest structures for enhancing biodetection performance in beads-based flow cytometry (FCM). The micro-sized MBs are employed as host spheres and the nano-sized spherical polyelectrolyte brushes (SPBs) are utilized as guest particles to achieve the covalent assembly of SPBs on MBs (that is BMBs). Our proposed BMBs carrier exhibits a remarkable signal to noise improvement of approximately 8-9 times in beads-based immunoassay on FCM compared to conventional MBs via preserving capture antibodies' activity and reducing detection antibodies' nonspecific adsorption. This study demonstrates the promising potential of our developed BMBs carrier for beads-based flow cytometry.

Our aim was to modulate magnetic cues to influence the differentiation of neural stem cell (NSC) into neuron during nerve repair and to explore corresponding mechanisms. Here, a magnetic hydrogel composed of chitosan matrices and magnetic nanoparticles (MNPs) with different content was prepared as the magnetic-stimulation platform to apply intrinsically-present magnetic cue and externally-applied magnetic field to NSC grown on the hydrogel. The MNP content had regulatory effects on neuronal differentiation and the MNPs-50 samples exhibited the best neuronal potential and appropriate biocompatibility in vitro, as well as accelerated the subsequent neuronal regeneration in vivo. Remarkably, the use of proteomics analysis parsed the underlying mechanism of magnetic cue-mediated neuronal differentiation form the perspective of protein corona and intracellular signal transduction. The intrinsically-present magnetic cues in hydrogel contributed to the activation of intracellular RAS-dependent signal cascades, thus facilitating neuronal differentiation. Magnetic cuedependent changes in NSCs benefited from the upregulation of adsorbed proteins related to "neuronal differentiation", "cell-cell interaction", "receptor", "protein activation cascade", and "protein kinase activity" in the protein corona. Additionally, magnetic hydrogel acted cooperatively with the exterior magnetic field, showing further improving neurogenesis. The findings clarified the mechanism for magnetic cue-mediated neuronal differentiation, coupling protein corona and intracellular signal transduction.

Digital bio-detection has become one of the most appealing methods in recent years due to its excellent performance with ultra-sensitivity in detection of low-abundance targets. Traditional digital bio-detection needs the utilization of micro-chambers for physical isolation of targets, while the recently developed beads-based micro-chamber free one is attracting extensive attention, although there exist the disadvantages of overlaps between positive ("1") and negative ("0") signals as well as the decreased detection sensitivity in multiplexed mode. Here we propose a feasible and robust micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads (EMMs) and tyramide signal amplification (TSA) strategy. An EMMs-based multiplexed platform is constructed by using a fluorescent encoding method, then a puissant signal amplification of positive events in TSA procedure is achieved via systematical revelation of key factors influences. For proof of concept, a three-plexed tumor markers detection is performed to evaluate our established platform. The detection sensitivity is comparable to the corresponding single-plexed assays and is also approximately 30-15,000 times improvement compared to the conventional suspension chip. Therefore, this multiplexed micro-chamber free digital bio-detection paves a promising way to be an ultrasensitive and powerful tool for clinical diagnosis.

Breast cancer affects more than 1 million women per year worldwide. Through this study, we developed a nanoparticle-based drug delivery system to target breast cancer cells. Aspirin has been found to inhibit thromboembolic diseases with its tumor-preventing activity. As a consequence, it relieves disease symptoms and severity. Here, mesoporous silica nanoparticles (MNPs) have been used to deliver aspirin to the tumor location. MNP-based aspirin in folic acid (F)-conjugated polydopamine (MNP-Asp-PD-PG-F) vehicles are prepared for targeted breast cancer therapy. The vehicle hinges on MNP altered with polymer polyethylene glycol (PG), polydopamine (PD), and F. The delivery vehicle was studied for in vitro drug release, cytotoxicity, and breast cancer cell proliferation. F-conjugated drug delivery vehicles let MNPs achieve an elevated targeting efficacy, ideal for cancer therapy. It was also observed that compared to free aspirin, our drug delivery system (MNP-Asp-PD-PG-F) has a higher cytotoxic and antiproliferative effect on breast cancer cells. The drug delivery system can be proposed as a targeted breast cancer therapy that could be further focused on other targeted cancer therapies. Delivering aspirin by the PD-PG-F system on the tumor sites promises a therapeutic potential for breast cancer treatment.