Antiviral agents are a cornerstone of therapeutic strategies against viral infections; however, the early-stage discovery and optimization of viral inhibitors remain time-consuming, resource-intensive, and mechanistically opaque. Here, we introduce a mechanism-guided strategy for rapid antiviral candidate identification and evaluation using confocal Raman microscopy (CRM) imaging across cellular and animal models. CRM enables label-free, quantitative, and spatially resolved assessment of drug efficacy at the single-cell level by correlating subcellular biochemical distributions, specifically of lipids, nucleic acids, and proteins, with virus-induced morphological alterations. To validate this approach, we evaluated α-linolenic acid (ALA) against influenza A virus (IAV) strain H1N1 PR8 in human non-small cell lung carcinoma A549 cells. CRM imaging revealed that ALA treatment restored lipid homeostasis, preserved nucleic acid integrity, and normalized protein expression profiles, changes those were spatially concordant with recovery of cellular architecture. These mechanistic insights directly informed the rational design of subsequent in vivo studies in murine models, markedly reducing reliance on empirical trial-and-error. Moreover, CRM was extended to ex vivo analysis of lung tissue, enabling high-resolution interrogation of alveolar architecture and surfactant layer composition in infected tissues. Collectively, our work establishes CRM as a rapid, mechanism-informed platform for prioritizing antiviral candidates, bridging molecular pharmacology and phenotypic response, and thereby accelerating the translation of promising leads into preclinical development.

Mechanical stimulation can remodel the physiological mechanical microenvironment of cardiomyocytes and influence their maturation and injury progression. However, the dynamic process by which cardiomyocytes transition from adaptive maturation to injury remains unclear, because conventional endpoint assays cannot continuously track cellular states. Here, we developed a heart-on-a-chip platform that reconstructs physiological strain and fluidic microenvironments. The chip integrates traction force microscopy (TFM) and a cardiac troponin I (cTnI) biosensor, enabling synchronous monitoring of cardiomyocyte contractility and injury-associated phenotypes. Under physiological cyclic stretch (20% strain), the platform induced a mature cardiomyocyte phenotype, with the gap junction protein CX43 and cytoskeletal protein F-actin increasing by 1.22- and 2.03-fold, respectively. Through 14 days of dynamic mechano-chemical monitoring, we identified 25% strain as a critical turning point in cardiomyocyte mechanical responses. Below this threshold (5%-20%), mechanical stimulation promoted cellular maturation and enhanced contractile stress, whereas above this threshold (30%), it induced an injury-associated phenotype. This heart-on-a-chip provides a controllable and quantitative in vitro platform for optimizing mechanical stimulation windows, evaluating cardiomyocyte injury, and screening cardioprotective therapeutics.

The immunosuppressive prodrugs azathioprine and 6-mercaptopurine necessitate therapeutic drug monitoring of their active metabolite, 6-thioguanine (6-TG), due to their narrow therapeutic window and substantial risk of severe adverse effects. Conventional chromatographic techniques are accurate but impractical for point-of-care (POC) applications. Conversely, existing electrochemical sensors, despite their widespread use, suffer from an over-reliance on nanomaterial modifications to enhance analytical performance and face persistent challenges associated with calibration. This work resolves a long-standing knowledge gap by providing the first experimental validation of the 6-TG redox mechanism on a Nafion-modified screen-printed carbon electrode (SPCE). Through dithiothreitol intervention and electrochemical kinetic analysis, we confirm that 6-TG oxidation proceeds via a disulfide bond-mediated, four-electron, four-proton multi-step pathway. Critically, elucidation of this mechanism reveals a consistent kinetic framework, which directly inspires a universal-slope (US) calibration-free strategy wherein the calibration slope becomes a stable parameter, obviating frequent recalibration for POC applications. Leveraging this mechanistic insight, we constructed a smartphone-assisted electrochemical sensor on the same Nafion/SPCE platform for on-site detection of 6-TG in erythrocyte lysate and serum. The sensor exhibits a linear response from 0.1 to 15 μM in erythrocyte lysate, effectively covering the clinically relevant therapeutic window, alongside excellent selectivity and reproducibility. Clinical validation using 50 patient samples demonstrates strong agreement with high-performance liquid chromatography results, and the US method yields outcomes statistically equivalent to those obtained via the standard addition approach. By resolving the redox mechanism and translating this insight into a calibration-free POC sensor, this work offers a clinically deployable solution for personalized thiopurine therapy.

Peptidylprolyl isomerase A (PPIA) catalyzes cis-trans isomerization of proline residues, a key process regulating protein folding and signal transduction. Its aberrant secretion is closely associated with tumor metastasis and progression, making it a promising biomarker. However, currently available immunoassays often suffer from insufficient sensitivity for low-abundance PPIA detection. Herein, we present an electrochemical biosensor that integrates self-propelled nanocarriers with DNA cascade amplification strategy for ultrasensitive PPIA detection. The biosensor employs PtNPs@COF particles that catalyze H2O2 decomposition to generate O2. This autonomous propulsion accelerates target binding in homogeneous solution and helps mitigate diffusion-limited binding kinetics. After PPIA binding, the liberated DNAzyme catalytically cleaves the co-immobilized substrate strands, generating numerous triggers for the catalytic hairpin assembly (CHA) reaction. The biosensor achieves a wide linear range from 1 pg/mL to 10 μg/mL with a detection limit of 0.330 pg/mL. It also exhibits excellent specificity and performs reliably in clinical lung adenocarcinoma serum specimens, demonstrating its promising applicability for early diagnosis of this malignancy.

CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.

African swine fever (ASF) is a highly contagious and lethal disease with mortality rates approaching 100% in pigs. Early and accurate detection remains critical as no approved vaccine or antiviral treatment exists. We identified ASFV phosphoprotein p32 as a promising early-stage biomarker. Unlike structural proteins such as p54 and p72 detected predominantly during later infection stages, p32 is expressed early during viral replication, highly conserved among ASFV isolates, and strongly immunogenic, making it a reliable target for early and broad-spectrum detection. The ASFV p32 gene was cloned, expressed, and purified using a prokaryotic expression system, with biological specificity validated by western blotting, ELISA and mass spectrometry. Epitope prediction and structural modelling using AlphaFold, PROCHECK and ERRAT identified key antibody-binding regions, and polyclonal antibodies were generated and characterized by SDS-PAGE. A gold nanowire and tungsten disulfide hybrid (AuNW-WS2) was synthesized and characterized by FESEM, HRTEM, SAED, FFT, XRD, XPS and AFM. Antibodies were immobilized onto hybrid-functionalized screen-printed carbon electrodes to fabricate the SPCE/AuNW-WS2/ASFV p32 Ab biosensor. Validated against clinical samples and benchmarked against qPCR, the sensor exhibited a linear detection range of 1 fM-1μM, a limit of detection of ∼1.55 fM, 92.31% sensitivity, 100% specificity and 95% accuracy, underscoring its potential for rapid and reliable ASF diagnostics.

Rapid on-site detection of polycyclic aromatic compounds (PACs), such as biphenyl and carbazole, remains challenging because of their poor aqueous solubility, weak electroactivity, and matrix interference. Here, we developed a biotransformation-enabled electrochemical decoding strategy that couples a biphenyl 2,3-dioxygenase (BphA)-expressing whole-cell module with a miniaturized reduced graphene oxide/colloidal gold (rGO/CG)-modified screen-printed electrode for biphenyl and carbazole quantification. BphA from Pseudomonas putida B6-2 was heterologously expressed in Escherichia coli to convert parent PACs into water-compatible, redox-active hydroxylated/dihydrodiol-bearing metabolites, while the geometry-optimized premixed rGO/CG interface enhanced faradaic responses and reduced charge-transfer resistance, yielding a peak current density of 169.53 μA cm[-2]. Molecular docking of BphA oxygenase α-subunit and electrochemically derived apparent kinetics indicated favorable binding of both substrates and an apparent preference for biphenyl (apparent Km 4.87 mg L[-1]; apparent Vmax 8.13 U mg[-1] DCW) over carbazole (apparent Km 6.64 mg L[-1]; apparent Vmax 6.29 U mg[-1] DCW), consistent with stronger biphenyl responses. The assay achieved detection limits of 0.021 mg L[-1] for biphenyl and 0.037 mg L[-1] for carbazole, enabled simultaneous quantification in mixtures (R[2] ≥ 0.995), and showed good reproducibility (deviation ≤3.81%). Results for lake water and petroleum wastewater agreed well with high-performance liquid chromatography (R[2] up to 0.996), demonstrating the potential of this platform for point-of-need monitoring of complex aromatic pollutants in environmental samples.

The escalating healthcare impact of chronic wounds has prioritized the development of smart dressings for precise diagnosis and treatment. While wearable colorimetric sensors offer convenient diagnostics, their clinical accuracy is severely bottlenecked by ambient light interference. In this work, we report a bioinspired Janus smart bandage with light-adaptive deep learning for precise wound monitoring and accelerated healing. The smart bandage utilizes its Janus substrate to drive unidirectional exudate drainage, maintaining a dry wound bed while continuously delivering fresh analytes to multiplexed colorimetric sensors. We also integrate an LSTM-based deep learning model that autonomously calibrates colorimetric signals under varying color temperatures, eliminating illumination-induced inaccuracies and enabling high-fidelity quantification of wound pH, temperature, and glucose concentration with >96% accuracy. In vivo results demonstrate that this smart bandage not only ensures precise wound monitoring but also significantly accelerates wound closure. The integration of a bioinspired Janus structure with the adaptive deep learning model offers a compelling strategy for smart dressings, potentially advancing high-precision therapeutics in digital healthcare.

Early warning of herb-induced liver injury (HILI) is hampered by the lack of biomarkers that are more sensitive and timelier than alanine/aspartate aminotransferase (ALT/AST). We previously identified miR-122 and miR-194 as a promising circulating miRNA biomarker panel for liver injury. To address the technical challenges of detecting such trace analytes, we developed a 3D-printed multifunctional metal-organic framework (MOF)-hydrogel microarray (3D-MHMA). The platform innovatively integrates UiO-66-NH2 MOF nanoparticles as dual-functional cores for fluorescence quenching and target enrichment, a catalytic hairpin assembly-hybridization chain reaction (CHA-HCR) cascade for enzyme-free signal amplification, and 3D bioprinting for fabricating a high-throughput, spatially encoded sensor array. The 3D-MHMA achieved ultra-sensitive detection of miR-122 and miR-194 with a detection limit of 2.61 fM. Validated in cellular and animal models of triptolide-induced liver injury, the platform reliably profiled these miRNAs, whose release significantly preceded the rise in serum ALT/AST levels, providing a superior early diagnostic window (area under the curve >0.9). This work presents an integrated solution coupling a high-performance sensing platform with a novel miRNA biomarker panel for the early warning of HILI.

Compartmentalization is a hallmark of cells, enabling parallel biochemical processes to proceed with high fidelity and minimal interference. Drawing inspiration from this spatial isolation principle, we developed a compartmentalization-inspired dual-chamber sensing platform for crosstalk-minimized multiplex miRNA analysis. In each physically isolated compartment, the target miRNA directs padlock-probe ligation to form a circular template, followed by rolling-circle-extension-driven loop-mediated isothermal amplification (R-LAMP). The resulting amplicons specifically activate the corresponding CRISPR/Cas12a-crRNA complex, triggering trans-cleavage of a hairpin-DNA biogate that seals Fe-MOF nanocontainers. Gate opening releases distinct electroactive reporters (3,3',5,5'-tetramethylbenzidine, TMB; or methylene blue, MB) from their respective chambers. After the compartmentalized reactions finish, the two supernatants are combined and read out on a screen-printed electrode modified with a Co-N-C single-atom catalyst, producing two well-resolved DPV peaks for simultaneous quantification. The platform achieves femtomolar detection limits in simultaneous assays (0.87 fM for miRNA-21 and 0.72 fM for miRNA-155), a broad linear range (1 fM-100 pM), and high discrimination against non-cognate or mismatched sequences. Accurate recoveries in diluted human serum and consistent trends in cell lysates (validated by RT-qPCR) confirm practical applicability. By integrating bioinspired compartmentalization with CRISPR precision and single-atom electrocatalysis, this platform provides a generalizable route to multiplex nucleic acid diagnostics with enhanced fidelity and sensitivity.

Digital nucleic acid amplification enables absolute molecular quantification by partitioning samples into thousands of micro-reactions, yet current analysis pipelines classify each partition from a single endpoint fluorescence image, discarding the temporal dynamics that distinguish true amplification from artifacts. This limitation is acute in portable point-of-care testing (POCT) instruments, whose simplified optics and low-cost sensors produce images with non-uniform illumination, elevated background noise, and low signal-to-noise ratios. This study presents TimeSight, an end-to-end spatiotemporal detection framework for time-resolved fluorescence image stacks acquired during digital recombinase polymerase amplification (dRPA) on a portable POCT instrument. Built upon factorised three-dimensional convolutions with a learnt temporal collapse mechanism, TimeSight jointly encodes spatial morphology and amplification kinetics without partition segmentation, spatial registration, or manual threshold tuning. On a held-out test set of 8248 ground-truth partitions, TimeSight achieved the highest F1 (0.968) and precision (0.977) among all single-frame and multi-frame baselines while adding <2% parameters. Feature activation analysis confirms that the network suppresses temporally invariant artifacts indistinguishable from true positives in endpoint images, maintaining consistent performance across normal, low-SNR, and artifact-dominated conditions. TimeSight matched reference concentrations across four orders of magnitude and agreed with commercial digital PCR on genuine S. aureus DNA, with edge deployment confirming portable feasibility at about 113 ms per stack. These results indicate that temporal amplification kinetics can compensate for the spatial information lost to hardware miniaturisation, lowering the barrier to point-of-care absolute quantification. TimeSight offers a spatiotemporal detection paradigm for digital nucleic acid quantification in resource limited settings.

Patients with Cystic fibrosis (CF) require frequent monitoring in evaluating lung infections, but current diagnostic approaches are invasive and resource-intensive. Ethyl acetate (EA) and pH changes in exhaled breath have emerged as promising non-invasive biomarkers. In this context, we report a smart face mask that integrates a dual-function paper-based analytical device (PAD) for sampling exhaled breath and detecting pH and EA after the mask is worn. The system combines molecularly imprinted polymers for selective EA adsorption, Candida antarctica lipase for enzymatic hydrolysis of EA into acetic acid, and an iridium oxide-modified electrode for sensitive pH monitoring. The (bio)sensing platform demonstrated high sensitivity and reproducibility across solution and aerosol, with linear ranges of pH 5.3-9 and EA concentrations from 0.01 to 2.5 mM, and a detection limit of 1.5 μM under breath-like conditions. Tests in human saliva aerosol confirmed high accuracy (89.7-106.8%recovery) and precision (<4.3% RSD), while selectivity studies showed no interference from common breath metabolites. By enabling rapid pH and EA quantification in a portable, low-cost format, this platform provides a robust and non-invasive tool for point-of-care CF diagnostics.

Rapid and selective detection of viral pathogens is essential for effective disease surveillance and outbreak control. To meet this demand, thermo-responsive epitope-imprinted nanogels were integrated into a quartz crystal microbalance (QCM) platform for the selective detection of Hepatitis A virus (HAV) in real samples. Two HAV-derived epitopes (Epitope 2 and Epitope 3), identified by in-silico analysis, were employed as templates for the solid-phase synthesis of molecularly imprinted polymer nanogels (MIP-NGs). Successful nanogel formation was confirmed by Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering, and electrophoretic light scattering. The binding affinities of the imprinted nanogels were comparatively evaluated using surface plasmon resonance, yielding dissociation constants (KD) of 2.49 × 10[-11] M and 5.07 × 10[-13] M for Epitope-2 MIP-NGs and Epitope-3 MIP-NGs, respectively. Based on their superior affinity, Epitope-3 MIP-NGs were selected as recognition elements and immobilized onto the QCM sensor surface for mass-based virus detection. Two immobilization strategies were investigated: drop-casting and electrochemical deposition via cyclic voltammetry in the presence of o-aminophenol and graphene quantum dots. Sensor surface formation was verified by atomic force microscopy and contact angle measurements. HAV detection was performed at 38 °C, under conditions where nanogel collapse supports binding via epitope-specific cavities. Real-time QCM measurements revealed concentration-dependent frequency shifts corresponding to HAV binding, achieving a detection limit of 1.05 pM over a working concentration range of 1.33-85 pM. Selectivity was assessed using non-imprinted polymer nanogels yielding an imprinting factor of 1.9, while specificity was confirmed against other pathogenic viruses. Finally, successful HAV detection in human serum demonstrated the applicability of the sensing platform in complex biological matrices.

The reusability of molecularly imprinted polymer (MIP)-based biosensors is severely limited by inefficient template removal suppressing in situ regeneration and surface fouling in complex matrices. While recent work demonstrated the feasibility of in situ electrostatic regeneration for MIPs, no study has yet integrated antifouling capability with regeneration ability in a single MIP platform. Here, we reported the first integration of antifouling and ultrahigh in situ regeneration capabilities in a single MIP biosensor for electrochemical cortisol detection in complex biological sweat and real pharmaceutical formulations. The interface exhibited robust antifouling capability against high concentrations of oppositely charged proteins and complex serum, which was primarily attributed to high hydrophilicity of oligochitosan-based surface. Meanwhile the highly hydrated surface effectively preserved structural integrity of biosensing surface and recognition cavities during repeated electrochemical template extraction. As a result, the biosensor achieved 23 regeneration cycles in buffer, a 4.6-fold improvement over previous report, and, more importantly, 12 cycles in sweat and over 15 cycles in pharmaceuticals. Unlike prior works that addressed only regeneration in buffer without antifouling consideration, this new platform simultaneously tackled both long-standing challenges within a single MIP architecture. The biosensor enabled sensitive cortisol detection with a linear range of 0.1 pM to 1 μM and a detection limit of 0.038 pM, with accurate quantification verified in human sweat and pharmaceutical samples. By integrating antifouling and regeneration, this work established a new strategy for robust and reusable MIP biosensors, addressing a key barrier to their practical application.

Accurate and reliable monitoring of nanoplastics-induced oxidative stress at the single-cell level is essential for revealing the relevant biotoxicity and environmental health risks. Herein, a novel nanopipette sensor coupling Prussian blue-ferroferric oxide (PB-Fe3O4) nanozyme with highly hydrophilic hydrogel (Gel/PB-Fe3O4) was fabricated for the spatiotemporal monitoring of hydrogen peroxide (H2O2) fluctuations in single cells. The Gel/PB-Fe3O4 networks confined inside nanopipette provided the nanochannels, which were featured of good catalytic activity, high ion transport efficiency, and excellent antifouling capacity. The Gel/PB-Fe3O4 catalyzed the decomposition of H2O2 to generate hydroxyl radicals, which mediated the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) substrate into the ABTS[+]. The directional migration of positively charged ABTS[+] within the hydrogel nanochannels caused a significant enhancement of ion current rectification, realizing highly sensitive response toward H2O2 with a detection limit of 0.2 μM. Moreover, the proposed nanosensor enabled the spatiotemporal monitoring of polystyrene nanoplastics-induced H2O2 fluctuations in single 293T cells and A549 cells, indicating different oxidative responses of the two cell lines at the single-cell level. The developed nanopipette sensor offers a reliable platform for in-depth investigation on the biotoxicity mechanisms of nanoplastics.