Circulating microRNAs (miRNAs) are colorectal cancer (CRC) biomarkers, but their short length, high sequence homology, and low abundance make sensitive and multiplexed detection difficult. Electrochemical biosensing offers a route for miRNA analysis, yet duplex detection remains limited by the need for reporter pairs with well-separated signals and minimal crosstalk. Here, we report an Ago-mediated Duplex Orthogonal Readout for microRNA Electrochemical detection (ADORE) for simultaneous detection of CRC-related miRNAs, miR-21 and miR-92a. ADORE uses methylene blue and L-012 as an orthogonal electrochemical code pair, validating L-012, a luminol derivative used in chemiluminescence and electrochemiluminescence, as an electrochemical reporter. The sensing interface integrates tetrahedral DNA nanostructures with reporter-loaded UiO-66-NH2 carriers to stabilize probe organization and amplify electrochemical responses. Target miRNAs first initiate an exponential amplification reaction (EXPAR) to generate guide DNAs. These guides then program Clostridium butyricum Argonaute (CbAgo) for sequence-specific cleavage at the interface, producing target-dependent signal-off responses. ADORE achieves limits of detection of 1.85 aM for miR-21 and 0.51 aM for miR-92a. To improve analysis of serum samples, multidimensional differential pulse voltammetry-derived features were integrated into a support vector machine model, increasing diagnostic performance from an area under the receiver operating characteristic curve (AUC) of 0.88 using single-feature analysis to 0.94 using multifeature classification in serum discrimination. This work provides an integrated strategy combining Ago-mediated signal transduction, orthogonal electrochemical coding, nanostructured interface engineering, and machine learning for duplex serum miRNA analysis and CRC-related biomarker detection, supporting development of multiplexed electrochemical liquid-biopsy assays for CRC screening.

CRISPR-Cas12a holds great promise for molecular diagnostics and gene editing due to its sequence-specific DNA recognition and robust trans-cleavage activity, yet precise tuning of its nuclease activity remains challenging. Herein, we report a simple temperature-regulation strategy using cost-effective ssDNA blockers, that enable hybridize with crRNA functional motifs at low temperatures to block and suppress Cas12a activation, while thermal dissociation at elevated temperatures restores Cas12a activity to realize thermoregulation of Cas12a activity, enabling precise thermal switching within 5 °C (37-42 °C). The cis- and trans-cleavage activity of Cas12a could be activated at desired temperature via different DNA blockers. Coupling this thermoregulated Cas12a system with recombinase polymerase amplification (RPA), we developed a one-tube thermal-start RPA-CRISPR/Cas12a assay, which avoids premature Cas12a activation. This method achieves a limit of detection (LOD) of 0.73 aM against Mycobacterium tuberculosis (MTB) with excellent specificity and reproducibility, and exhibits 96.67% concordance with qPCR in clinical specimens. An all-in-one THERMAL-CRISPR POCT platform was developed by using a 3D-printed portable device for streamlined clinical testing, with clinical sensitivity of 90.48% and 100% specificity. This programmable thermal modulation strategy provides a novel and simple technical route for constructing robust, engineerable CRISPR tools for point-of-care diagnostic and gene editing.

MicroRNAs (miRNAs) are important biomarkers for cancer diagnosis and prognosis, but their short length, low abundance, and high sequence similarity make rapid and sensitive detection challenging. Split crRNA-based CRISPR/Cas12a systems have emerged as a promising amplification-free strategy for RNA detection, yet the design principles governing their performance remain largely unexplored. Here, we report a rationally engineered split crRNA-Cas12a platform for direct detection of breast cancer-associated miRNAs. Using miR-155 and miR-10b as model targets, the influence of spacer split position on assay performance was systematically evaluated and further interpreted through structural prediction analysis. The results revealed distinct effects of spacer split position on signal output, reaction kinetics, and background signal, providing guidance for the rational design of split crRNA-Cas12a systems. DNA activator design, Cas12a/crRNA ratio, and reaction conditions were further optimized to improve assay performance. The system enabled sensitive detection of miR-155 and miR-10b, achieving limits of detection of 68 and 98 fM, respectively, while maintaining excellent specificity against non-target miRNAs and strong discrimination of mismatched sequences. The assay supported dual-target screening within a single reaction and demonstrated reliable performance in serum-spiked samples. These findings establish key design principles for split crRNA-Cas12a systems and provide a rational framework for engineering amplification-free RNA detection platforms. The proposed platform extends the application of Cas12a for direct RNA detection and provides a promising strategy for rapid biomarker screening in point-of-care and resource-limited settings.

CRISPR-Cas13a enables programmable RNA recognition without target nucleic-acid preamplification, but conventional assays encode collateral RNase activity by cleaving fluorescent reporters. Here, we developed a cleavage-gated strategy that converts transient Cas13a cleavage into a sequence-defined, strand-displacement-competent DNA actuator, shifting signal encoding from cumulative reporter intensity to a discrete scaffold topology. A chimeric masking hairpin (CMH) links Cas13a to a DNA nanostructure sensor (DNA-NS) by using a cleavable RNA loop to constrain a DNA-key bearing a pre-exposed toehold. Target-activated Cas13a cleaves the loop, converting the partially masked key into a strand-displacement-competent intermediate that reconfigures an M13mp18 scaffold from a linear to a looped state, producing a distinct electrophoretic mobility signature. Screening of 40 CMH-sensor-handle combinations identified a 4-nt toehold paired with an 18-nt sensor handle as the best-performing configuration within the tested design space (component-level ΔΔG = -1.19 kcal/mol at 37 °C). Using pooled crRNAs targeting the S, M, and L genomic segments of severe fever with thrombocytopenia syndrome virus (SFTSV), the assay detected the lowest tested input concentration of 10.38 fM in all three replicates. In a retrospective case-control evaluation of 87 plasma-derived RNA samples, the assay agreed with RT-qPCR for 45 of 47 positive specimens and 40 healthy controls, yielding a positive percent agreement of 95.7% and a negative percent agreement of 100%. Replacing the crRNAs redirected recognition to Hantaan virus reference RNA without redesigning the CMH or DNA-NS. This work establishes a modular framework for topology-resolved viral RNA detection without target preamplification.

The functional decline of aging skeletal muscle is primarily driven by myoblast senescence, a process that severely impairs regenerative capacity. While mitochondrial dysfunction is a hallmark of this state, traditional senescence assays rely on static, late-stage markers requiring destructive cell processing, making them unsuitable for real-time monitoring. To bridge this diagnostic gap, we developed a non-destructive electrochemical (EC) platform utilizing a Matrigel-functionalized gold nanostructure electrode to predict the myogenic potential of senescent myoblasts in their undifferentiated state. The platform resolved a pronounced bioenergetic deficit in undifferentiated senescent cells and, during myogenic induction, tracked in real-time their failure to execute the metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Notably, the basal EC signal correlated with the senescence regulators p53 and p21 and predicted subsequent myogenic competence, linking metabolic state to differentiation fate. Furthermore, we validated the platform's utility for senotherapeutic screening by quantifying the biphasic metabolic recovery induced by metformin, distinguishing effective recovery range from supra-pharmacological toxicity. This study establishes EC metabolic profiling as a rapid, label-free, and predictive framework for assessing stem cell quality and accelerating the discovery of anti-aging interventions.

Accurate monitoring of doxorubicin (DOX) is important for individualized chemotherapy, yet conventional fiber-optic surface plasmon resonance (SPR) sensors are often limited by large probe dimensions and temperature cross-sensitivity. In this paper, a compact fiber-tip microprobe that integrates SPR and Fabry-Perot interference (FPI) for synchronous detection of DOX concentration and temperature is reported. The microprobe is composed of a square frustum-similar structure, which is directly printed on the end face of a single-mode fiber by femtosecond laser-induced two-photon polymerization. By optimizing the structural parameters, SPR and FPI are simultaneously excited within the 24 μm-high microprobe. The sensing surface is further functionalized with the DOX-specific aptamer to enable selective molecular recognition. In the range of 0-100 μM DOX, the SPR and FPI channels exhibit sensitivities of 0.075 and 0.060 nm/μM, respectively, with corresponding limits of detection of 8.8 and 19.1 μM. Over 28-40 °C, the SPR and FPI temperature sensitivities are -0.66 and -0.61 nm/°C, respectively. A dual-parameter decoupling matrix enabled simultaneous determination of DOX concentration and temperature. The microprobe also showed satisfactory stability and selectivity, demonstrating its potential for miniaturized therapeutic drug monitoring.

Staphylococcus aureus is a major foodborne pathogen associated with dairy products; however, rapid and reliable detection remains challenging due to complex food matrices. Herein, we developed a one-step isothermal biosensing strategy based on an artificial nucleic acid circuit-assisted Argonaute system (PfAgo-ANCA) for rapid and sensitive detection of S. aureus. By targeting the species-specific nuc gene, the platform utilizes the high-temperature endonuclease activity of Pyrococcus furiosus Argonaute (PfAgo). The strategy integrates programmable PfAgo-mediated target recognition with downstream artificial nucleic acid circuit-based signal amplification, converting target cleavage into an amplified fluorescence signal without conventional nucleic acid extraction, purification, or amplification. Under optimized conditions, the PfAgo-ANCA platform achieved a detection limit of 10[0] CFU/mL for pure S. aureus cultures and exhibited excellent specificity against S. aureus. In artificially contaminated milk and dairy beverage samples, the assay maintained a detection limit of 10[0] CFU/mL. Owing to the high-temperature reaction characteristics of PfAgo, the platform integrates bacterial thermal lysis and enzymatic signal amplification within a sealed single-tube format, thereby eliminating the need for conventional DNA extraction and purification procedures. Furthermore, the modularity of the PfAgo-ANCA platform enabled adaptation to different genetic targets through guide DNA redesign, supporting dual-gene detection of S. aureus and enterotoxin-associated genes. Overall, PfAgo-ANCA provides a distinct and modular strategy that combines programmable target recognition, circuit-based signal amplification, and simplified sample processing, showing considerable potential for rapid food safety monitoring.

Multiplexed small extracellular vesicles (sEVs) biomarker detection holds great promise for breast cancer diagnosis and subtyping; however, its clinical application is often hampered by the complex isolation and detection processes. Herein, we propose a temperature-responsive sensing platform (PDDNFs@QDs) utilizing poly(N-isopropylacrylamide) (PNIPAM)-linked, dumbbell-shaped DNA nanoflowers (DNFs) loading quantum dots (QDs). The sensing mechanism is based on the disassembly of DNFs triggered by aptamer-target recognition, which releases the loaded QDs. This process enables simultaneous quantitative analysis of human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) on the surfaces of breast cancer-derived sEVs. Importantly, the PNIPAM-mediated thermo-responsive strategy promotes the aggregation of unreacted probes, thereby reducing matrix-associated background signals. This approach addresses matrix interference in crude membrane filtrates, allowing for the direct detection of sEVs isolated through simple filtration. Additionally, the platform was successfully applied to 45 clinical samples, achieving an area under the curve (AUC) of 0.97 for breast cancer diagnosis and an AUC of 0.85 for HER2-status discrimination, with results closely aligned with clinical imaging findings and pathological assessments. Overall, this strategy provides a simple and sensitive approach with significant potential for breast cancer diagnosis and subtyping.

PIWI-interacting RNAs (piRNAs) are pivotal regulators of physiological and pathological processes, and their aberrant expression is tightly linked to cancer development and prognosis. Nevertheless, their ubiquitous presence in both tumor and normal cells poses a major challenge to achieving cancer-specific detection and regulation of cancer-associated piRNAs. Herein, we design a smart multi-activatable DNAzyme nanoamplifier for spatiotemporal imaging of multiple piRNAs and on-demand gene therapy. This nanoamplifier integrates three functional modules: an APE1-gated recognition module, a target-activated orthogonally controlled catalytic DNA (CCD) circuitry module, and a DNAzyme-mediated module with the integration of signal output and gene therapy. Upon cellular internalization of the nanoamplifier, APE1 within the tumor microenvironment triggers the release of intelligent-responsive elements. These elements can specifically bind to piRNAs, initiating the CCD circuitry to generate two active double-ended DNAzyme assemblies. Leveraging their high-turnover efficiency to cleave molecular beacons (MBs) and specifically knock down VEGF mRNA, these double-ended DNAzyme assemblies can simultaneously restore Cy5/Cy3 fluorescence signals in MB1/MB2 and achieve target-stimulated in situ on-demand gene therapy. Notably, the liberated quencher-labeled fragments can serve as piRNAs analogues to re-activate the CCD circuitry, significantly improving detection sensitivity. This nanoamplifier can overcome the kinetic bottleneck of traditional CHA systems, facilitating sensitive detection and in situ visualization of attomolar-level multiple piRNAs without pre-amplification. Moreover, this nanoamplifier can not only quantify piRNAs levels across various human cells and precisely discriminate expression differences between breast cancer and healthy tissues, but also induce cancer cell apoptosis through synergistic gene therapy, providing a robust integrated theranostic platform.

Salmonella contamination in poultry meat remains a major global food safety concern, necessitating rapid and reliable viability-targeted detection methods. Here, we developed a platform of phage amplification (PA)-assisted lateral flow biosensor for rapid screening of viable Salmonella in chicken meat. The platform combines a short selective enrichment step in Rappaport-Vassiliadis soya broth with infection by the Salmonella-specific phage SE-W109 (multiplicity of infection of 0.1). In the presence of viable Salmonella cells, progeny phages were produced and subsequently detected via lateral flow immunoassay (LFI). The entire procedure was completed within approximately 7 h, providing an instrument-free visual readout suitable for point-of-need testing. PA-LFI detected major Salmonella serovars, including S. Enteritidis and S. Typhimurium, without cross-reactivity toward non-Salmonella bacteria. The detection limit was approximately 100 colony-forming units per 25 g of chicken meat. Field evaluation of PA-LFI using 30 retail chicken samples, compared with ISO 6579-1:2017 as the reference method, showed that Salmonella was present in 36.7% (11/30) of the samples. PA-LFI correctly identified 8 of the 11 culture-positive samples and 17 of the 19 culture-negative samples, thus achieving a sensitivity of 72.7%, a specificity of 89.5%, and overall agreement of 83.3%. This work demonstrates a phage-amplified lateral flow biosensing platform that enables rapid, viability-targeted detection, complements culture-based methods as a practical screening tool for routine food safety monitoring, and can be readily adapted to other bacterial pathogens through appropriate phage selection.

Extracellular vesicles (EVs) are promising non-invasive biomarkers, but their clinical application is limited by inefficient isolation and downstream analysis. Here, we developed a biomimetic magnetic cilia microfluidic chip (MagCilia-chip) for EV capture and proteomic profiling from urine. Tim4-functionalized magnetic microcilia specifically captured phosphatidylserine-positive EVs and underwent magnetic-field-driven three-dimensional rotation to enhance EV-microstructure interactions. Compared with ultracentrifugation, the MagCilia-chip operated in 3D rotation mode achieved a recovery rate of 85% versus 27%, with 5.32-fold higher EV yield and 3.13-fold higher purity. On-chip lysis and digestion followed by mass-spectrometry analysis identified 2903 EV-derived proteins from 50 μL of urine. In the discovery cohort of 12 prostate cancer (PCa) and 8 post-treatment (PT) samples, 368 differentially expressed EV proteins were identified, yielding a five-protein candidate panel. Independent parallel reaction monitoring analysis of 19 PCa and 9 PT samples confirmed differential expression of C9, COL6A3, and VTN. A three-protein panel achieved area-under-the-curve values of 0.78-0.95 across ten machine-learning classifiers using leave-one-out cross-validation. This platform provides an efficient strategy for urinary EV isolation and proteomic analysis and supports further evaluation of EV-derived candidate biomarkers for PCa treatment.

Rapid and accurate identification of pathogenic Vibrio in complex aquatic product matrices is critical for food safety. Herein, an entropy-driven detection platform (SEFAM) based on triple fluorescence-coded quantum dots (QDs) probes encapsulated in metal-organic framework (MOF) and mesophilic Clostridium butyricum Argonaute (CbAgo) is developed, which enables multiplexed and DNA extraction-free detection of Vibrio in aquatic products. The SEFAM platform utilizes programmable magnetic aptamer switches to achieve targeted bacterial recognition, thereby initiating the entropy-driven circuit (EDC) response and releasing large quantities of guide DNA (gDNA) to stimulate the specific cleavage activity of CbAgo. The multicolor quantum dots@MOF-5 with different emission bands (430 nm, 525 nm, and 635 nm) were modified with single-stranded DNA (ssDNA) and conjugated with magnetic graphene oxide (mGO), serving as triple fluorescent probes to distinguish three Vibrio species. The platform achieved the limits of detection (LOD) for Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae as 6.8, 4.3 and 6.2 CFU/mL respectively, and the linear range is 10[1]-10[8] CFU/mL. Furthermore, the practical applicability of the SEFAM platform was successfully demonstrated through validation using 44 real samples. This study has presented a highly specific, stable and repeatable biosensor, providing a promising method for the on-site multiplex detection of pathogenic Vibrio in complex matrices.

PIWI-interacting RNA (piRNA) as a type of small non-coding RNA holds great promise for managing disease occurrence and progression. Herein, we construct a ligation-assisted DNA strand displacement strategy for enhanced CRISPR/Cas14 cleavage, which provides remarkable SERS and electrochemical responses to indicate trace piRNA information. The upstream reaction is rationally designed to integrate traditional strand displacement amplification (SDA) and ligation-mediated target recycling, which benefits improvement of the efficiency. The generated single-stranded DNA products can activate trans-cleavage activity of Cas14, enabling the DNA structural changes on the sensing interface and subsequent signal measurements. By coupling ligation-assisted SDA (LSDA) and CRISPR/Cas14 system, ultrahigh sensitivity is achieved with low background signal. This method not only reports consistent results comparing with standard qRT-PCR technique, but can discriminates piRNA expressions in samples from breast cancer patients. Therefore, a reliable and practical tool is provided for piRNA related clinical applications.

MicroRNA signatures provide an informative readout of host-response dysregulation in pediatric sepsis, but their clinical translation remains limited by low target abundance and multiplexing requirements. Here, a target amplification-free CRISPR biosensing platform was established. The sensing mechanism couples Cas13a target recognition to promoter release, template reconstruction, and transcription-driven Cas13a reactivation, enabling fluorescence amplification through internal molecular cycling without amplifying the target miRNAs. Then, an electrowetting-based digital microfluidic chip was designed to compartmentalize parallel reactions and automate droplet generation, reaction assembly, isothermal incubation, magnetic separation, and fluorescence readout. Under optimized conditions, four sepsis-associated biomarkers (miR-15a, miR-21, miR-146a, and miR-155) and the reference control (snRNA U44) were detected at attomolar levels (expected mean input of 1.67 molecules per reaction) with good specificity and minimal inter-droplet interference. The resulting miRNA profiles were then used as input features for machine learning-enhanced sepsis classification and risk assessment. In 252 clinical samples, the resulting model achieved a test-set AUC of 0.973 and an overall accuracy of 92.1%. The model-derived risk score was associated with the Phoenix Sepsis Score and, in a representative longitudinal case, showed preliminary potential to reflect disease evolution. Overall, this platform enables automated analysis of disease-associated miRNA signatures and demonstrates its potential for in-vitro diagnosis of pediatric sepsis.

Herein, a self-powered biosensor (SPB) was constructed using a biomass-derived bioanode and a Zn/Fe zeolitic imidazole framework (Zn/Fe-ZIF) derived-abiotic cathode. Specifically, a natural plant biomass (Juncus effuses) was carbonized to prepare a three-dimensional hierarchical carbon scaffold named as CJE, on which carbon nanotubes (CNTs) were grown under the catalysis of Co, Ni, and Co/Ni catalysts, respectively. The length, diameter and coverage of CNTs on CJE were regulated via adjusting the type and mass proportion of the metal catalysts. The optimum signal output (3.61 μW cm[-2]) is achieved by the SPB with the CJE anode substrate catalyzed by the maximum Co loading (Juncus effuses immersed in10 mM Co[2+]), which enables the uniform growth of high-density, small-diameter CNTs on the CJE. The highly conductive CNTs effectively promote electron transfer from glucose oxidase (GOx) to the electrode and increase the specific surface area of the CJE. The GOx-modified bioanode exhibits excellent catalytic activity toward glucose oxidation under mild neutral conditions. Meanwhile, Zn/Fe-ZIF was calcined to obtain a composite cathode catalyst composed of iron oxides, iron carbide, and N-doped carbon, which electrochemically catalyses O2 reduction at a relatively positive potential (E1/2 ≈ 0.862 V vs RHE). Based on an enzyme activity inhibition strategy, this SPB can sensitively monitor a vitally important cellular metabolite H2O2, which is closely associated with the occurrence and development of cancer cells. The application of abiotic non-precise metal cathode with high O2 reduction activity greatly reduces the preparation cost and improves the stability of the SPBs. More importantly, this work also provides an effective strategy for constructing portable and sensitive sensors.