Flexible piezoresistive sensors are extensively applied in human physiological monitoring and dexterous hand motion detection. However, they often suffer from a trade-off between high sensitivity and wide sensing range, as well as limited linearity induced by the intrinsic microstructural drawbacks. In this study, we propose a flexible piezoresistive sensor featuring a well-ordered and three-level pyramidal microstructure (OTPm) fabricated by laser direct writing and wet etching to overcome such limitations. Benefiting from stress concentration at pyramid tips and stepwise interfacial contact, the OTPm sensor achieves progressive conduction behavior, which effectively enhances sensitivity and suppresses pressure saturation. Notably, the OTPm sensor exhibits three linear sensitivity stages of 77.80, 39.84, and 19.20 kPa-1, a broad detection range up to 800 kPa, a low limit of detection of 98 Pa, and a response time of 91.2 ms, coupled with stable sensing performance over 10,000 cycling tests. These sensing features enable the sensor to effectively detect various physiological signals and dynamic human motion changes. Furthermore, the developed 3×3 sensor array realizes three-dimensional(3D) pressure distribution imaging and validates reliable mechanical sensing capability during grasping motions. With the balanced sensing performance, the proposed sensor presents great application potential for medical rehabilitation, robotic control, and human-machine interaction.
Thermal nociceptive sensors capable of distinguishing innocuous and noxious thermal stimuli are crucial for self-protective humanoid robotics, adaptive prosthetics and intelligent human-machine interactions. However, most artificial thermal nociceptors lack the threshold-like, nonlinear and ion-mediated required to emulate biological thermal nociception mediated by transient receptor potential vanilloid 1(TRPV1), a key heat-activated ion channel protein in humans. Here, we report a neuromorphic thermal nociceptive sensing system consisting of an ion-regulated thermal sensor (iRTS) and an ion-gated synaptic transistor (iGST) based on layered semiconductors. Through ionic modulation of nonlinear thermally activated charge transport in the layered WSe2/MoS2 heterojunction, the iRTS exhibits a clear turning point at ∼320 K, close to the human noxious-heat perception threshold, beyond which the thermally evoked current increases nonlinearly with a high sensitivity of up to ∼35% K-1, while maintaining an ultrahigh resolution of 0.005 K and an mA-level thermally evoked output current. By integrating the iRTS with the iGST, these thermal nociceptive signals are further converted into post-synaptic outputs that exhibit adaptive threshold modulation and sensitization behaviors, including hyperalgesia and allodynia-key features of biological thermal nociception. In addition, the iGST output is quantitatively mapped to a projected robotic finger-angle response, extending the artificial nociceptive pathway from thermal sensing and synaptic processing to behavior-level output. These results establish a high-performance neuromorphic thermal nociceptive sensor platform and offer a device-level strategy for intelligent robotics and adaptive human-machine interfaces.
The development of rapid, sensitive, and specific nucleic acid assays is pivotal for advancing molecular detection in clinical diagnosis, food safety, and environmental monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems, renowned for their programmable and signal amplification capacity, have emerged as efficient tools for meeting these challenges. However, the intrinsic sensitivity of CRISPR/Cas assays relying on a single Cas effector is typically confined to the picomolar level, often necessitating complex nucleic acid preamplification. Cascaded CRISPR systems, which integrate sequential enzymatic reactions or multiple CRISPR effectors, can address this limitation by achieving nucleic acid preamplification-free signal enhancement. This review starts with the introduction of the design principles and working mechanisms of cascaded CRISPR strategies, encompassing Enzyme-Coupled cascades, Multi-effector class 2 CRISPR cascades, and Type III CRISPR-mediated cascades. Then, we highlight the deployment of these techniques across diverse bio-sensing scenarios, ranging from disease diagnosis to food and environmental surveillance. Finally, critical challenges and emerging frontiers are discussed, including integration with digital detection platforms and AI-assistant algorithms.
Tumor-derived exosomes (TEXs) hold immense promise as biomarkers for cancer diagnosis, but sensitive and accurate identification is hindered by their intrinsic heterogeneity and extreme scarcity. Co-profiling of exosomal protein and microRNA (miRNA) at the single-molecule level may greatly enhance the precision of cancer diagnosis and prognosis. Herein, we present a single microbead-confined dual-mode detection system that selectively captures TEXs for concurrent quantification of protein and miRNA biomarkers. Exosomal proteins are quantified by measuring fluorescence signal concentrated on a single microbead-based enrichment platform. After on-bead lysis, individual miRNA is recognized by the two peptide nucleic acids (PNAs) to form a strict one-to-one correspondence with the intense light-scattering signal of gold nanoparticles (AuNPs), enabling single-molecule counting analysis. Through comprehensive profiling, a panel of four conjoint biomarkers achieve the highest diagnostic accuracy of 98.33%, outperforming any individual markers or alternative combinations. Importantly, this is the first demonstration of a universal PNA modification strategy that harnesses a DNA mediator to prevent AuNPs self-aggregation. This platform can also be adapted to other cancers by simply changing the aptamers and PNAs to target corresponding biomarkers, showing significant potential for advancing exosome-based liquid biopsies and accelerating the development of next-generation clinical diagnostic tools.
Uranium, a radioactive and toxic pollutant, poses significant threats to human health. The development of sensitive platforms for uranium detection and bioimaging remains challenge. Herein, a pyridine-based covalent organic framework (COF) was synthesized via a Schiff-base reaction using 2,2'-bipyridine-5,5'-dicarbaldehyde (Bpydah) and 1,3,5-tris(4-aminophenyl)benzene (TAPB). Subsequently, europium ions (Eu3+) were anchored to the pyridinic nitrogen sites of the COF framework via post-synthetic coordination, enhancing the intrinsic fluorescence and yielding a europium-functionalized sensor (COF@Eu). Attributed to the combined contributions of photoinduced electron transfer (PET) and Förster resonance energy transfer (FRET), COF@Eu exhibits a fluorescence "turn-on" response toward uranyl ion (UO22+), with a detection limit of 3.3 nM and good reliability in complex sample matrices (RSD < 2.9%). Moreover, the "turn-on" fluorescence response, coupled with favorable biocompatibility and low cytotoxicity of COF@Eu, facilitates UO22+ imaging in cells and mice. This in vivo imaging capability highlights the potential of COF@Eu for visualizing internal uranium exposure. This work establishes a multifunctional platform, offering a potential strategy for environmental monitoring and nuclear toxicology.
Precise regulation of nanozyme activity is crucial for expanding their practical applications in complex multi-domain environments, yet designing a bidirectional precision regulation strategy remains a significant challenge. Herein, this study developed a novel strategy based on photoinduced and phosphate-mediated of nanozymes, enabling bidirectional and dynamic regulation of enzymatic activity. Specifically, UV-responsive nitrogen-doped carbon quantum dots (NCDs) served as photosensitive materials were introduced with 2D nanosheets copper-iron layered double hydroxide (CuFe-LDH) to form a composite structure (NCDs/LDH, NL). Photo-induced method could enhance peroxidase-like (POD) activity by facilitating interfacial electron transfer, which accelerates the valence state cycling of copper and iron centers. In addition, phosphate-mediated coordination suppressed the catalytic reaction by forming metal-oxygen-phosphorus coordination bonds that alter the electron density of the active sites. Both regulatory modalities operate by modulating the electron transfer and valence transition processes at the copper and iron active centers. Based on this mechanism, the constructed nanozyme-based sensor platform demonstrates effective performance in the accurate screening of multiple antioxidants. Principal component analysis of the response dataset revealed well-defined clustering of all six antioxidants, with 95% confidence ellipses being sharply separated, demonstrating its high discrimination capability. This work not only elucidates the regulatory principles of photo-induced method and phosphate-mediated coordination in the bidirectional control of nanozyme activity, but also provides a new design pathway for developing intelligent nanozymes applicable to complex biological and environmental systems.
Diabetes, hyperuricaemia, dyslipidaemia, and related metabolic disorders often coexist, creating a need for point-of-care testing (POCT) that can analyse multiple biomarkers from a small whole-blood specimen. Most disposable electrochemical strips remain single-analyte devices and therefore require repeated sampling and separate strips. Here, we developed a disposable five-channel electrochemical test card based on a fivefold rotationally symmetric stepped radial microfluidic architecture. A 10 μL aliquot of whole blood is introduced once through the central inlet and passively distributed to four amperometric channels for glucose (Glu), uric acid (UA), total cholesterol (TC), and β-hydroxybutyrate (β-HB), together with an impedance channel for independent haematocrit (HCT) estimation. The injection-moulded substrate integrates embedded copper traces with gold-plated sensing areas and requires no pump or valve. A glucose-preloading challenge produced a mean maximum receiver-channel crosstalk of 0.675 ± 0.077%, with a highest individual value of 0.815% under the nominal loading condition. In 20 clinical whole-blood samples, test-card results correlated with the corresponding reference methods (R2 = 0.9484-0.9959), and analyte-specific Bland-Altman analysis supported preliminary agreement within the tested ranges. This disposable microfluidic electrochemical test card enables comprehensive multi-metabolite analysis under POCT conditions using only 10 μL whole blood, and offers a promising technical solution for next-generation precision management of metabolic diseases and multiplex POCT systems.
Wearable gel electronics require soft material platforms capable of supporting autonomous tactile sensing and physiological monitoring within a compact system. Here, we report a raccoon-paw-inspired porous hydrogel electronic skin for self-powered slip perception and artificial sweat fingerprint recognition. A sacrificial sucrose-template strategy was used to construct an interconnected porous hydrogel network with low volume density, high compressibility, rapid liquid uptake, and continuous ionic transport. After loading with the [Fe(CN)6]3-/4- redox couple, the hydrogel generates a thermogalvanic output under a temperature difference. Benefiting from the active piezoresistive effect based on thermoelectricity, the electronic skin enables self-powered pressure sensing with a sensitivity of 161.3 kPa-1, a detection limit of 2.5 Pa, and response and recovery times of 65 and 75 ms, respectively. It is further coupled with a honeycomb-structured contact layer to capture sliding-induced current signals for surface texture perception, achieving 95.23% accuracy in recognizing nine surface textures. In addition, the hydrogel network converts ion-dependent diffusion dynamics of mixed sweat electrolytes into time-resolved sweat fingerprints, allowing eight artificial sweat compositions to be identified with 95.0% accuracy. The recognized compositions are further mapped to three electrolyte-status levels for electrochromic visual feedback. This porous hydrogel platform provides a simple strategy for integrating self-powered tactile perception with artificial sweat classification in wearable electronics.
We report MetaVision-SPR, a 96-well chromatic metasurface plasmon resonance (Meta-SPR) imaging platform for serum biomarker quantification and apparent kinetic analysis. The system integrates a nanocup-array Meta-SPR chip, light-emitting diode (LED) illumination, an industrial camera, and computational correction including automated well recognition, sucrose-reference-based optical flat-field correction, and Δ(R-G) chromatic readout. Spectral simulation and optical response matching identified the 20/75/25 nm Ti/Ag/Au structure and Δ(R-G) signal as the optimal chip-readout combination for quantitative imaging. Flat-field correction reduced inter-well variation in a Protein A/immunoglobulin G (IgG) assay from 26.74% to 6.74%, while programmable shaking improved mass transport in the open-well format. Using gold nanoparticle (AuNP)-enhanced sandwich assays, the platform enabled sensitive detection of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) with limits of detection (LoDs) of 5.0 pg/mL for AFP, 10.0 pg/mL for CEA and 0.49 U/mL for CA19-9. Clinical serum measurements showed reference-method consistency with hospital electrochemiluminescence immunoassay (ECLIA) and commercial enzyme-linked immunosorbent assay (ELISA) results. In addition, the system supported real-time measurement of binding curves for antibody-drug conjugate and lipopolysaccharide-related interactions, yielding apparent kinetic parameters suitable for high-throughput screening. These results support MetaVision-SPR as a low-hardware-cost 96-well chromatic Meta-SPR workflow for serum biomarker measurement and apparent interaction ranking.
Nanozymes with dual enzyme-like behaviors have broad application prospects in the field of biosensing, but their catalytic activity is usually restricted to acidic conditions. Therefore, developing bifunctional nanozymes with high activity under neutral pH conditions is particularly important. Here, a histidine-functionalized 2-aminoterephthalic acid-Cu (Cu-BDC-NH2@His) nanozyme was fabricated via a defect engineering approach. Relative to Cu-BDC-NH2, the obtained Cu-BDC-NH2@His nanozyme exhibited higher peroxidase-like (POD) and laccase-like (LAC) activities under neutral pH conditions. Given that different sulfides exerted divergent regulatory impacts on the dual enzyme-like behaviors of Cu-BDC-NH2@His, the array units were constructed using three unique response signals from the nanozyme sensing system (POD-370, POD-652 and LAC-510), and a "fingerprint" analysis spectrum of sulfides was further established. In addition, the robust discrimination model with simultaneous concentration- and matrix-independent performance was constructed by combining machine learning (ML) with the array system to classify multiple sulfides spiked in three food samples. The sulfide identification accuracy of the array was increased from 55.17% to 100%. Finally, a visualized on-site intelligent sensing platform was established using chromaticity imaging and deep learning algorithms to further improve the practicality of sulfide identification. This study not only provides a novel approach to overcome the pH limitation of nanozymes, but also offers an innovative intelligent sensing paradigm for the analysis of complex food matrix samples under neutral pH conditions.
CRISPR/Cas12a combined with nucleic acid amplification enables highly specific and sensitive detection. However, its broader deployment is constrained by protospacer adjacent motif (PAM) dependence, multistep workflows, and limited reagent practicality. Here, we identify a PAM-independent Cas12a activator, termed mosaic DNA, which exhibits structural features intermediate between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Building on this finding, we develop a PAM-independent mosaic switch triggered by a solid-liquid phase transition, thereby addressing these limitations within a single platform. Distinct from previous reports, our experiments show that this activator is generated prior to the digestion of dsDNA into ssDNA by the lambda exonuclease, forming the basis of lambda exonuclease-driven Cas12a activation, a process we refer to as the mosaic switch. Mosaic switch can detect arbitrary dsDNA with sensitivity comparable to that of PAM-containing dsDNA, and maintain single-nucleotide discrimination. Lyophilizing mosaic switch reagents and encapsulating them in paraffin improve stability and usability, which also enables straightforward one-pot integration with recombinase polymerase amplification (RPA) via a solid-liquid phase transition. Applied directly to 58 extraction-free mpox clinical samples, this platform showed complete concordance (100%) with quantitative PCR. This CRISPR/Cas12a platform maintains analytical performance while broadening the range of targets, simplifying the workflow, and enhancing reagent practicality, showing great potential for clinical deployment.
Accurate monitoring of intracellular glutathione (GSH) fluctuations at the single-cell level is significant to gain an in-depth understanding of related pathological events and facilitate disease diagnosis. However, conventional sensing interfaces are susceptible to nonspecific interference and surface passivation in complex biological systems, severely restricting the development of high-performance platforms for single-cell analysis. Herein, an antifouling nanoelectrochemical sensor integrating sulfonated cobalt phthalocyanine (CoPcS) and a zwitterionic poly(dopamine-co-sulfobetaine methacrylate) layer fabricated via one-step electropolymerization was developed for GSH detection. The zwitterionic sensing interface exhibited prominent anti-interference and antifouling performance in high-concentration protein media, retaining over 90% of its initial electrochemical signals after incubation with 10 mg/mL BSA for 2 h. Benefiting from the highly selective electrocatalytic capability of CoPcS and the hydration protection effect of the robust zwitterionic polymer layer, the proposed nanosensor achieved reliable electrochemical detection of GSH. The results of single-cell analysis indicated that the proposed nanosensor successfully distinguished the different GSH levels in A549 cells and HeLa cells, and accurately responded to drug-induced intracellular GSH fluctuations. This nanosensing platform provides a powerful tool for revealing cellular heterogeneity and exploring disease pathogenesis, holding great promise for biological research and clinical therapeutic applications.
Traditional CRISPR-Cas12a mutation detection systems are limited by poor single-base specificity, target-specific crRNA redesign, and insufficient sensitivity for low-abundance mutations, restricting their clinical liquid biopsy applications. Herein, we developed a crRNA-universal, sensitive and specific CRISPR-Cas12a detection platform, termed DESIC (double-end blocker and split-input mediated CRISPR-Cas12a system), for single-base mutation detection. The DESIC system adopts two key structural designs: double-end blocker (DEB) and duplicated split-input (SIN). The DEB spatially isolates crRNA recognition and target-binding regions, enabling universal detection of various mutation sites without crRNA redesign. The SIN strategy amplifies thermodynamic differences from single-base mismatches, greatly improving single-nucleotide discrimination. We targeted four prevalent pancreatic cancer KRAS mutations (G12D, G12R, G12V, Q61H) and optimized the system to achieve optimal discrimination. The optimized DESIC system exhibited ultra-low limits of detection down to 0.01% mutant allele fraction with reliable linear quantitative performance. Clinical validation using 15 pairs of pancreatic cancer tissue and peripheral blood samples confirmed that DESIC results were highly consistent with gold-standard NGS data. With a flexible modular design, this low-cost, easy-operated platform can be readily extended to multiple tumor mutations, holding great potential for tumor liquid biopsy and early molecular diagnosis.
Split-NanoLuc biosensors for homogeneous small-molecule detection are often limited by structural heterogeneity arising from random conjugation, which compromises enzyme complementation efficiency and analytical reproducibility. Here, a site-specifically labeled split-NanoLuc biosensor was developed for rapid detection of 3-phenoxybenzoic acid (3-PBA), a representative biomarker of pyrethroid insecticide exposure. A site-specific chemoenzymatic labeling strategy integrating sortase A-mediated ligation with oxime-based bioorthogonal chemistry was employed to generate a structurally homogeneous LgBiT-3-PBA tracer. Combined with a SmBiT-nanobody recognition probe, the system enabled efficient proximity-induced NanoLuc complementation in a competitive assay format. Structure-informed linker optimization yielded a signal-to-noise ratio > 28, a half-maximal inhibitory concentration (IC50) of 2.3 ng/mL, and a detection limit of 0.41 ng/mL, with a total assay time of 10 min and no washing steps required. This represents an approximately seven-fold improvement in sensitivity compared with the previously reported 3-PBA homogeneous detection method. The sensor demonstrated satisfactory recoveries in urine and water samples and good agreement with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (R2 = 0.98). This work establishes a site-specific conjugation strategy for constructing homogeneous, high-performance split-NanoLuc biosensors, which holds potential for broader applications in small-molecule detection.
Non-invasive urine-based cancer biomarker detection holds significant clinical value in cancer screening, treatment efficacy monitoring, recurrence warning, and auxiliary diagnosis. To address the issues of poor sensitivity and selectivity in photoelectrochemical (PEC) biosensing for the detection of cancer biomarkers in urine, oxygen vacancies (OVs)-induced dual-interface modulation strategy is proposed for constructing a TiO2/BiOI-based PEC sensor. The introduction of OVs not only induces the in-situ formation of bismuth nanoparticles at the solid-solid interface but also generates positive surface charge at the solid-liquid interface, which promotes the formation of the Z-scheme heterojunction and the modification with a negatively charged anti-fouling polymer layer (C-PEG), thereby effectively enhancing the photoelectric conversion efficiency and antifouling performance of the PEC sensor. Meanwhile, functionalization with aptamers further improves its selectivity. Leveraging the synergistic effects of the Z-scheme, anti-fouling polymer layers, and aptamers, the developed PEC sensor achieves highly sensitive and selective detection of alpha-fetoprotein (AFP), with a linear relationship in the range of 1.0 pg/mL∼150.0 ng/mL, and a detection limit of 0.3 pg/mL (LOD = 3s/k). In addition, the sensor successfully measures AFP levels in serum and urine samples from healthy individuals and hepatocellular carcinoma patients, and statistical analysis reveals a strong correlation between the serum and urinary AFP concentrations (r = 0.934, P < 0.001, n = 9). These findings indicate the preliminary feasibility of the proposed PEC sensor for non-invasive urine-based AFP detection, which may provide insights for future research.
A high-performance glucose biosensor based on a U-shaped Sagnac optical microfiber coupler (OMC) functionalized with a polyethylene glycol/chitosan-glucose oxidase (PEG/CS-GOD) composite coating is presented. The sensor is designed by integrating the Sagnac interferometer's common-mode noise rejection with the U-shaped microfiber's strong evanescent field, effectively addressing environmental instability in micro-structured optical sensors, while the multifunctional coating simultaneously provides antifouling properties, biocompatibility, and enzymatic specificity. The sensor exhibits a high refractive index sensitivity of ∼1717 nm/RIU, glucose sensitivities of 3.17 and 3.09 nm mL·mg-1 for two interference dips with excellent linearity (R2 > 0.99), a detection limit of 0.224 mg/mL, and a response time of 13 s in simulated urine. Operational stability is demonstrated by minimal hysteresis error (<2%), approximately 81% response retention over 14 days, and satisfactory selectivity against key interferents at physiologically relevant concentrations. Consistent performance in simulated urine (3.03 nm mL·mg-1) and artificial serum (3.03 nm mL·mg-1) confirms dual-matrix detection capability. A dual-dip demodulation matrix enables real-time temperature compensation. With its balanced combination of high sensitivity, rapid response, long-term stability, and dual-matrix compatibility, this biosensor represents a promising platform for future point-of-care glucose detection.
MicroRNAs (miRNAs) in blood are promising liquid biopsy biomarkers, yet their short length, low abundance, and high intra-family homology hinder sensitive and specific detection. Combining rolling circle amplification (RCA) with CRISPR-Cas12a enables isothermal detection, but existing methods typically depend on auxiliary enzymes or in-assay ligation and rarely encode sequence discrimination within the template itself. Here, we report a one-pot dual-toehold RCA (dtRCA)-Cas12a biosensor driven by a preassembled three-arm toehold-gated (3TG) DNA template for ultrasensitive and selective miRNA detection. The 3TG template adopts a three-arm dumbbell conformation, eliminating the need for a ligase during the assay, and presents two target-complementary toehold domains with a Cas12a-recognition sequence. Target binding triggers strand displacement, initiating dtRCA via a single polymerase. The resulting amplicons activate Cas12a trans-cleavage for fluorescence or lateral flow assay (LFA) readouts. Crucially, a single-base mismatch within the toehold suppressed amplification, whereas a topology-matched circular template lacking the toehold gate failed to distinguish the target, demonstrating that selectivity arises from the template structure. The one-pot dtRCA-Cas12a system achieved attomolar sensitivity, detecting miR-21, miR-375, and let-7a at 2.5, 114.9, and 8.0 aM, respectively. The paper-based LFA maintained femtomolar sensitivity and enabled an instrument-light readout. In plasma, this platform discriminated breast cancer patients (n = 17) from healthy donors (n = 10) with AUC values of 0.97-0.98. Three-marker classification demonstrated robust performance in leave-one-out cross-validation and correctly classified 30 samples in an independent validation cohort, showing performance comparable to RT-qPCR. By embedding selectivity into a preassembled template, this 3TG-driven dtRCA-Cas12a platform provides a highly sensitive and specific strategy for multi-marker miRNA analysis with simplified readout.
