To address the growing challenge of bacterial antibiotic resistance, this study designed and synthesized chiral carbon quantum dots (h-DCQDs) from levofloxacin and D-histidine. The h-DCQDs exhibit strong broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria, while also inhibiting the development of bacterial resistance and promoting wound healing. The antibacterial mechanism of h-DCQDs involves reactive oxygen species generation and specific interaction with bacterial cell walls/membranes, leading to structural disruption, content leakage, and cell death. Owing to their excellent biocompatibility and fluorescence, h-DCQDs can also serve as effective bacterial imaging probes. This work highlights the innovation of using chiral carbon quantum dots, which achieve bacteria-specific targeting and integrate antimicrobial and reporting functions. This offers a promising strategy for tracking and treating bacterial infections.

Establishing robust pharmacokinetics-pharmacodynamics (PK-PD) correlations remains a major challenge owing to high selectivity and low permeability of the blood-brain barrier (BBB), which limits the predictive power of conventional plasma pharmacokinetics on specific brain tissue. Here, we present a highly biomimetic microfluidic BBB-brain organ-on-a-chip combined with liquid chromatography-mass spectrometry (LC-MS) and electrochemical sensing technology for micro PK-PD monitoring with target cells. The platform incorporates human cerebral microvascular endothelial cells and neuron-like cells cultured on opposite sides of a collagen/fibronectin-modified porous membrane under physiological shear stress. This configuration reinforces the physical, metabolic and physiological barrier functions of BBB, as evidenced by the high expression of tight junction proteins, low apparent permeability, expression of efflux transporters, and reversible response to hypertonic stimuli. A neurodegenerative disease model is induced using 1-methyl-4-phenylpyridinium iodide (MPP+) to recapitulate key pathological features of early-stage Parkinson’s disease. The pharmacokinetic profile of pramipexole (PPX) is monitored using both LC-MS and an integrated, regenerable electrochemical sensor. The sensor enables in situ, real-time and online detection of PPX with high sensitivity and specificity, showing strong concordance with LC-MS. Furthermore, neurotransmitter (norepinephrine) exocytosis level is quantified as a pharmacodynamic indicator, enabling micro PK-PD correlation within the disease-on-a-chip model. Collectively, the proposed new method for micro PK-PD study is expected to provide great prospects for the preclinical screening and action mechanism research of novel anti-Parkinson’s disease drugs.

Sepsis diagnosis and prognosis are hindered by the limitations of traditional single-parameter assays, which are costly, slow, and impractical for resource-limited settings. To overcome this, we developed a novel combined nanozyme-linked immunosorbent assay (NLISA) kit for the multiplexed detection of three critical sepsis biomarkers: C-reactive protein, serum amyloid A, and D-dimer. Our approach leverages a unique flower-like CoFe2O4/MoS2 nanosheet with dual substrate affinity, which serves as a highly stable and efficient nanozyme label. This sandwich-type immunocomplex enabled us to achieve robust sensitivity, specificity, and reproducibility at clinically relevant concentrations. Clinical validation using plasma samples from sepsis patients demonstrated strong concordance with the collective data from three separate enzyme-linked immunosorbent assay tests, confirming a significant improvement in the diagnostic reliability. Our NLISA kit not only simplifies multibiomarker detection but also provides a more comprehensive and accessible tool for sepsis diagnosis and monitoring, offering a powerful alternative to conventional methods.

The growing threat of bacterial adaptive resistance against nanomaterials necessitates an in-depth understanding of the molecular mechanisms underlying nano-bactericidal effects. Carbon quantum dots (CQDs) have emerged as promising functional nanomaterials for bioimaging, biosensing and biomedical detection, whereas high-performance CQDs eliminating drug-resistant bacterial infections remain greatly limited. Herein, quaternized tartaric acid-based carbon quantum dots (TDAQDs) with prominent antibacterial potency and strong ability to restrict bacterial drug resistance were synthesized using tartaric acid and diallyldimethylammonium chloride (DDA). The average particle diameter (1.21 nm), zeta potential measurement (+35.5 mV), and the MIC (at 5 μg/mL for S. aureus and clinical multidrug-resistant MRSA, and 15 μg/mL for E. coli) were the core quantitative physicochemical and biological parameters of TDAQDs. TDAQDs bind to bacteria through electrostatic interaction and induce reactive oxygen species overproduction, which disrupts bacterial membrane structure and triggers massive cytoplasmic leakage. TMT-based quantitative proteomics analysis revealed that TDAQDs markedly disturbed core biological pathways of S. aureus, including ribosome function, RNA degradation and substance metabolism, while suppressing ABC transporter-associated bacterial pathogenic processes. In vivo wound healing quantification illustrated superior therapeutic performance (91.35% and 88.88% for S. aureus and E. coli-infected wounds), confirming prominent infection elimination and accelerated skin regeneration in TDAQDs-treated groups. In vitro and in vivo biosafety tests verified the good biocompatibility of TDAQDs with negligible cytotoxicity to H9C2 cells, erythrocytes and major mouse organs. Collectively, these findings establish TDAQDs as a robust, biocompatible nanoplatform that simultaneously combats drug-resistant bacteria and mitigates resistance evolution, representing a meaningful step toward developing next-generation antimicrobial strategies to address the global crisis of antibiotic failure.

Wearable sweat sensor systems, which monitor target biomarkers in sweat, play a crucial role in sports and health monitoring. However, there are two major challenges: first, the difficulty of simultaneously monitoring multiple biomarkers; and second, the inability to dynamically adjust sensitivity, which limits their detection range. To address the complexity of sweat composition and the wide variation in concentration ranges, we propose an adaptive amplification circuit for extremely weak current signals, combined with a time-division detection strategy tailored for sweat sensor systems. This system enables dynamic adjustment of current sensitivity and simultaneous detection of multiple biomarkers in sweat, such as glucose, lactate, Na+, and K+. We utilize an integrated, numerically controlled, switchable feedback resistor network transimpedance amplifier (TIA) circuit architecture to achieve dynamic adjustment of current sensitivity over a broad range from 10 nA to 2 mA and to enhance the linear correlation coefficients for glucose and lactate detection, reaching 0.998 and 0.995, respectively. To further realize multi-channel synchronous acquisition, the system employs a numerically controlled switch circuit combined with a time-division multiplexing strategy and dual TIA channels to simultaneously monitor 4 types of sweat biomarkers. During in vivo cycling experiments, the sensor system successfully demonstrated in vivo monitoring of 4 biomarkers in human sweat. This study provides an effective technical solution for multi-biomarker detection and health monitoring with a wide detection range.

Analyte-induced self-enhanced electrochemiluminescence (ECL) is a powerful strategy for simplifying ECL sensing and improving the overall sensitivity without the addition of external coreactants. However, most Ru(bpy)(3)(2+)-based ECL systems still rely on external coreactants such as tri-n-propylamine (TPrA) or oxalate, which introduce additional reagents and increase assay complexity. Herein, we report that the moxifloxacin (MFX) can intrinsically function as an efficient coreactant for Ru(bpy)(3)(2+), enabling an external-coreactant-free and self-enhanced ECL system for its sensitive detection. Electrochemical and ECL investigations reveal that MFX participates directly in the ECL pathway through anodic oxidation, generating reactive intermediates that efficiently trigger the excited Ru(bpy)(3)(2+)* state. As a result, the Ru(bpy)(3)(2+)-MFX system generates similar to 17-fold higher ECL intensity than the classical oxalate system and a comparable ECL response to TPrA (similar to 66%) under identical conditions. Leveraging this intrinsic self-amplification, an ECL method was developed for sensitive and selective detection of MFX. The developed method exhibits two linear dynamic ranges (0.05-200 mu M and 200-800 mu M) with a limit of detection (LOD, S/N = 3) of 36 nM. The proposed method offers excellent sensitivity, reproducibility, and selectivity against common interferents and ophthalmic excipients. Moreover, validation using commercial ophthalmic formulations demonstrated excellent agreement with HPLC results, supporting the accuracy of the developed method for pharmaceutical quality control. By integrating analyte recognition and signal generation into a single molecular entity, this work opens a new pathway toward simple and green self-enhanced ECL sensing platforms.

Chemiluminescence (CL) ranks among the most intrinsically sensitive signal-generation strategies in analytical chemistry, distinguished by its near-zero optical background and excitation-free operation. Nevertheless, classical CL architectures remain constrained by transient, diffusion-limited kinetics and ensemble-averaged signal outputs, which limit spatiotemporal precision and suppress analytical information depth. Recent advances are reshaping the analytical paradigm of CL, transforming it beyond conventional detection into a programmable, reconfigurable, and information-centric modality. This review critically examines the foundational design principles enabling this transition. The evolution from passive intensity-based readouts to engineered kinetic and spatial regulation is explored through advances in reaction and excited-state reprogramming, temporal control, spatial confinement, and non-equilibrium system design. These strategies enable sustained, delayed, oscillatory, cyclic, and pattern-forming emission profiles; spatial resolution from nanoscale confinement to single-entity imaging; and the emergence of autonomous and stimuli-responsive chemiluminescent systems. Concurrently, multicolor encoding, ratiometric analysis, logic gating, signal amplification, and machine-learning–assisted interpretation expands CL from a scalar intensity signal into a multidimensional analytical language capable of intelligent sensing and dynamic information processing. Persistent challenges including kinetic robustness, reproducibility, system complexity, scalability, and device integration are evaluated to clarify translational barriers and practical implementation pathways. By articulating a systems-level framework for programmable CL, this review outlines a forward-looking roadmap toward autonomous and integrated analytical technologies.

The RET M918T mutation is a critical biomarker for early screening of medullary thyroid carcinoma (MTC). However, its ultralow abundance amid excess wild-type sequences poses challenges for accurate detection and limits the suitability of conventional methods for rapid point-of-care testing (POCT). Herein, a paper-based bipolar electrode-electrochemiluminescence (BPE-ECL) and colorimetry dual-modal biosensor integrating 2'-fluorinated nucleotide (2'-FN)-modified Y-shaped junctions with g-C3N4@FeMOFs nanozyme-mediated amplification was designed to enable sensitive and single-base resolution detection of M918T. The rigid 2'-FN Y-shaped junctions enhanced mismatch discrimination, while the g-C3N4@FeMOFs nanozyme exhibited strong peroxidase-like catalytic activity to simultaneously strengthen the ECL emission of Ru(bpy)32+/tripropylamine (Ru(bpy)32+/TPrA) and the colorimetric image of oxidizing 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue chromogenic product in paper-based device readily quantified by smartphone imaging. Under optimal conditions, the proposed dual-mode strategy enabled detection of M918T with limits of 0.76 pM (ECL, S/N = 3) and 1.4 pM (colorimetry, S/N = 3) and demonstrated successful applicability in human serum samples. Both signals served as mutual self-validation for avoiding false-positives. This integrated, low-cost and portable sensing platform offers a rapid and reliable strategy for clinical biomarker detection, showing great potential for early diagnosis and on-site screening of MTC and related diseases.

BACKGROUND:Cluster of differentiation 123 (CD123) prominently overexpress in various hematological malignancies and plays a crucial role in diagnosis and prognosis of leukemia. Clinical studies have demonstrated that cell-free CD123 levels also significantly influence leukemia immunotherapy outcomes. The development of novel electrochemical immunosensors addresses the need for point-of-care detection tools, thereby facilitating advancements in clinical monitoring technologies. METHODS:This study presented a practical electrochemical immunosensor constructed using sandwich strategy for CD123 detection based on the modification of gold nanoparticles and poly (2-aminoterephthalic acid). Common proteins and tumor-related biomarkers found in human were selected as interference factors to evaluate the detection specificity of the electrochemical immunosensor. Further, the electrochemical immunosensor was utilized to directly detect CD123 in bone marrow supernatant from leukemia patients. RESULTS:The constructed electrochemical immunosensor exhibited good linearity for CD123 detection over a range of 0.02 to 2.5 µg/mL, with a detection limit of 12.8 ng/mL, alongside satisfactory specificity and repeatability. Furthermore, the immunosensor was successfully employed to detect CD123 levels in the bone marrow supernatant of leukemia patients, demonstrating results that were highly consistent with those obtained via ELISA. CONCLUSIONS:The developed approach is anticipated to provide robust technical support for the long-term monitoring of leukemia patients during their diagnosis and treatment.

Alkaline phosphatase (ALP) is an important biomarker that reflects osteoblast activity and bone growth. Accurately detecting ALP activity is of pivotal clinical significance for the diagnosis and differentiation of bone system diseases. In this work, an enhanced bipolar electrode electrochemiluminescence (BPE-ECL) biosensor based on enzyme catalysis and electrocatalysis was proposed for ultrasensitive detection of ALP in osteoblasts. Carbon nitride nanosheets (CNNS) were utilized as electrocatalysts to facilitate the electrochemical reaction of bipyridine ruthenium (Ru(bpy)(3)(2+)) and tripropylamine (TPrA) in the reporting cell. Meanwhile, in the sensing cell, the target ALP catalyzed the conversion of p-aminophenyl phosphate monohydrate into p-aminophenol (p-AP). The p-AP was subsequently oxidized at the anode of the driving electrode, producing electrons and increasing the Faradaic current of BPE. The dual-signal amplification strategy, combining electrocatalysis and enzyme catalysis, resulted in a 10-fold enhancement of the ECL intensity of Ru(bpy)(3)(2+)/TPrA. The BPE-ECL biosensor achieved ultrasensitive detection of ALP with a detection limit as low as 1.9x10(-6) U/L, and was successfully applied to monitor ALP activity in osteoblast cells. Additionally, a portable smartphone imaging was developed for the visual detection of ALP. This research introduces an innovative BPE-ECL biosensor for the monitoring of ALP activity and point-of-care testing (POCT) in osteoblasts in clinical settings.

Detecting multiple tumor markers is of great importance. It helps in early cancer detection, accurate diagnosis, and monitoring treatment. In this work, gold nanoparticles–toluidine blue–graphene oxide (AuNPs-TB–GO) and gold nanoparticles–carboxyl ferrocene–tungsten disulfide (AuNPs–FMC–WS2) nanocomposites were prepared for labeling Carcinoembryonic antigen (CEA) antibody and Carbohydrate antigen 72–4 (CA72-4) antibody, respectively, and used as two kinds of probes with different electrochemical signals. With the excellent magnetic performance of biotin immune magnetic beads (IMBs), the biofunctional IMBs were firmly deposited on the magnetic glassy carbon electrode (MGCE) surface by applying a constant magnetic field, and then the CEA and CA72-4 antibody were immobilized on the IMBs by the avidin–biotin conjugation. The assay was based on the change in the detection peak current. Under the optimum experimental conditions, the linear range of detection of CEA is of the two-component immunosensor is from 0.01 to 120 ng/mL, with a low detection limit of 0.003 ng/mL, and the linear range of detection of CA72-4 is from 0.05 to 35 U/mL, with a detection limit of 0.016 U/mL. The results showed that the proposed immunosensor enabled simultaneous monitoring of CEA and CA72-4 and exhibited good reproducibility, excellent high selectivity, and sensitivity. In particular, the proposed multiplexed immunoassay approach does not require sophisticated fabrication and is well-suited for high-throughput biosensing and application to other areas.

Helicobacter pylori (H. pylori) have garnered increasing attention due to its high infection rate and potential links to a variety of diseases. Achieving a radical cure is of significant importance for public health. However, drug resistance resulting from single nucleotide variations (SNVs) of H. pylori poses a challenge. Accurate identification of these SNVs is essential for effective diagnosis and treatment. In this study, we designed a single-base precision DNA-driven theranostics test strip, and proposed a novel responsive treatment strategy targeting H. pylori. These probes were fixed in separate regions on a carbon-dot-modified strip substrate, onto which samples can be loaded. Two probes loaded with oxytetracycline (OTC) and silver clusters (Ag NCs) that can be activated by either SNV or wild-type (WT) sequences of H. pylori. Fluorescent signals were utilized for genotype identification, while WT or SNV sequences triggered distinct modes of drug release, leading to a multiple treatment. A simulated theranostics assay was conducted on H. pylori-infected mice while the efficacy and impact were thoroughly characterized. Histopathological and immunohistochemical staining results indicated no significant side effects. Further analysis, including 16S rRNA gene sequencing of intestinal microflora and metabolite profiling in the kidney, demonstrated that the combination treatment was both efficient and safe. These findings confirm the potential application of this strategy in medical care.

Background: Lamotrigine plays a crucial role in the treatment of epilepsy and bipolar disorder in adults and children. However, its pharmacokinetic (PK) behavior in first or long-term treatment in pediatric patients and the changes in drug exposure in patients with renal impairment are not well characterized. The purpose of the research was to build a robust physiologically based pharmacokinetic (PBPK) model of lamotrigine for the prediction of drug exposure in diverse populations to facilitate therapeutic drug monitoring (TDM) and guide dosing regimens. Methods: The physicochemical parameter values of lamotrigine were integrated to establish and validate the model in an adult population in PK-sim. This adult PBPK model can be extrapolated to children and patients with renal impairment to predict PK changes. Results: Most of the observed data were within the 5th and 95th percentile intervals of the variability around the predicted plasma concentrations. The model predicted pharmacokinetic thresholds and exposure values for clinically safe and effective doses recommended by the FDA for initial and long-term treatment of epilepsy in adults and children aged 2–12 years. Notably, patients with severe renal impairment and end-stage renal disease experienced an average increase in the area under the curve of 1.51 folds and 1.62 folds, respectively. This scenario necessitates further lamotrigine dose adjustments. Conclusions: The developed lamotrigine PBPK model offers a strategy for assisting clinicians in TDM and dose adjustment for special populations, thereby offering a reference (PK parameters, as well as peak and valley concentrations to reach a steady state) for a safer administration regimen in clinical treatment.

Developing sensitive analytical techniques for multiple biomarkers detection are crucial for improving clinical diagnosis and guiding medical research. However, typical detection methods, especially photoelectrochemical (PEC) biosensing, often involve natural enzymes onto (photo-)electrode surfaces, which limit electron transfer due to the insulating nature of proteins, leading to poor sensing efficiency. Herein, we report a dual-mode bioanalytical platform using Prussian blue-tungsten disulfide nanocomposites (PB-WS2 NCs) as nanozymes for colorimetric detection of cholesterol, and PEC detection of hydrogen peroxide (H2O2), and the lung cancer biomarker cytokeratin 19 fragment (CYFRA21-1), respectively. By combining the peroxidase-mimicking activity of PB with the photoelectrocatalytic properties of WS2, we achieve synergistic amplification of both catalytic and PEC signals. This PB-WS2 nanozyme enhances colorimetric sensing sensitivity for cholesterol detection and a sensitive PEC immunosensor for CYFRA21-1 in serum. Importantly, the synergistic nanozyme and PEC effects of PB-WS2 nanozyme enabled sensitive detection of H2O2 released from living cells. Moreover, it demonstrated excellent anti-interference capability for detection of multiple biomarkers in real samples. This dual-mode sensing platform demonstrates high selectivity, sensitivity, and stability, with significant potential for clinical diagnostics and nanozyme-based PEC biosensors.

Dopamine (DA) is a catecholamine neurotransmitter in the brain, and changes in its concentration are closely related to neurological diseases. Thus, the development of a sensitive, cost-efficient and reliable method for DA monitoring has important application value. Herein, a photoelectrochemical (PEC) sensor based on TiO2 and polydopamine-modified TiO2 (PDA@TiO2) was constructed to achieve highly sensitive detection of DA secreted from living nerve cells. Upon DA introduction, PDA@TiO2 has a wide range of visible light absorption, promoting light absorption. The sensitization of PDA effectively improve the charge carrier transport efficiency, promote the photocatalytic activity of TiO2. The coating of PDA promotes its hydrophilic properties, effectively excluded the biofouling macromolecules and interference signals, ensuring sensor reproducibility. The result reflected that the current reponse increase of PDA@TiO2 is 3.5 times higher than the TiO2 after DA reaction due to the PDA@TiO2 can promote efficient carrier separation. The PDA@TiO2 sensor has superior sensitivity, a wider detection linear range and long-term stability. Notably, this PEC sensor is characterized by its simplicity in fabrication, low production cost, and portability. Under optimized experimental conditions, the PDA@TiO2 sensor showed a favorable linear response to DA using PBS incubated with pheochromocytoma (PC12) cells concentration ranging from 2 to 500 μM with a detection limit of 24.7 nM (S/N = 3). Meanwhile, the sensor was effectively applied to detect DA levels in PC12 cells. This work not only provides a new and efficient strategy for signal amplification, but also provides a promising candidate platform for sensitive detection of DA, offering a valuable tool for neuroscience and clinical diagnostics.

Amidst multiple epidemics, a rapid, sensitive, economical, and portable infection diagnosis strategy is crucial for primary medical care, particularly through the analysis of pathogen sources to determine appropriate antibiotic use. C-reactive protein (CRP) and serum amyloid A (SAA) are host-related biomarkers, and their combined detection can effectively distinguish between bacterial and viral infections, which holds great significance for the diagnosis of unknown pathogens. In this work, a portable dual-channel electrochemical biosensor based on a one-step assembly of immunomagnetic beads was proposed for the on-site combined detection of plasma CRP and SAA, which streamlined the operation and shortened the minimum detection time to less than 3 min. The biosensor exhibited excellent linearity in the detection of 3.125-1250 ng/mL CRP and 31.25-1250 ng/mL SAA, with detection limits of 0.91 and 12 ng/mL, respectively, falling within the clinically relevant reference range. Through simulated sample tests, the biosensor effectively distinguished between bacterial infection, viral infection, and healthy plasma samples. The actual sample tests demonstrated a high correlation and comparable medical value to enzyme-linked immunosorbent assay. Overall, this proposed strategy showed potential to aid in infection diagnosis and enable rapid combined detection of multiple biomarkers.

Since cartilage injury is often accompanied by subchondral bone damage, conventional single-phase materials cannot accurately simulate the osteochondral structure or repair osteochondral injury. In this work, a gradient gelatin-methacryloyl (GelMA) hydrogel scaffold was constructed by a layer-by-layer stacking method to realize full-thickness regeneration of cartilage, calcified cartilage and subchondral bone. Of note, to surmount the inadequate mechanical property of GelMA hydrogel, nanohydroxyapatite (nHA) was incorporated and further functionalized with hydroxyethyl methacrylate (nHA-hydroxyethyl methacrylate, nHAMA) to enhance the interfacial adhesion with the hydrogel, resulting in better mechanical strength akin to human bone. Specifically, the biomimetic nHAMA/GelMA (B-nHAMA) scaffold involved a pure GelMA top layer for cartilage, a 30/70% (w/w) nHAMA/GelMA intermediate layer for calcified cartilage, and a 70/30% (w/w) nHAMA/GelMA bottom layer for subchondral bone. This B-nHAMA scaffold exhibited optimal porosity (continuous-gradient pore size), mechanical performance (Young's modulus, 181.48 ± 29.94 kPa), biodegradability (degradation rate in 25 day, 66.04 ± 7.19%) and swelling properties (swelling ratio in 25 h, 424.8 ± 9.9%) that cater to osteochondral tissue environment. It also showed excellent biocompatibility, cell adaptability, chondrogenic and osteogenic properties, leading to effective osteochondral regeneration. Collectively, the developed B-nHAMA scaffold with similar osteochondral microenvironment of trilayered structure could facilitate simultaneous osteochondral regeneration, providing a promising strategy to improve the full-thickness cartilage injury regeneration. STATEMENT OF SIGNIFICANCE: This research presents a significant advancement in osteochondral repair with the development of a biomimetic B-nHAMA scaffold. The scaffold's design overcomes the inadequate stiffness of gelatin-methacryloyl (GelMA) by incorporating hydroxyethyl methacrylate-functionalized nanohydroxyapatite (nHAMA), enhancing interfacial adhesion and achieving mechanical strength equivalent to human bone. Through a layer-by-layer stacking approach, the B-nHAMA scaffold features a gradient composition that replicates the anisotropic nature of osteochondral tissue, with distinct layers tailored for cartilage, calcified cartilage and subchondral bone. Its biomimetic structure, closely resembling native cartilage in physicochemical and osteogenic properties, positions the B-nHAMA scaffold as a potent therapeutic candidate for the full-thickness repair of osteochondral defects, offering a clinically viable solution.

Accurate and sensitive detection of microRNAs (miRNAs) in tumorigenesis has posed a great challenge, as its characteristics of ultra-low abundance and high sequence homology. In this approach, a highly sensitive and selective Y-shape-structured photoelectrochemical (PEC) biosensor coupled with dual signal amplification strategy was constructed with introduction of heterojunction nanocomposite and in situ enzymatic generation of electron donor. Specifically, Bi4NbO8Cl/TiO2 heterojunction allowed for efficient electron transport, further achieved the electrons-holes pairs separation, and thus greatly improved photocurrent intensity. Alkaline phosphatase (ALP) modified with auxiliary probes catalyzes hydrolysis of ascorbic acid 2-phosphate (AAP) to in situ generate ascorbic acid (AA) as electron donors for further signal amplification. Furthermore, hairpin capture probes with rigid structure were immobilized upright on electrode surface and stable Y-shaped junction configurations were formed away from the electrode surface, which was beneficial to avoid probes' selfentanglement, improve the hybridization efficiency and enhance the recognition ability. As a proof-ofconcept, the Y-shape-structured PEC biosensor coupled with dual signal amplification tactic achieves accurate quantification of miRNA-222 (papillary thyroid carcinoma-relevant biomarker) with salient selectivity, rapid response and low detection limit (0.15 pM, S/N = 3). The proposed PEC biosensor displays potential applications in tumor-related miRNAs assay for tumor prediction, diagnosis and prognosis.