Liver fibrosis can arise from diverse etiologies, including metabolic stress and nutritional deficiencies. The methionine- and choline-deficient (MCD) diet induces liver fibrosis independent of metabolic syndrome, offering a model to study non-metabolic drivers of hepatic fibrogenesis. SUMO-specific protease 2 (SENP2) has been implicated in metabolic liver disease, but its role in nutritionally induced fibrosis remains unknown. Here, we investigated how hepatic Senp2 regulates MCD-induced liver fibrosis, with a focus on vascular remodeling. Liver-specific Senp2 knockout mice and wild-type littermates were fed an MCD diet to establish fibrosis. Liver injury, fibrosis, inflammation, and angiogenesis were assessed. Hepatic Senp2 deficiency markedly attenuated MCD-induced liver injury, fibrosis, and inflammation. Notably, Senp2 loss triggered a distinct hepatic vascular remodeling pattern: it promoted portal angiogenesis while suppressing hepatic sinusoidal capillarization, as evidenced by altered Cd31 expression and vascular architecture. Mechanistically, Senp2 ablation significantly reduced leukocyte cell-derived chemotaxin 2 (LEC2) expression and secretion, alongside downregulation of both active non-phospho-β-catenin and total β-catenin. In the MCD-induced fibrosis model, hepatic Senp2 drives pathological vascular remodeling through a β-catenin/Lect2 axis. Loss of Senp2 restrains this pathway, rebalances hepatic angiogenesis, and ultimately mitigates fibrosis progression. This reveals a nutrition-specific, pro-fibrotic mechanism of Senp2 centered on vascular regulation, highlighting a potential therapeutic target for non-metabolic fibrotic liver diseases.

MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are critical regulators in cancer development, making them promising diagnostic biomarkers and therapeutic targets. However, conventional single-biomarker detection often fails to meet the demands of precision medicine. This study developed an AND logic gatecontrolled "dual-lock" detection platform based on a CRISPR/Cas13a-Cas12a cascade system, enabling highly sensitive and specific joint detection of lncRNA HOTAIR and miRNA-155. The core design involves: (1) lncRNA HOTAIR acting as the "first key" to activate Cas13a trans-cleavage, unlocking the 12a-crRNA template for transcription by T7 RNA polymerase; (2) miRNA-155 serving as the "second key" to release target DNA through CHA-SDR cascade reactions; (3) only when both biomarkers are present can Cas12a bind 12a-crRNA and target DNA, activating nuclease activity to cleave a probe that restrains Au@PtNPs nanozyme-immobilized magnetic beads, thereby significantly enhancing TMB catalytic signal. Experimental results show the biosensor offers excellent specificity, stability, and repeatability, with detection limits reaching the fM level for both lncRNA and miRNA. This work establishes a novel molecular diagnostic tool for early cancer screening and precision diagnosis.

Urinary tract infection (UTI) requires rapid and accurate pathogen identification, yet conventional culture-based methods are time-consuming and many molecular assays remain dependent on sophisticated instrumentation. Here, we developed a multiplex visual biosensing platform for uropathogen detection by integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a recognition, a CRISPR-responsive DNA hydrogel gate, and a δ-MnO2 nanozyme-mediated colorimetric module. In this system, target-derived amplicons activate pathogen-specific Cas12a, which degrades the DNA hydrogel and releases encapsulated δ-MnO2, thereby catalyzing the TMB/H2O2 reaction to produce a visible and spectrophotometrically measurable signal. The platform was applied to five representative uropathogens, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, and Enterococcus faecalis. Under optimized conditions, the assay showed good linearity for all five targets, with R2 values of 0.9955, 0.9958, 0.9948, 0.9974, and 0.9956, respectively, and limits of detection of 2.60, 3.15, 2.44, 20.40, and 45.47 copies/μL, respectively. The platform also exhibited satisfactory specificity, multiplexing capability, reproducibility, and storage stability. To improve quantitative interpretation, an image-recognition- and machine-learning-assisted workflow was further introduced, in which KernelRidge-RBF showed the best overall performance among the tested models. Clinical urine sample testing further supported the applicability of the platform, with AUC values of 1.000, 0.976, 0.994, 0.947, and 0.939 for the five target pathogens, respectively. Collectively, the RPA-Cas12a-DNA hydrogel-δ-MnO2 platform enables visual and parallel multi-target detection of uropathogens within approximately 3 h and provides a proof-of-concept framework for the further development of portable nucleic acid biosensors.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that can cause severe illnesses such as food poisoning and toxic shock syndrome, posing a significant threat to public health. Therefore, rapid and highly sensitive detection of S. aureus is crucial for ensuring food safety. In this study, a Meso-UiO-66(Zr)@PtPdNPs nanozyme with triple enzyme activities was synthesized and combined with the RPA-CRISPR/Cas12a system to construct a novel colorimetric platform for S. aureus detection. This platform utilizes the oxidase and peroxidase activities of Meso-UiO-66(Zr)@PtPdNPs to achieve self-driven catalytic cascade signal amplification without the need for external H2O2. During the assay, RPA first amplifies the target DNA to activate the trans-cleavage function of CRISPR/Cas12a. The activated CRISPR/Cas12a then degrades the magnetic bead probe (SMBs-S2), preventing it from coupling with the nanozyme probe (Meso-UiO-66(Zr)@PtPdNPs-S1). After magnetic separation, the nanozyme in the precipitate efficiently catalyzes the color development of TMB through the OXD-POD cascade effect. The resulting color intensity reflects the S. aureus levels. This biosensor shows excellent sensitivity and specificity by integrating the specific recognition and cleavage capabilities of the CRISPR system with the robust catalytic performance of the nanozyme. It displays a linear range of 1.5 × 101-1.5 × 108 CFU/mL, alongside a 2.6 CFU/mL detection limit. This study provides a novel strategy for the detection of S. aureus in food.

The abnormally high expression of MUC1 in various malignant tumors makes it a crucial biomarker for early cancer diagnosis. This study synthesized a Cu-MOF@PtPdNPs nanocomposite exhibiting robust peroxidase-like activity via an in-situ reduction method. By integrating this nanomaterial with a dual-aptamer recognition sandwich strategy, an efficient colorimetric sensor was constructed for MUC1 detection. The nanozyme modified with aptamer2 (Cu-MOF@PtPdNPs-Apt2) was used to specifically recognize the target MUC1. Then, magnetic beads modified with aptamer1 (MB-Apt1) were used to enrich and separate the Cu-MOF@PtPdNPs-Apt2@MUC1, forming sandwich complexes. The sandwich complexes were separated and removed using magnets, and the remaining Cu-MOF@PtPdNPs-Apt2 in the supernatant was used for colorimetric detection. This sensor exhibits a linear range from 1 ng/mL to 100 μg/mL, alongside a limit of detection (LOD) of 0.211 ng/mL. Benefiting from the ingenious sandwich architecture, the sensor demonstrates high selectivity toward MUC1 and shows a good response in human serum, with recovery rates between 92.00% and 97.82%. In conclusion, this study provides a fast and sensitive technical platform for MUC1 detection.

Background The precise detection of tumor markers is crucial for early cancer diagnosis and monitoring. Existing unimodal detection methods are susceptible to interference in complex biological samples, making it difficult to simultaneously achieve high sensitivity and reliability. MicroRNAs (miRNAs), as a key class of cancer-related biomarkers, necessitate novel detection methods capable of multi-layered verification. This study aims to develop an biosensing platform with multi-signal outputs to address the critical challenge of balancing sensitivity, specificity, and result credibility in current tumor marker detection. Results We successfully constructed a tri-modal biosensor based on a dual CRISPR/Cas12a system for the highly sensitive and specific detection of miRNA let-7a. The sensor generates a trigger strand via an exponential amplification reaction (EXPAR), which concurrently regulates three independent signaling pathways: ① It initiates the first CRISPR/Cas12a to suppress G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme) formation, turning off the ABTS colorimetric signal. ② It also activates duplex-specific nuclease (DSN) to inhibit the hybridization chain reaction (HCR), thereby blocking FAM fluorescence coupling to streptavidin-coated magnetic nanoparticles (SMBs) and turning off the fluorescence signal. ③ The absence of HCR products on the SMBs inhibits the second CRISPR/Cas12a system, thereby preserving the electrode's P1 probe for binding with P2-3D-CdCo-ONSs@AuNPs and maintaining a high “turn-on” electrochemical signal from the nanocomposite. Thus, the concentration of miRNA let-7a, ranging from 50 fM to 1 pM, can be precisely quantified and validated through colorimetric, fluorescent, and electrochemical signals. Significance This study integrates a dual CRISPR/Cas12a system with a tri-modal output strategy encompassing colorimetric, fluorescent, and electrochemical detection, thereby constructing a detection platform featuring a cross-verification mechanism. This design not only significantly enhances detection accuracy and anti-interference capability but also lays a solid foundation for developing next-generation, highly reliable molecular diagnostic tools. It holds considerable application potential in the fields of early cancer screening and precision medicine.

Early and accurate diagnosis of cancer relies on the detection of highly sensitive and specific biomarkers. MicroRNAs (miRNAs), as key regulatory factors, are ideal diagnostic markers for cancer. However, their extremely low abundance and susceptibility to degradation in biological samples pose significant challenges for traditional detection methods. To address this challenge, this study developed a dual-mode synchronous detection biosensor for the combined detection of breast cancer-related biomarkers miRNA-96 and miRNA-21. The biosensor integrates an AND-logic-gate colorimetric detection (CRISPR/Cas12a-nanozyme) with a NOT-logic-gate fluorescence detection (RCA-based), enabling multiplexed target discrimination. The entire system forms a dual-input (miRNA-96, miRNA-21), triple-output (colorimetric, FAM, ROX) molecular logic circuit. Its output patterns are: (1,1) to (1,0,0); (1,0) to (0,0,1); (0,1) to (0,1,0); (0,0) to (0,1,1). The biosensor demonstrated high performance, with colorimetric detection limits of 29.70 fM (miRNA-96) and 47.81 fM (miRNA-21), and fluorescence limits of 103.54 fM and 366.06 fM, respectively. It exhibits outstanding specificity against similar miRNA sequences, demonstrates good stability, and achieves accurate recovery rates in clinical serum samples, enabling reliable differentiation between plasma samples from healthy individuals and breast cancer patients. This cost-effective, modular strategy provides a sensitive tool for early breast cancer screening.

Developing portable, accurate, cost-effective hydrogen peroxide (H2O2) detection platforms is essential for industrial applications and early disease diagnosis. Metal–organic frameworks (MOFs) based composites integrated with metal nanoparticles have been intensively investigated in electrochemical sensing, attributable to their unique architecture and performance characteristics. Herein, PdNPs@NH2-MIL-101(Fe) nanocomposites were synthesized via a green reduction process using tannic acid and applied in the development of an enzyme-free electrochemical sensor for H2O2 detection. The synergistic effect of the Pd nanoparticles and the MOFs matrix provided abundant active sites and enhanced electron transfer capability, enabling the sensor to exhibit excellent electrocatalytic performance. Under optimal conditions, the sensing platform exhibited a wide linear response from 10 μM to 15 mM, with a detection threshold of 3.6 μM (S/N = 3). Furthermore, the sensor achieved reliable detection of exogenous H2O2 in commercial mouthwash samples and intracellularly generated H2O2 by cancer cells (HepG2), underscoring its effectiveness in practical scenarios. This work presents a novel strategy for synthesizing high-performance composite nanomaterials and offers valuable insights into the large-scale application of electrochemical sensors for H2O2 detection.

The dual-mode sensors can cross-verify the presence of target analyte with the help of two mutually independent output signals, which effectively reduces the probability of false-positive or false-negative results, improves the sensitivity of the sensors and expands the detection range. With its large specific surface area, good biocompatibility, and rich hydrophilic functional groups on the surface, MXene can be used to immobilize natural bio-enzymes and prepare nano-enzymes. CRISPR/Cas system has the function of specific recognition and cis/trans cleavage, and can effectively recognize, amplify and output detection signals when combined with different nucleic acid amplification technologies. Based on the above features, MXene nanoenzymes and CRISPR/Cas system have been used to construct dual-mode sensors in recent years. Therefore, this study summarized the preparation of MXene and its application in natural enzyme immobilization and nano-enzyme preparation, as well as the types of CRISPR/Cas system and its role in sensor construction, focusing on the application of dual-mode sensors based on MXene nanoenzyme or CRISPR/Cas system in the analysis and detection of nucleic acids, pathogenic bacteria, and small molecular substances.

The development of portable exosome detection platforms is crucial for noninvasive early diagnosis of lung cancer, particularly in resource-limited settings. Herein, we engineered a paper-based colorimetric biosensor utilizing a MnO2/CeO2 heterojunction nanozyme for visual quantification of plasma exosomes. The heterojunction synergistically integrates the high surface area of CeO2 (45.689 m2/g) with the nanorod shaped MnO2, generating an interfacial electron transfer pathway that enhances peroxidase-like activity by 1.77 and 5.87 fold (Km = 1.197 mM, Vmax = 0.884 M/min) compared to individual components. Molecular recognition was achieved through dual-target immobilization: exosome-specific aptamers and anti-CD63/FGG/FGB antibodies conjugated on a chitosan-modified cellulose substrate. A smartphone-based hue-saturation-value (HSV) analysis system embedded in the platform enabled rapid on-site screening within 15 min, followed by microplate scanning of suspected high-risk samples, achieving a detection range of 102-108 particles/μL with an ultralow detection limit of ∼2 particles/μL. Integration of a logistic regression model incorporating CD63, FGG, and FGB absorbance values further improved classification robustness. Clinical validation using 194 plasma samples demonstrated exceptional diagnostic accuracy in discriminating malignant from benign pulmonary nodules (AUC = 0.996), with a 95 % confidence interval ranging from 0.797 to 1.05, with 98.51 % sensitivity and 95.52 % specificity. This work establishes a multimodal strategy combining heterojunction nanozyme catalysis, paper-based microfluidics, smartphone-assisted imaging, and machine learning, offering a scalable paradigm for liquid biopsy applications in primary healthcare.

Early diagnosis and treatment can significantly enhance the prognosis of patients with non-small cell lung cancer (NSCLC); however, current diagnostic methods remain plagued by inadequate sensitivity or excessive invasiveness. This study describes the innovative construction of a dual-fluorescence, dual-target biosensor based on copper nanoclusters (CuNCs) and hollow mesoporous silica nanospheres (HMSNs) for highly sensitive detection of CD91 and CD151 on the surface of NSCLC exosomes. Specifically, HMSNs regulate the fluorescence signals of red and blue glutathione-CuNCs by loading Ce3+ and Fe3+ and conjugating a pH-responsive chitosan coating: Ce3+ significantly enhances the fluorescence signal of red CuNCs by inducing their aggregation, whereas Fe3+ quenches the fluorescence signal of blue CuNCs through oxidation. Exosomes are captured using a CD63 aptamer conjugated to streptavidin magnetic beads. HMSN-Ce3+-CS-antiCD151 and HMSN-Fe3+-CS-antiCD91 probes are then employed to target exosome surface proteins CD151 and CD91, respectively, allowing synchronous detection of dual-fluorescence and dual targets. We then measured and analyzed the change ratio of fluorescence signals, and optimized the detection parameters while evaluating the sensitivity, stability, and linear range of the system. The detection limits of red and blue fluorescence systems were 1058 particles center dot mL- 1 and 239 particles center dot mL- 1, and ranges of linearity were 4 x 103-4 x 108 particles center dot mL- 1 and 7 x 102-7 x 106 particles center dot mL- 1, respectively. Overall, this study introduces a novel strategy for the early screening of NSCLC, with high sensitivity and minimal invasiveness. The complementary design of the dual-fluorescence system extends its detection range and holds potential for clinical application.

A novel electrochemical sensor was developed for the determination of uric acid (UA) by modifying a screenprinted carbon electrode (SPCE) with a graphene/zirconium dioxide/graphene quantum dots (graphene/ZrO2/ GQDs) nanocomposite. The physicochemical properties of the synthesized nanocomposite were systematically characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and electrochemical impedance spectroscopy (EIS). The sensor's electrocatalytic performance was optimized, revealing that a graphene:ZrO2:GQDs ratio of 1:1:1 yielded the highest activity towards UA oxidation. The optimal voltammetric response was achieved in a 0.1 M phosphate buffer solution (PBS) at pH 6.5. Under these optimized conditions, the sensor exhibited a wide linear detection range for UA from 20 to 500 mu M with a high correlation coefficient (R2 = 0.997). The limits of detection (LOD) and quantification (LOQ) were calculated to be 1.07 mu M and 3.55 mu M, respectively. Interference studies confirmed the sensor's excellent selectivity against common co-existing species. The practical applicability of the sensor was successfully demonstrated by quantifying UA in human serum samples, yielding satisfactory recovery rates. This work presents a robust and sensitive platform for UA analysis with significant potential for clinical applications.

A multi-signal amplification system using an ITO electrode printed with disposable screen ink was developed for the rapid detecting des-gamma-carboxy prothrombin (DCP). Gold nanoparticles, uniformly shaped, were electrodeposited on the ITO electrode to improve conductivity and biocompatibility. To boost the sensor's sensitivity, isoreticular metal-organic framework-3 (IROMF-3) and methylene blue (MB) were employed to expand the electrode's surface area and intensify the current signal. Additionally, the avidin-loaded silver nanoelectrode, capable of binding more biotinylated DCP antibodies, further heightened sensitivity. The developed immunosensor effectively detected trace DCP in serum, with a linear range of 0.31 ng/mL to 40 ng/mL, a detection limit of 0.138 ng/mL, and a recovery rate of 97.9-109.2 % under optimal experimental conditions. This study offers a promising approach for the rapid field detection of trace DCP in serum.

microRNA (miRNA) has drawn significant attention as a potential clinical biomarker for cancer. However, traditional amplification methods often face specificity issues, while next-generation sequencing technologies typically involve lengthy analysis times and high costs, making them unsuitable for routine clinical diagnostics. Therefore, it is essential to develop an analytical system that is simple, economical, and capable of rapidly, selectively, and accurately detecting miRNAs. To address this need, we propose a multistage amplification strategy that combines catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), CRISPR/Cas12a, and strand displacement reaction (SDR) switch for the ultrasensitive detection of miRNA-375. This system enhances the Cy5 fluorescence signal through the CHA-HCR reaction. At the same time, the CRISPR/Cas12a activates its ability to cleave single-stranded DNA, thereby inhibiting the SDR reaction and reducing the displacement of ferrocene (Fc) on the electrode. We subsequently measured and analyzed the Cy5 fluorescence and Fc electrochemical signals, optimizing the detection parameters while evaluating the system's sensitivity, stability, and linear range. The Cy5 fluorescence signal biosensor can detect miRNA-375 from 1 fM to 10 nM, with a detection limit of 143 aM. In contrast, the Fc electrochemical signal biosensor has a detection range of 100 aM to 10 pM, with a detection limit of 27 aM. With an ideal recovery rate in detecting human serum, this "DualSignal-On" biosensor enhances the detection range, lowers detection limits, and improves accuracy, highlighting its potential application value in cancer diagnosis.

Mucin 1 (MUC1), a pivotal tumor biomarker overexpressed in breast cancer, is conventionally detected using laborious, costly, and technically demanding methods. To overcome these limitations, we present a novel labelfree electrochemical aptasensor for rapid, sensitive, and cost-efficient MUC1 quantification. The sensor employs a nanohybrid platform composed of gold nanoparticle-decorated three-dimensional cadmium-cobalt oxide nanostructures (3D-CdCo-ONSs@AuNPs), which synergistically amplify the differential pulse voltammetry (DPV) signal. The 3D-CdCo-ONSs provide a high-surface-area scaffold for dense AuNP deposition, enabling efficient immobilization of thiolated aptamers via Au-S bonds. Comprehensive material characterization via scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) confirmed the structural and compositional integrity of the nanocomposite. Under optimized conditions, the sensor exhibited a broad linear detection range from 10 pg/mL to 100 mu g/mL (R2 = 0.998) with an ultralow limit of detection (LOD) of 0.96 pg/mL. The assay demonstrated excellent reproducibility, stability, and selectivity in complex matrices, facilitating precise MUC1 quantification in clinical samples. This work advances the development of portable, high-performance biosensors for early cancer diagnosis and monitoring.

Circulating tumor cells (CTCs), as a critical biomarker for early diagnosis and real-time monitoring of tumor progression, play a significant role in early cancer diagnosis, treatment monitoring, and prognosis evaluation. However, existing CTCs detection methods are limited by low purity, long processing time, high cost, and insufficient specificity, making them inadequate for clinical needs. This study developed an ultrasensitive and visually detectable CTCs sensing platform. The platform integrates nucleic acid aptamer-functionalized dopamine-coated magnetic nanoparticles as capture probes for the specific isolation and enrichment of CTCs, and MXenes@MnCoZDH@Au NPs as signal-amplifying nanoprobes with exceptional peroxidase-like activity. The colorimetric sensing mechanism relies on the linear increase in absorbance with CTCs concentration, enabling quantitative detection. Under optimized experimental conditions, the sensor demonstrated a broad detection range of 10 to 106 cells/mL and an ultra-low 2 cells/mL detection limit. Compared to traditional methods, this detection approach exhibited high specificity, sensitivity, and reproducibility, highlighting its potential for clinical translation. This innovative method provides a reliable and practical tool for liquid biopsy-based cancer diagnostics, offering significant advancements in precision medicine.

Accurate and sensitive detection of the neurotransmitter dopamine (DA) plays a significant role in medical diagnosis and human health assessment. Herein, an electrochemical sensing platform for ultrasensitive determination of DA was constructed based on porous Co3O4 nanocubes anchored to three-dimensional Ti3C2 MXenereduced graphene oxide aerogel (Co3O4/3D MX-rGO). The 3D MX-rGO aerogel with a high specific surface prevents aggregation of Co3O4 nanocubes and exposes more active sites, resulting in excellent electrocatalytic activity and outstanding sensing properties. The electrochemical reaction kinetic was investigated in detail, a possible sensing mechanism was proposed, and the interaction mechanism between the composites and DA was revealed combined with density functional theory (DFT) calculations. Under optimized experimental conditions, the sensor developed using Co3O4/3D MX-rGO modified glassy carbon electrodes (GCE) exhibited low detection limits (40 nM) in the range of 0.1-300 mu M for DA with good selectivity, reproducibility, and stability. The sensor has been successfully use to detect DA in human serum and urine samples with recoveries of 95.3 %-103.7 %, which has great potential for practical applications.

Fluorescent sensor is an important tool to reliaze qualitative or quantitative detection of target analyte based on the fluorescence principle. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has been utilized to develop as a precise, efficient, and highly sensitive molecular diagnostic tool due to its efficient targeting and gene editing ability. At present, CRISPR/Cas system-based fluorescent sensors have shown excellent performance in the field of analysis and detection, and have received widespread attention. Therefore, this paper reviews the mechanism of the CRISPR/Cas system, the characteristics of different Cas proteins, and the principle and characteristics of the fluorescent sensor, with a focus on summarizing the application of the CRISPR/Cas system-based fluorescent sensor for analysis and detection.

Bloodstream infection (BSI) is a critical condition with extremely high mortality. Rapid and accurate diagnosis is crucial for effective treatment. The traditional blood culture (BC) method has issues, such as long testing times and limited sensitivity, making it challenging to meet the need for timely diagnosis. To address this problem, various molecular biology methods for directly detecting blood samples (whole blood, plasma, serum, and positive BC samples) have emerged. These include Raman spectroscopy, mass spectrometry, nucleic acid amplification, and hybridization techniques (such as the CRISPR/Cas system, digital droplet PCR (ddPCR), and T2 magnetic resonance (T2MR)), biosensors, and next-generation sequencing (NGS). These methods can quickly identify pathogens and their drug-resistant markers, significantly reducing diagnostic delays and helping to provide earlier targeted treatment. This article systematically analyzes the principles, advantages, and disadvantages of these advanced techniques, explores their value in revolutionizing the BSI diagnostic model, and looks ahead to future development directions, providing a reference for research and clinical applications in this field.

Survivin serves as a crucial biomarker for bladder cancer, so exploring the detection method is essential. Therefore, a sensitive ratiometric electrochemical immunosensor was developed. In brief, a double-layered amino graphene oxide (NH2-GO) sandwich enhanced by Fe-Ni@Ti3C2Tx (NH2-GO/Fe-Ni@Ti3C2Tx/NH2-GO) significantly amplified the detection signal. ferrocene@Au nanoparticles (Fc@AuNPs) provided a reference signal, and Ab1 was anchored via an Au-N bond. Moreover, AuPt-methylene blue (AuPt-MB) served not only as a secondary signal but also anchored Ab2 through Au-NH2 and Pt-NH2 bonds. Black phosphorus nanoplates (BPNPs) improved conductivity and provided additional binding sites for AuPt-MB. The immunosensor exhibited a broad detection range (0.001-1000 ng/mL) and a low detection limit (0.00065 ng/mL), facilitating successful survivin detection in human urine samples with promising clinical analysis applications.