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DNA Tetrahedron-enhanced single-particle counting integrated with cascaded CRISPR Program for ultrasensitive dual RNAs logic sensing

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JOURNAL OF COLLOID AND INTERFACE SCIENCE 2025年第Pt 2期683卷 521-531页

作者： Zhan, Xiaohui Jiang, Yujia Lei, Jiahui Chen, Hao Liu, Tangyuheng Lan, Fang ying, binwu Wu, Yao Sichuan Univ Natl Engn Res Ctr Biomat Chengdu 610064 Peoples R China Sichuan Univ West China Hosp Dept Lab Med Chengdu 610041 Peoples R China

CRISPR-Cas-based technology, emerging as a leading platform for molecular assays, has been extensively researched and applied in bioanalysis. However, achieving simultaneous and highly sensitive detection of multiple nucleic acid targets remains a significant challenge for most current CRISPR-Cas systems. Herein, a CRISPR Cas12a based calibratable single particle counting-mediated biosensor was constructed for dual RNAs logic and ultra-sensitive detection in one tube based on DNA Tetrahedron (DTN)-interface supported fluorescent particle probes coupled with a novel synergistic cascaded strategy between CRISPR Cas13a system and strand displacement amplification (SDA). As expected, our platform enables dual RNA molecules intelligent detection using only one crRNA of Cas13a, achieving a sensitivity enhancement of three orders of magnitude assisted with multiple signal amplification and accurate fluorescence particle counting with DTN mediated nano-biointerface enhancement, compared to traditional bulk Cas13a assays. Moreover, the effectiveness and universality of our strategy are experimentally investigated and demonstrated through the detection of mRNAs (cervical cancer swab clinical samples and cultured cancer cells) and bacterial 16s rRNAs. This work not only proposes a highly promising avenue for designing CRISPR-based multiplex detection systems that excel in ultra-sensitivity, specificity, and clinical molecular diagnostics, but also provide new insights into the potential applications of nanotechnology in molecular diagnostics, functional surface engineering, and interface-mediated bioreactions.

关键词： CRISPR Cas Cascaded logic gates Single particle counting DNA tetrahedron Multiplex detection

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High-activity PtIr@Fe-MOF nanozyme with low-hindrance PEA-CRISPR strategy for ultrasensitive lateral flow assay of AMI diagnosis

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CHEMICAL ENGINEERING JOURNAL 2025年 511卷

作者： Jiang, Yujia Zhan, Xiaohui Shui, Shaoxuan Liu, Yicheng Li, Zhiyu Peng, Yong ying, binwu Yi, Qiangying Lan, Fang Wu, Yao Sichuan Univ Natl Engn Res Ctr Biomat Chengdu 610064 Peoples R China Sichuan Univ West China Hosp Dept Cardiol Chengdu 610064 Peoples R China Sichuan Univ West China Hosp Dept Lab Med Chengdu 610064 Peoples R China

Colorimetric lateral flow assay (CLFA) has emerged as an essential tool for clinical diagnostics due to its convenience and rapid response. However, the traditional CLFA technique has failed to achieve comprehensive breakthroughs in sensitivity and specificity. This study, based on the concepts of multiscale design and system integration. To start with, by leveraging the spatial manipulation capability of the Fe-MOF framework for PtIr nanoparticles, we constructed a novel PtIr@Fe-MOF nanozyme with exceptional peroxidase-like catalytic activity, which significantly enhances its performance as a colorimetric label for CLFA at the nanoscale level. Concurrently, we developed a homogeneous protein detection strategy with minimal spatial hindrance by integrating proximity extension assay (PEA), exponential amplification reaction (EXPAR), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies through a molecular-level ssDNA-antibody conjugated probe. Integrating this high-performance nanozyme colorimetric label with homogeneous protein detection strategy into CLFA achieved ultrasensitive detection of cardiac troponin I (cTnI) (10 fg/mL) within 30 min. Subsequently, combined this strategy with smartphones or scanners for precise quantitative detection of cTnI in serum samples, achieving results highly consistent with ELISA assays. This approach provides a systematic solution for the development of universal, high-sensitivity protein detection platforms.

关键词： Point-of-care Lateral flow immunoassay Catalytic nanozyme Protein-nucleic acid signal transduction CRISPR Cas12a cTnI detection

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Energy-Confinement 3D Flower-Shaped Cages for AI-Driven Decoding of Metabolic Fingerprints in Cardiovascular Disease Diagnosis

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ACS NANO 2025年第6期19卷 6180-6194页

作者： Li, Zhiyu Zhang, Shuyu Xiao, Qianfeng Shui, Shaoxuan Dong, Pingli Jiang, Yujia Chen, Yuanyuan Lan, Fang Peng, Yong ying, binwu Wu, Yao Sichuan Univ Coll Biomed Engn Natl Engn Res Ctr Biomat Chengdu 610064 Peoples R China Sichuan Univ Coll Comp Sci Machine Intelligence Lab Chengdu 610064 Peoples R China Sichuan Univ West China Hosp Dept Cardiol Chengdu 610064 Peoples R China Sichuan Univ West China Hosp Dept Lab Med Chengdu 610064 Peoples R China

Rapid and accurate detection plays a critical role in improving the survival and prognosis of patients with cardiovascular disease, but traditional detection methods are far from ideal for those with suspected conditions. Metabolite analysis based on nanomatrix-assisted laser desorption/ionization mass spectrometry (NMALDI-MS) is considered to be a promising technique for disease diagnosis. However, the performance of core nanomatrixes has limited its clinical application. In this study, we constructed 3D flower-shaped cages based on controllable structured metal-organic frameworks and iron oxide nanoparticles with low thermal conductivity and significant photothermal effects. The elongation of the incident light path through multilayer reflection significantly enhances the effective light absorption area of the nanomatrixes. Concurrently, the alternating layered structure confines the thermal energy, reducing thermal losses. Moreover, the 3D structure increases affinity sites, expanding the detection coverage. This approach effectively enhances the laser ionization and thermal desorption efficiency during the LDI process. We applied this technology to analyze the serum metabolomes of patients with myocardial infarction, heart failure, and heart failure combined with myocardial infarction, achieving cost-effective, high-throughput, highly accurate, and user-friendly detection of cardiovascular diseases. Subsequently, deep analysis of detected serum fingerprints via artificial intelligence models screens potential metabolic biomarkers, providing a new paradigm for the accurate diagnosis of cardiovascular diseases.

关键词： flower-shaped cages detection of cardiovascular disease metabolite profiling mass spectrometry artificialintelligence

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Clinical evaluation of droplet digital PCR in suspected invasive pulmonary aspergillosis

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CLINICA CHIMICA ACTA 2025年 569卷 120153页

作者： Liu, Yang Tang, Qiuping Tang, Sishi Huang, Hengjian Kou, Lanxi Zhou, Yi Ruan, Hongxia Yuan, Yu He, Chao ying, binwu Sichuan Univ West China Hosp Dept Lab Med Chengdu 610041 Sichuan Peoples R China Sichuan Univ West China Hosp West China Precis Med Ind Technol Inst Chengdu 610041 Sichuan Peoples R China

Invasive pulmonary aspergillosis (IPA), the most common fungal infection, is associated with high mortality of affected patients. Traditional diagnostic methods exhibit limited sensitivity and specificity, raising big challenges for precise management of the patients. There is thus an urgent need to find out a timely and accurate diagnostic method in clinical practice. In this study, 163 patients suspected with IPA were enrolled. The medical data of the patients were retrieved from hospital information system. The 158 patients with complete data were classified into an IPA group with 122 cases (58 putative IPA, 19 probable IPA, and 45 possible IPA cases) and a non-IPA group with 36 cases. Cell-free DNA (cfDNA) of bronchoalveolar lavage fluid (BALF) or plasma samples was detected via a droplet digital PCR (ddPCR) assay targeting Aspergillus spp. Overall, this ddPCR assay demonstrated a higher sensitivity of 50.8 % for IPA diagnosis, compared with that of fungal culture (44.3 %) and smear test (10.7 %). Moreover, its sensitivity was higher in the IPA group (73.1 %) and putative IPA subgroup (88.2 %) when using BALF samples, compared with those using plasma samples (P < 0.01). It achieved a high specificity of 94.4 % for IPA diagnosis, with significant variations in cfDNA copy numbers across the subgroups (P < 0.05). In addition, the ddPCR results were associated with the prognosis of the patients at the discharge (P < 0.05). In conclusion, ddPCR assay demonstrated a good performance for IPA diagnosis when using BALF samples, especially for putative IPA. The ddPCR results could be integrated with clinical data to improve prognostic prediction.

关键词： Invasive pulmonary aspergillosis Droplet digital PCR Diagnose

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Dual-mode sensing of Mycobacterium tuberculosis with DNA-functionalized gold nanoparticles and asymmetric RPA-triggered PAM-free CRISPR system

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SENSORS AND ACTUATORS B-CHEMICAL 2025年 424卷

作者： Zhang, Limei Bai, Hao Zhang, Chuyan He, Xun Zou, Jie Bai, Weiyi Tang, Jianchao Zhuang, Weihua Hu, Jie Yao, Yongchao ying, binwu Hu, Wenchuang (Walter) Sichuan Univ West China Hosp Precis Med Translat Res Ctr PMTRC Medx Ctr MfgDept Lab MedState Key Lab Resp Hlth Chengdu 610041 Peoples R China Univ Elect Sci & Technol China Inst Fundamental & Frontier Sci Chengdu 610054 Peoples R China

Mycobacterium tuberculosis (MTB) causes the ancient infectious disease of tuberculosis, and its rapid, specific, and sensitive detection has long been a challenging endeavor. Herein, we propose integrating DNA-functionalized gold nanoparticles and asymmetric RPA-triggered PAM-free CRISPR for sensing of MTB. Upon activating the CRISPR/Cas12a complex by specific single-stranded products of asymmetric RPA and subsequent DNA degradation between AuNP and fluorophore, metal-enhanced fluorescence occurred with visible color changes. Therefore, this method has a dual signal output mode for point-of-care pathogen detection. Significantly, the entire detection process occurs at a constant temperature within a single tube, thereby minimizing the risk of aerosol contamination. In the experiment, the detection limit for MTB is remarkably low at 1 copy mu L-1, and importantly, this method exhibits no cross-reactivity with other common respiratory pathogens. Notably, we observed a 100 % concordance rate with clinical qPCR results in 32 sputum samples, underscoring the reliability and accuracy of this method. This biosensing platform not only effectively overcomes the limitations posed by PAM requirements but also holds great promise for point-of-care applications.

关键词： CRISPR system Isothermal Amplification Metal-Enhanced Fluorescence Mycobacterium tuberculosis POCT

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Advances in nanozymes with peroxidase-like activity for biosensing and disease therapy applications

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JOURNAL OF MATERIALS CHEMISTRY B 2025年第5期13卷 1599-1618页

作者： Yuan, Xiaohua He, Xun Fan, Jiwen Tai, Yunze Yao, Yongchao Luo, Yao Chen, Jie Luo, Han Zhou, Xingli Luo, Fengming Niu, Qian Hu, Wenchuang (Walter) Sun, Xuping ying, binwu Sichuan Univ West China Hosp Clin Lab Med Res Ctr Dept Lab Med Chengdu 610041 Sichuan Peoples R China Sichuan Univ West China Hosp Frontiers Sci Ctr Dis Related Mol Network Chengdu 610041 Sichuan Peoples R China Sichuan Univ West China Hosp Ctr High Altitude Med Chengdu 610041 Sichuan Peoples R China Sichuan Univ West China Hosp Dept Pulm & Crit Care Med Chengdu 610041 Sichuan Peoples R China Shandong Normal Univ Coll Chem Chem Engn & Mat Sci Jinan 250014 Shandong Peoples R China

Natural enzymes are crucial in biological systems and widely used in biomedicine, but their disadvantages, such as insufficient stability and high cost, have limited their widespread application. Since discovering the enzyme-like activity of Fe3O4 nanoparticles, extensive research progress in diverse nanozymes has been made with their in-depth investigation, resulting in rapid development of related nanotechnologies. Nanozymes can compensate for the defects of natural enzymes and show higher stability with lower costs. Among them, peroxidase (POD)-like nanozymes have attracted extensive attention in biomedical applications owing to their efficient catalytic performance and diverse structures. This review explores different types of nanozymes with POD-like activity and discusses their activity regulation, particularly emphasizing their latest development trends and advances in biosensing and disease treatment. Finally, the challenges and prospects for the development of POD-like nanozymes and their potential future applications in the biomedical field are also provided.

关键词： Nanoclay

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Deciphering the molecular basis of lipoprotein recognition and transport by LolCDE

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Signal Transduction and Targeted Therapy 2025年第1期10卷 434-445页

作者： Wen Qiao Chongrong Shen Yujiao Chen Shenghai Chang Xin Wang Lili Yang Jie Pang Qinghua Luo Zhibo Zhang yingxin Xiang Chao Zhao Guangwen Lu Bi-Sen Ding binwu ying Xiaodi Tang Haohao Dong Department of Laboratory Medicine State Key Laboratory of BiotherapyNational Clinical Research Center for GeriatricsWest China HospitalSichuan University and Collaborative Innovation Center of BiotherapyChengduChina Frontiers Medical Center Tianfu Jincheng LaboratoryWest China HospitalSichuan UniversityChengduChina Center of Cryo Electron Microscopy Zhejiang UniversityHangzhouChina

Outer membrane(OM)lipoproteins serve vital roles in Gram-negative bacteria,contributing to their pathogenicity and drug *** these lipoproteins to function,they must be transported from the inner membrane(IM),where they are assembled,to the OM by the ABC transporter *** have previously captured structural snapshots of LolCDE in multiple states,revealing its dynamic conformational ***,the exact mechanism by which LolCDE recognizes and transfers lipoprotein between domains remains ***,we characterized the *** LolCDE complex bound with endogenous lipoprotein or ATP to explore the molecular features governing its substrate binding and transport *** found that the N-terminal unstructured linker of lipoprotein is critical for efficient binding by LolCDE;it must be sufficiently long to keep the lipoprotein’s main body outside the complex while allowing the triacyl chains to bind within the central *** assays identified key residues that mediate allosteric communication between the cytoplasmic and transmembrane domains and in the periplasmic domain to form a lipoprotein transport pathway at the LolC-LolE *** study provides insights into the OM lipoprotein relocation process mediated by LolCDE,with significant implications for antimicrobial drug development.

关键词： functions outside chains

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MB based RT-qPCR increase the clinical application of cfEBV DNA for NPC in non-endemic area of China

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SCIENTIFIC REPORTS 2025年第1期15卷 1-12页

作者： He, Qiao Tuo, Yi Zhou, Yi Yan, Yue Liu, Xin Zhao, Dan Wang, Qiuju Luo, Hao Zhang, Zhengyao Meng, Fanping ying, binwu Wang, Dongsheng Yang, Mu Huang, Yecai Univ Elect Sci & Technol China Sichuan Canc Hosp & Inst Sichuan Clin Res Ctr Canc Sichuan Canc CtrDept Clin LabSch Med Chengdu 610000 Peoples R China Sichuan Univ West China Hosp Dept Lab Med Chengdu 610041 Peoples R China Chengdu Univ Tradit Chinese Med Coll Med Technol Chengdu 611137 Peoples R China Chong Qing Univ Dept Clin Lab Three Gorges Hosp Chongqing Peoples R China Univ Elect Sci & Technol China Sichuan Canc Hosp & Inst Sichuan Clin Res Ctr Canc Sichuan Canc CtrDept Expt Res Chengdu 610000 Peoples R China Univ Elect Sci & Technol China Dept Radiat Oncol Radiat Oncol Key Lab Sichuan Prov Sichuan Clin Res Ctr CancSichuan Canc Hosp & Inst Chengdu 610000 Peoples R China Univ Elect Sci & Technol China Sichuan Canc Hosp & Inst Sichuan Canc Ctr Dept Clin LabSichuan Clin Res Ctr Canc Chengdu 610000 Sichuan Peoples R China

To compare the performance of magnetic bead (MB) and the concentrated precipitation (CP) based RT-qPCR to qualify cell free EBV DNA (cfEBV DNA) for nasopharyngeal carcinoma (NPC) in non-endemic area of China. From January 2014 to June 2024, a retrospective analysis of 2 cohort studies on cfEBV DNA in NPC patients was conducted to assess the diagnostic value, positive detection rate and clinical application. cfEBV DNA detection with CP based RT-qPCR in cohort 1 and MB based RT-qPCR method in cohort 2. The MB based RT-qPCR for the quantitative measurement of cfEBV DNA load was higher than the CP based RT-qPCR in the same plasma samples from NPC patients (P < 0.001). CP based RT-qPCR measured cfEBV DNA in 1405 NPC and 244 healthy controls in cohort 1 with 40.8% sensitivity (AUC = 0.704, 95% CI: 0.676-0.731). In cohort 2(683 naive NPC and 303 controls), cfEBV DNA had a sensitivity of 75.84% (AUC = 0.879, 95% CI: 0.86-0.90). There were no significant differences in TNM stage among NPC between the two cohorts (P > 0.05). The MB method considerably increased the positive detection rate of cfEBV DNA in NPCs at stages III-IV, T2-T4, N1-N3, and M0 (P < 0.001). At the end of treatment, 97.51% of patients had no detectable EBV and just 2.49% had detectable cfEBV DNA. Those who received <= 2 or >= 3 cycles of NAC had a median t(1/2) clearance rate of 9.8 days and 12.6 days, respectively. MB based RT-qPCR increased the quantity of cfEBV DNA. MB based RT-qPCR demonstrated superior sensitivity and positive detection rates for cfEBV DNA. cfEBV DNA can be more positively noticed, with a higher diagnostic value and a broader variety of clinical applications among NPC in non-endemic areas.

关键词： RT-qPCR MB CP cfEBV DNA NPC

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Enhanced diagnosis of multi-drug-resistant microbes using group association modeling and machine learning

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NATURE COMMUNICATIONS 2025年第1期16卷 1-15页

作者： Saliba, Julian G. Zheng, Wenshu Shu, Qingbo Li, Liqiang Wu, Chi Xie, Yi Lyon, Christopher J. Qu, Jiuxin Huang, Hairong ying, binwu Hu, Tony Ye Tulane Univ Ctr Cellular & Mol Diagnost Sch Med New Orleans LA 70112 USA Tulane Univ Sch Sci & Engn Dept Biomed Engn New Orleans LA USA Tulane Univ Sch Med Dept Biochem & Mol Biol New Orleans LA 70112 USA Shenzhen Third Peoples Hosp Dept Clin Lab Shenzhen Guangdong Peoples R China Natl Clin Res Ctr Infect Dis Shenzhen Guangdong Peoples R China Sichuan Univ West China Hosp Dept Lab Med Chengdu Sichuan Peoples R China Capital Med Univ Beijing Chest Hosp Natl Clin Lab TB Beijing Peoples R China

New solutions are needed to detect genotype-phenotype associations involved in microbial drug resistance. Herein, we describe a Group Association Model (GAM) that accurately identifies genetic variants linked to drug resistance and mitigates false-positive cross-resistance artifacts without prior knowledge. GAM analysis of 7,179 Mycobacterium tuberculosis (Mtb) isolates identifies gene targets for all analyzed drugs, revealing comparable performance but fewer cross-resistance artifacts than World Health Organization (WHO) mutation catalogue approach, which requires expert rules and precedents. GAM also reveals generalizability, demonstrating high predictive accuracy with 3,942 S. aureus isolates. GAM refinement by machine learning (ML) improves predictive accuracy with small or incomplete datasets. These findings were validated using 427 Mtb isolates from three sites, where GAM inputs are also found to be more suitable in ML prediction models than WHO inputs. GAM + ML could thus address the limitations of current drug resistance prediction methods to improve treatment decisions for drug-resistant microbial infections.

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A sample-to-answer digital microfluidic multiplexed PCR system for syndromic pathogen detection in respiratory tract infection

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LAB ON A CHIP 2025年第6期25卷 1552-1564页

作者： Bai, Hao Hu, Jie Liu, Tangyuheng Wan, Liang Dong, Cheng Luo, Dasheng Li, Fei Yuan, Zhanxin Tang, Yunmei Chen, Tianlan Wang, Shan Gou, Hongna Zhou, Yongzhao ying, binwu Huang, Jin Hu, Wenchuang (Walter) Sichuan Univ West China Hosp Clin Lab Med Res Ctr Dept Lab Med Chengdu 610041 Peoples R China Sichuan Univ West China Hosp Precis Med Translat Res Ctr PMTRC Chengdu 610041 Peoples R China Sichuan Univ West China Hosp Med Equipment Innovat Res Ctr Med X Ctr Mfg Chengdu 610041 Peoples R China Livzon Diag Inc Zhuhai 519000 Peoples R China Cent South Univ Coll Chem & Chem Engn Changsha 410000 Peoples R China Jinan Univ Sch Intelligent Syst Sci & Engn JNU Ind Sch Artificial Intelligence Zhuhai 519000 Peoples R China Digifluid Biotech Ltd Zhuhai 519000 Peoples R China Sichuan Univ West China Hosp Dept Integrated Care Management Ctr Chengdu 610041 Peoples R China

Timely identification of infectious pathogens is crucial for the accurate diagnosis, management, and treatment of syndromic respiratory diseases. Nevertheless, the implementation of rapid, precise, and automated point-of-care testing (POCT) remains a significant challenge. This study introduces an advanced digital microfluidic (DMF) POCT testing system designed for the rapid molecular syndromic testing of multiple respiratory pathogens from a single untreated sample. The system seamlessly integrates magnetic beads-based nucleic acid extraction, PCR amplification, and real-time fluorescence analysis in an automatic run, facilitating sample-to-answer detection within 80 min. It accommodates various sample types, including nasopharyngeal swabs, oropharyngeal swabs, bronchoalveolar lavage fluid, and sputum. A facile sample loading method has been developed to reduce hands-on time to less than 1 min. The system exhibits high sensitivity (200-628 copies per mL) for 15 pathogens and has the capacity for up to 32 multiplexed tests per run. Validation with 255 clinical samples confirms its high sensitivity and specificity. The DMF-based system significantly reduces manual labour, enhances rapid POCT for respiratory infections, and, with optimized manufacturing processes, lowers costs for large-scale production. The system can be applied and improve clinical management near the patients as well as in resource-limited settings.

关键词： Digital microfluidics

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