ACS Sensors
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2026
·Vol. 11
·期 7
·5553-5565
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IF 10.9
Qiu Zhiqiang,Chen Jiabo,Wu Jinxian,Wang Jujing,Hu Yuting,Feng Wei,Zhao Jun,Minamoto Toshifumi,Chen Xiao,Zhou Lei,Liu Min,Wang Chen,Lin Mei,Hu Menglu,Zhou Xiaoming,Wang Junjie
Developing a rapid, sensitive, and field-deployable assay for on-site environmental DNA (eDNA) detection of endangered species is crucial, as current PCR-based assays are slow and expensive and rely on laboratory-based thermal cycling. Here, we adapted a previously reported optically controlled one-pot RPA-CRISPR-Cas12a (OORC) assay to detect eDNA of the critically endangered Bahaba taipingensis . The assay combines isothermal recombinase polymerase amplification (RPA) with the high specificity of a CRISPR-Cas12a trans -cleavage reaction in a single tube. The entire process is operated at a constant temperature, avoiding thermal cycling. Low-template replicate experiments conservatively redefined the OORC limit of detection as 6 copies/reaction. To support near-field application, we further integrated the assay with a portable handheld fluorescence detector capable of 365 nm photoactivation and fluorescence readout. This portable and highly sensitive workflow extends the potential of eDNA monitoring, offering a practical tool for the conservation of endangered species.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Li Li,Yuanqian Xiong,Yuqian Guo,Hong Duan,Yuankui Leng,Xiaolin Huang,Gang Chen,Yonghua Xiong
DNA-templated silver nanoclusters (tDNA-AgNCs) show considerable promise as fluorescence reporters for the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system because of their ease of synthesis, strong resistance to photobleaching, and large Stokes shift. However, the weak luminous intensity and low cleavage efficiency of tDNA-AgNCs limit the sensitivity of CRISPR assays. In this study, we developed a novel approach by introducing activator DNA (aDNA) with a G-quadruplex structure to enhance the luminous intensity of the tDNA-AgNCs. As a result of the high cleavage efficiency of free aDNA by activated CRISPR/Cas12a, the G-quadruplex-enhanced tDNA-AgNCs (GED-AgNCs) were integrated into recombinase polymerase amplification and CRISPR/Cas12a system for the ultrasensitive detection of Salmonella typhimurium. By optimizing the synthesis of tDNA-AgNCs and GED-AgNCs, our developed G-quadruplex-enhanced DNA-AgNC CRISPR assay (G-DACA) platform enabled the sensitive determination of S. typhimurium with a detection limit as low as 1 CFU/mL and a wide dynamic range of 10–108 CFU/mL. Moreover, our proposed method demonstrated good accuracy and reliability for the quantitative analysis of S. typhimurium in real pasteurized milk samples, with recovery rates ranging from 81.06% to 102.33% and relative standard deviations between 7.17% and 14.84%. Overall, our innovative G-DACA platform offers an economical and versatile solution for food safety and clinical diagnostics.
研究领域:
生物传感器
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分子诊断
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纳米技术
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食品安全
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CRISPR技术
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分析化学
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Analytical Methods
·
2025
·
IF 2.6
Jicong Hao, Xueyan Gong, Xueyuan Duan, Weiwei Qin, Haiying Ren, Xuping ShenTu, Zihong Yea ,Xiaoping Yu
The rapid and ultrasensitive detection of parvovirus B19 (B19V) DNA is critical for preventing severe complications in high-risk populations, such as fetal hydrops in pregnant women and aplastic crisis in immunocompromised patients. The absence of clinically approved vaccines or antivirals against B19V thus mandates the urgent development of accessible in vitro diagnostics to enable time-critical interventions and contain community transmission. Herein, we developed an electrochemical biosensor as a proof-of-concept for B19V by integrating CRISPR-Cas12a with a multivalent framework nucleic acids (FNAs), namely, 12 nm tetrahedral DNA nanostructures (TDNs). Target B19V DNA activates Cas12a to indiscriminately cleave the biotin-modified ssDNA protruded from the four vertexes of the TDNs, while the TDNs precisely orient ssDNA probes on electrodes, minimizing nonspecific adsorption. This method takes advantage of the target-specific cleavage ability of CRISPR-Cas12a (Cas12a-crRNA complex, 10-12 nm) and the unique structural and functional features of 12 nm TDNs. The comparable dimensions of the Cas12a-crRNA complex and the TDN suggest a potential synergistic effect, which contributes to the observed signal amplification and high detection sensitivity. The developed platform is user-friendly, has a low detection limit (2.19 fM), and shows high selectivity. This work establishes a foundational biosensing platform, demonstrating potential for ultrasensitive nucleic acid detection. By combining the accuracy of CRISPR-Cas12a and the benefits of FNAs, this method provides a more efficient, amplification-free, and reliable approach that holds promise for future development in point-of-care diagnostics and other applications.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Qisheng Luo,Chuanhua Zheng,Yongning Huang,Linghuan Liang,Yuanxun Gong,Jiayi Zhang,Qianli Tang,Kai Zhang,Xianjiu Liao
Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb 2+ safety concerns. We construct a CsPbBr 3 @PDA–Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr 3 , suppressing trap-mediated quenching and mitigating Pb 2+ release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide–DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from “off” to “on.” Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr 3 ; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Wen Tian,Tao Liu,Xiaoying Niu,Po Li,Zhehuang Li,Songcheng Yu,Peng Zhang
DNA methylation serves as a critical epigenetic biomarker for early disease screening and clinical diagnostics, exemplified by the FDA-approved detection of methylated Septin9 gene in peripheral blood for colorectal cancer (CRC) screening. Conventional CRISPR/Cas12a-based detection systems rely on fluorophore-quencher-labeled ssDNA probes to transduce trans-cleavage activity into fluorescence signal, which yet suffers from limitations including complex probe synthesis, background noise induced by incomplete quenching and high cost. In this study, we developed a cascaded and label-free CRISPR/Cas12a system for Septin9 methylation detection using DNAzyme and split DAP-10. The method employed methylation-sensitive Aci I digestion to selectively hydrolyze non-methylated DNA, leaving methylated Septin9 DNA fragment intact to activate CRISPR/Cas12a system. Activated Cas12a triggered trans-cleavage of DNAzyme, a bifunctional molecule that acted as both trans-cleavage substrate of Cas12a and hydrolysis regulator of linker DNA that facilitated split DAP-10 reassembly to light-up Auramine O (AO) fluorescence proportionally to methylation levels. The assay achieved a linear range from 2 to 200 nM with a detection limit of 1.74 nM and robust performance in accuracy, precision, and selectivity. The proposed method was successfully applied in methylated Septin9 detection in cell lines, clinical tissues and blood samples, which indicated application potential for CRC screening.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Junlan Liu,Yuan Gao
The exceptionally sensitive and specific CRISPR-Cas systems have revolutionized molecular diagnostics, but their application remains largely confined to nucleic acid targets. To overcome this limitation and enable one-pot, mix-and-read detection of non-nucleic acid analytes, we present a generalizable CRISPR-Cas13a sensing platform that utilizes a hybrid DNA:RNA strand displacement-based switch to conditionally activate Cas13a. The switch leverages Cas13a's strict requirement for single-stranded RNA activators to mediate non-RNA target-triggered activation while protecting functional DNA elements from collateral cleavage. By integrating the modular Cas13a switch with various functional DNAs, including DNAzyme, aptazyme, and duplexed aptamer, we constructed a suite of one-pot sensors capable of detecting non-RNA targets such as lead ion, ATP, and thrombin. These CRISPR-Cas13a sensors demonstrate enhanced sensitivity, high selectivity, and robust performance in complex biological matrices. The high modularity of the CRISPR-Cas13a sensor allows rapid reconfiguration for new targets by simply altering the output sequence of functional DNA without extensive re-engineering. This versatile and robust sensing strategy expands the application landscape of CRISPR-Cas systems and lays the foundation for flexible, sensitive, and one-pot detection of a wide array of clinically and environmentally relevant targets.
研究领域:
分子诊断学
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合成生物学
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生物传感器
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CRISPR技术
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核酸化学
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生物分析化学
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SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 8.3
Weiwei Jiang,Shuyu Zhou,Shaoqi Dong,Xinyi Gu,Liuyu Pan,Zhaohui Qiao,Danting Yang
Nucleic acid amplification-free detection of Salmonella Typhimurium ( S. Typhimurium ) remains challenging for decentralized food safety monitoring. Herein, we report a multivalent aptamer–mediated competitive electrochemical CRISPR platform for amplification-free detection of S. Typhimurium . A hybridization chain reaction (HCR) was employed to construct HCR-multiApt, a multivalent aptamer scaffold containing repeated crRNA-complementary activator regions. In the absence of bacteria, HCR-multiApt interacts with the Cas12a–crRNA complex and activates trans -cleavage of surface-immobilized electrochemical reporters. Upon target binding, S. Typhimurium competitively occupies the aptamer domains and reduces the accessibility of the crRNA-complementary activator regions, thereby suppressing Cas12a activation and modulating the electrochemical signal. A three-dimensional reduced graphene oxide/gold nanoparticle (3D-rGO/AuNPs) interface was used to enhance probe immobilization and electron transfer, while Hg 2 + -mediated T–Hg²⁺–T coordination enabled conformational regulation of the methylene blue-labeled reporter for improved signal transduction. Mechanistic studies, including microscale thermophoresis, enzyme kinetic analysis, and electrophoretic mobility shift assay, supported the target-dependent competitive regulation mechanism and indicated that HCR-multiApt enhanced the apparent Cas12a activation–cleavage efficiency mainly by increasing productive activation rather than directly altering the intrinsic catalytic activity of activated Cas12a. Under optimized conditions, the platform exhibited a linear detection range of 10 0 –10 8 CFU mL −1 with a detection limit of 3.75 CFU mL −1 . Selective discrimination against non-target bacteria and acceptable recoveries of 83.0–105.7% in spiked milk samples were achieved, with results consistent with conventional plate counting. This competitive multivalent EC-CRISPR strategy provides a programmable framework for amplification-free electrochemical pathogen detection.
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DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
·
2026
·Vol. 116
·期 3
·117523
·
IF 1.8
Huifeng Yang,Xiaoyi Li,Yimin Su,Jianxin Guo,Tengfei Wang,Zhigang Wang,Hai Fu,Zhi Chen,Zhongjian Xie,Defa Li
Purpose Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection. Materials and methods A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis , SARS-CoV-2, Streptococcus pneumoniae , and human adenovirus) using real-time fluorescence and gel electrophoresis. Results This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR–Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR—preliminarily supporting the system's potential diagnostic accuracy—we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance. Conclusion The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.
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Journal of Materials Chemistry B
·
2025
·
IF 5.7
Siyue Wang,Xiaotian Guan,Shuqing Sun
C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections, autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng/mL and a quantifiable range of 0.1 -150 µg/mL. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical application.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Wenjia Dong,Xiaoyan Wu,Hongsheng Li,Junjie Yu,Siqi Xu,Yayun Jiang
Methicillin-resistant Staphylococcus aureus (MRSA) infections progress rapidly, posing a significant threat to patient health. Culture-based drug susceptibility testing and nucleic acid amplification methods are time-consuming and complex, requiring specialized instruments and technicians. To address these limitations, we present a novel, visual POCT platform for MRSA detection with the naked eye. This visualization method uses CRISPR/ Ca s12a to accurately identify target genes, cleave the strand displacement amplification ( SD A) template, and regulate the aggregation of DNA-functionalized gold nanoparticles ( Au NPs), with the resulting color change serving as a signal (CaSD@Au). The presence of the target gene activates CRISPR/Cas12a enzymatic activity, which cleaves the SDA template. This cleavage suppresses the SDA reaction, prevents DNA-AuNP aggregation, and maintains nanoparticle dispersion, resulting in a visible red color. In the absence of the target gene, the SDA reaction proceeds, and the gold nanoparticles aggregate, turning purple. Sensitivity analysis confirmed that CaSD@Au detects MRSA with a limit of detection of 5 cfu mL −1 . When combined with recombinase polymerase amplification (RPA), CaSD@Au showed detection performance comparable to qPCR in the actual detection of 20 clinical strains. Furthermore, this platform enables results to be determined simply by observing color changes with the naked eye, without the need for complex instruments. The CaSD@Au platform offers reliable, sensitive, and specific detection, enhancing the applicability of CRISPR/Cas12a-based assays for pathogen detection, especially in resource-limited settings.
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Advanced Science
·
2020
·Vol. 7
·期 14
·1903381
·
IF 15.84
Hang Yin,Xiaoling Yuan,Lihua Luo,Yichao Lu,Bing Qin,Junlei Zhang,Yingying Shi,Chunqi Zhu,Jie Yang,Xiang Li,Mengshi Jiang,Zhenyu Luo,Xinyu Shan,Dawei Chen,Jian You
The development of gene delivery has attracted increasing attention, especially when the introduction and application of the CRISPR/Cas9 gene editing system appears promising for gene therapy. However, ensuring biosafety and high gene editing efficiency at the same time poses a great challenge for its in vivo applications. Herein, a pardaxin peptide (PAR)-modified cationic liposome (PAR-Lipo) is developed. The results are indicative that significantly enhanced gene editing efficiency can be obtained through the mediation of PAR-Lipos compared to non-Lipos (non-PAR-modified liposomes) and Lipofectamine 2000, owing to its protection toward carried nucleotide by the prevention of lysosomal capture, prolongation of retention time in cells through the accumulation in the endoplasmic reticulum (ER), and more importantly, facilitation of the nuclear access via an ER-nucleus route. Accumulation of PAR-Lipos in the ER may improve the binding of Cas9 and sgRNA, thus further contributing to the eventually enhanced gene editing efficiency. Given their high biosafety, PAR-Lipos are used to mediate the knockout of the oncogene CDC6 in vivo, which results in significant tumor growth inhibition. This work may provide a useful reference for enhancing the delivery of gene editing systems, thus improving the potential for their future clinical applications.
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ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Jiuying Cui,Qisheng Luo,Cheng Wei,Xiandong Deng,Hongqu Liang,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
Background Escherichia coli ( E.coli ) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract , posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. Results We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli . Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. Significance With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 10 2 - 1 × 10 7 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.
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