ANALYTICA CHIMICA ACTA
·
2026
·
IF 6.1
Lingjun Cheng,Qingmei Yang,Yuanyuan Yang,Yufeng Wang,Qingfei He,Zhiyan Zou,Canping Su,Jiaming Chen,Yinhuan Liu,Guolin Hong
Background Tuberculosis (TB) nucleic acid diagnosis urgently requires rapid, amplification-free methods to overcome limitations of quantitative real-time polymerase chain reaction (qPCR), including instrument dependency, prolonged time, and contamination risks from nucleic acid amplification. Results Here, an amplification-free electrochemiluminescence (ECL) biosensor based on peptide-templated gold nanoclusters and the clustered regularly interspaced short palindromic repeats-Cas12a system (CRISPR-Cas12a) has been constructed for the detection of Mycobacterium tuberculosis (MTB)-specific IS6110 sequences. Peptide-templated gold nanoclusters with low background and high ECL response can be used as sensitive signal probes. When CRISPR-Cas12a recognizes the target IS6110 DNA, it exhibits non-specific cleavage activity, repeatedly cleaving ferrocene-labelled DNA. This results in the restoration of ECL signals quenched by ferrocene, thereby achieving signal amplification. There is a linear relationship between the signal and the target concentration range from 10 CFU/mL to 10 4 CFU/mL, with a detection limit of 7 CFU/mL (S/N = 3). Clinical validation ( n = 40) showed strong agreement with qPCR ( κ = 0.90). Significance Critically, this amplification-free strategy eliminates the need for temperature cycling equipment, reduces detection time to 1 h, and completely avoids the risk of amplicon contamination, providing an ideal solution for rapid screening of highly infectious diseases such as TB.
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Analytical Methods
·
2025
·Vol. 17
·期 25
·5298-5307
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IF 2.6
Yang Cao,Chun Huang,Kui Li,Xi Zhang,Mengfei Tong,Jiale Tu,Kun Yang,Shuai Yuan,Hong Zhang
To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2′-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)32+/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL−1 in a linear detection range of 10−5–100 ng mL−1. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.
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高影响因子
Biosensors-Basel
·
2025
·Vol. 15
·期 10
·700
·
IF 5.6
Yu Qin,Jiali Xie,Shujun Zhen
Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5–9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.1
Meilin Liu,Yi Duan,Yiyao Luo,Xuhui Xia,Mingyi Guo,Danqun Huo,Changjun Hou
Ochratoxin A (OTA), a prevalent cereal mycotoxin, exhibits carcinogenic and teratogenic properties that threaten public health. Consequently, sensitive and reliable OTA detection methods are critically needed. A CRISPR-Cas12a-mediated colorimetric biosensor was developed, integrating Cas12a regulation via modular hairpin structures with MOF@Pt@MOF nanozyme amplification. Hairpin structures incorporate target-specific sequences; target binding induces rearrangement, preventing Cas12a recognition while enabling dsDNA activators to restore Cas12a function. Magnetic beads (MBs) were coupled with MOF@Pt@MOF nanozymes via ssDNA to construct MBs-MOF@Pt@MOF probes. Following OTA-induced activation, Cas12a cleaves ssDNA, releasing nanozymes into the supernatant, catalyzing TMB oxidation to generate a blue chromophore. A detection limit of 0.055 ng/mL was achieved with excellent selectivity. This approach utilizes inhibitory aptamer conformational changes for precise Cas12a regulation and nanozyme signal amplification to enhance sensitivity and specificity. The modular design demonstrates versatility for diverse target detection in point-of-care testing (POCT) applications.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 7.7
Huikai Lin,Caiyi Du,Ningyi Xu,Yiru Wang,Chunyan Sun
CRISPR/Cas12a can activate trans-cleavage activity upon single-stranded DNA binding, making it widely applicable in highly sensitive fluorescent biosensors. Sensors utilizing monochromatic fluorescence output modes are susceptible to interference from multiple factors, resulting in unstable detection methods. To address this, we designed a ratio-based fluorescence sensor for detecting tetracyclines (TCs) based on the synergistic signal amplification of nucleic acid aptamer (42TET) and zirconium-based metal-organic framework (PCN-224) with CRISPR/Cas12a. PCN-224 adsorbs single-stranded DNA, quenching its FAM-labeled fluorescence while simultaneously emitting its own fluorescent signal. This dual-fluorescence output reduces background interference and enhances detection accuracy. By designing an activator as a complementary sequence (cDNA) to the nucleic acid aptamer, the system binds to 42TET in the presence of tetracycline, releasing the cDNA to activate the CRISPR/Cas12a system. Through this system's signal amplification capability, highly sensitive detection of tetracycline is achieved, with a detection limit reaching 8.7 nM. It possesses the same detection capabilities as traditional HPLC detection methods. This method is simple, rapid, efficient, and highly quantitative, offering broad prospects for future applications.
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ANALYTICAL CHEMISTRY
·
2026
·
IF 6.7
Xiang Chen,Chunxiang Mao,Yuanji Gao,Chaoting Shi,Yu Wang,Zhuolin Jin,Bing Xia,Yan Zhou
Direct quantitative analysis of low-abundance protein biomarkers by electrospray ionization mass spectrometry (ESI-MS) remains challenging due to poor ionization efficiency and matrix interferences. Herein, we report an ultrasensitive analytical platform, termed CRISPR/Cas12a-mediated liposomal amplification coupled with electrospray ionization mass spectrometry (CMLA-MS), that overcomes this limitation by integrating CRISPR/Cas12a-mediated dual-cascade signal amplification with an ESI-MS readout. The strategy converts the detection of poorly ionizable protein molecules into the quantification of numerous, highly ionizable small-molecule reporters: proteins trigger Cas12a trans-cleavage (first amplification), which subsequently cleaves single-stranded DNA (ssDNA) probes anchored to signal-loaded liposomes, causing the burst release of thousands of MS-detectable reporters (second, physical amplification). This dual-amplification strategy enabled an exceptionally low limit of detection (LOD) of 10.8 fg/mL, and the method successfully quantified cardiac troponin I (cTnI) in clinical serum samples with high recoveries (90.3–101.6%).
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ANALYTICAL CHEMISTRY
·
2026
·
IF 6.7
Kun Wang,Wanli Wang,Yufeng Hu,Xiaoyang Li,Haoyu Yu,Xin Xia,Tianhan Kai,Mei Wen
Staphylococcus aureus (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.
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SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2026
·
IF 4.8
Wenjie Liao,Shiqiang Li,Shaoyong Wu,Xiaosong Yang,Bo Liu,Liansheng Ling,Qiuyao Zeng,Ji Zhang
Prostate Cancer Antigen 3, a type of long non-coding RNA, exhibits outstanding specificity as a biomarker for the diagnosis of prostate cancer, offering a highly effective diagnostic indicator, whereas the currently used prostate specific antigen exhibits low specificity, leading to reduced accuracy in prostate cancer diagnosis. Herein, we designed a novel fluorescent sensing platform for targeted detection of PCA3, which integrates the non-specific trans-cleavage activity of the CRISPR/Cas12a with the remarkable fluorescence quenching effect of Gold Nanorods. The Cas12a recognizes and binds to specific sequences of PCA3, thereby activating nonspecific cleavage activity, which cleaves fluorescent reporter probes adsorbed on AuNRs, thus leading to the recovery of fluorescence signals and enabling sensitive detection. The proposed fluorescent sensor exhibits excellent accuracy and convenience for the detection of PCA3 in urine, and a detection limit as low as 1.65 pM was obtained. This sensing system has achieved effective detection of clinical samples of prostate cancer and is expected to provide significant assistance in the screening and therapeutic feedback of prostate cancer in clinical diagnosis.
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BIOMATERIALS
·
2025
·
IF 12.9
Ting Fang,Chang Peng,Rui Ding,Yinzhen Fan,Jiajia Jia,Jiajie Chen,XinHang Zhang,Dongkai Wang,Ji Li
Head and neck squamous cell carcinoma (HNSCC) continues to exhibit a poor prognosis, largely due to late diagnosis and the development of cisplatin resistance. Tumor proliferation in HNSCC is closely associated with upregulation of key glycolytic enzymes. However, monotherapeutic targeting of glycolysis paradoxically enhances compensatory glutaminolysis via glutamate overproduction. To overcome this metabolic adaptation, we developed biomimetic nanoparticles P-T-p@CM, fabricated from the pH/reduction dual-responsive copolymer poly(lactic acid)-polyhistidine-polyethylenimine (PLA-pHis-ss-PEI). This nanoplatform enables coordinated co-delivery of telaglenastat (a glutaminase 1 (GLS1) inhibitor) and a CRISPR-Cas9 plasmid encoding sgRNA targeting HIF-1α. This system utilizes homologous cancer cell membrane coating for precise tumor homing, with stimuli-responsive release enabling simultaneous dual metabolic blockade: CRISPR-mediated HIF-1α knockout attenuates glycolysis while telaglenastat suppresses glutamine-to-glutamate conversion. Metabolic analyses confirmed significant reduction in Glycolysis proton efflux rate (GlycoPER), the oxygen consumption rate (OCR) and ATP generation, as well as the related metabolites including the lactate production and glutamate. This dual-starvation strategy depleted energy reserves and biosynthetic precursors, inducing severe metabolic disruption. Notably, in vivo studies showed a 90% tumor inhibition rate (TIR) after 15 days of treatment, through enhanced apoptosis, reduced proliferation, and tumor glucose/glutamate depletion. Collectively, P-T-p@CM establishes a paradigm-shifting approach to disrupt metabolic compensation in the treatment of HNSCC.
研究领域:
肿瘤学
·
纳米医学
·
癌症代谢
·
基因治疗
·
药物递送系统
·
分子生物学
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Yuqian Guo,Yuanqian Xiong,Weipeng Tong,Xiaolin Huang,Yonghua Xiong
Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.
研究领域:
分子诊断
·
食品安全监测
·
纳米生物技术
·
CRISPR技术
·
生物传感
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ACS Omega
·
2025
·
IF 4.3
Na Du,Guochao Xu,Yao Zhang,Jing Hou,Ying Wang,Yiran Li,Bing Xu
Nosocomial infection is a major global cause of mortality, posing significant public health challenges. Recent studies have identified RNA biomarkers, including microRNA (miRNA) and circular RNA (circRNA), as promising diagnostic tools for nosocomial infection, providing essential information for timely clinical intervention. In this study, we developed a versatile and label-free circRNA and miRNA detection platform based on a Cas13a/crRNA complex-based target recognition that triggers a primer exchange reaction (PER)-driven DNA walker. In this method, the Cas13a/crRNA complex is used to specifically identify target RNA biomarkers and to induce subsequent signal amplification strategies following cleavage of uracil (UUU, trU)-containing locked “sensing probe” based on trans-cleavage of Cas13a. Taking the merit of the high specific target recognition capability of Cas13a/crRNA, false signals or unintended circRNA degradation can be greatly avoided. The PER performed on the surface of streptavidin-coated magnetic beads (sMBs) motivated the walking of primer sequences, greatly amplifying fluorescence signals. Based on this design, the proposed method exhibits high sensitivity and precision, with detection limits as low as 0.41 fM for miRNA-21 and 3.12 fM for cZNF292. These findings suggest that this strategy could advance the identification of RNA biomarkers and hold considerable potential for future applications in diagnosing nursing infections.
研究领域:
分子诊断
·
CRISPR技术
·
核酸检测
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传染病监测
·
生物传感器
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SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Kai Zhang,Meina Yang,Helin Sun,Xueyin Wang,Hao Li,Jinxiang Han,Zhongwen Zhang
Using Peptide nucleic acid (PNA), this work provides a unique approach for controlling CRISPR/Cas13a activity by RNA Inhibitory PNA (RIP). This method reduces the action of CRISPR/Cas13a by inhibiting the binding of crRNA to CRISPR/Cas13a, which was subsequently utilized to detect metalloproteinase-2 (MMP-2) activity. We carefully constructed a PNA sequence that includes a peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) that is particularly cleaved by MMP-2 in the middle. When MMP-2 is not present in the system, the PNA/crRNA complex reduces crRNA binding to CRISPR/Cas13a, hence inhibiting CRISPR/Cas13a cleavage activity. When MMP-2 is present in the system, it cleaves the peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) selectively and so prevents PNA from binding to crRNA, enabling crRNA to reach the "free" form. Then, in the presence of an RNA activator, crRNA may bind to CRISPR/Cas13a and stimulate its activity. CRISPR/Cas13a triggered in this manner may precisely cleave the DNA sequence containing modified -UU- bases on the electrode surface . Thus, the ferrocene (Fc) is changed away from the electrode surface at one end of the DNA , enabling for the recovery of the Electrochemiluminescence (ECL) signal from the two-dimensional material transformed on the electrode surface. As a result, MMP-2 activity was detected utilizing this technique of inhibiting crRNA activity with a detection limit of 15.56 fM. Our suggested method of inhibiting CRISPR/Cas13a activity via PNA has a broad variety of potential therapeutic applications in gene editing and the development of CRISPR/Cas13a-based biosensors.
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