Biosensors-Basel
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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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LWT-FOOD SCIENCE AND TECHNOLOGY
·
2025
·
IF 6.6
Xingyue Tang,Yafang Shen,Jiangqi Wang,Zhaohui Qiao,Fangkai Han,Yanna Cui,Qiuyue Hu,Yuxin Wu,Jiahua Wang,Xiaodan Liu,Huang Dai,Guijie Hao
Sensitive and accurate detection of enrofloxacin (ENR) residues is essential for food safety. Herein, a competitive immunosensor was developed for the detection of ENR, using a gold nanoparticle (AuNP)-enhanced CRISPR/Cas12a system. In this method, bovine serum albumin-ENR- and single-stranded DNA-modified AuNPs (AuNPs-BSA-ENR/ssDNA) was used to act as both a competitor and CRISPR/Cas12a activator. The use of AuNPs-BSA-ENR/ssDNA helped transduce the target recognition information to the cleavage activity of the CRISPR/Cas12a system. Moreover, benefiting from the high ssDNA loading capability of AuNPs, it enabled one-to-multiple CRISPR/Cas12a activation. The synergetic signal amplification from AuNPs and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a limit of detection of 1 ng/mL. Its feasibility for the detection of food and environmental samples was also investigated with recovery rates ranging from 91.0% to 115.2%. The developed immunosensor offered a promising alternative for ENR detection.
研究领域:
分析化学
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生物传感器
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食品安全
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纳米技术
·
分子诊断
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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
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CRISPR技术
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生物传感
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ANALYST
·
2022
·Vol. 147
·期 24
·5687-5693
·
IF 5.227
Yuyuan Zhang,Jing Lei,Wei Liu,Yan Jin,Baoxin Li
Various CRISPR/Cas12a-based biosensing systems have been developed in the past few years, and most of these systems used Taqman probes to report signals through fluorescence resonance energy transfer (FRET). In this study, we explored chemiluminescence resonance energy transfer (CRET) as the readout mode for CRISPR/Cas12a-based biosensing. The chemiluminescence (CL) reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) and H2O2 was used to excite the fluorophore dye of the Taqman probe. Different from FRET, CRET does not need external excitation light, which can effectively avoid autofluorescence and photobleaching. The detection limit of this CRET readout mode was estimated to be 10 pM for target DNA, which was about 8 times lower than that of the widely used FRET readout mode. These results suggest that CRET can serve as a rapid, sensitive and simple readout mode of CRISPR/Cas12-based biosensing, and can further enrich the toolbox of CRISPR/Cas12-based biosensing.
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FOOD CONTROL
·
2022
·
IF 6.652
Baoqing Zhou,Qinghua Ye,Moutong Chen,Chufang Wang,Xinran Xiang,Ying Li,Jumei Zhang,Youxiong Zhang,Juan Wang,Shi Wu,Qihui Gu,Yu Ding,Qingping Wu
Rapid and accurate screening of foodborne pathogens in contaminated food is with great significance for food safety. AuNPs-based colorimetric assay is an attractive strategy in the field of food safety inspection, but is limited by tedious probe preparation, low color rendering resolution, and narrow color gamut. Herein, we proposed an e nhanced c olorimetric p latform based on the C RISPR/Cas12a system and label-free D NA-AuNP p robe (CDP-ECP) for high-throughput detection of Staphylococcus aureus ST398. In this strategy, a novel and specific gene targeting S. aureus ST398 was identified and efficient AuNP-bioprobe was successfully prepared. Through a PCR/RAA amplification, the trans -ssDNA cleavage ability induced by target DNA product triggered an aggregation performance for the prepared AuNP-bioprobe pairs, which could realize target colorimetric detection within 50 min. Under optimal conditions, the colorimetric assay could detect as low as 6.4 × 10 2 cfu/mL of S. aureus ST398 in pure culture. Specific gene mining and target-induced trans -cleavage character gave this method a superior specificity and anti-interference. More importantly, this colorimetric method can be extended to a high-throughput platform for rapid detection of spiked and natural sample by using a common microplate reader. Since it is simple to operate, low cost, high sensitivity and specificity, this new platform is fully qualified for clinical diagnosis, environmental monitoring onsite testing requirements.
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BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Feng Qiu,Xiaoyan Gan,Jianglong Yao,Bingying Jiang,Ruo Yuan,Yun Xiang
Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.
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LWT-FOOD SCIENCE AND TECHNOLOGY
·
2023
·
IF 6
Yingchun Li,Yaqin Zhang,Fange Kong,Chunxia Wang,Shanshan Chen,Jiasi Wang,Di Wang
Fusarium temperatum ( F. temperatum ) causes maize stalk rot disease, reducing grain yield and producing multiple harmful mycotoxins that threaten food safety and quality. However, current detection methods are limited by the requirement of well-trained personnel, complicated operations and expensive instruments. To address this challenge, gold nanoparticles (AuNPs)-based colorimetric assay using loop-mediated isothermal amplification (LAMP) coupled with CRISPR-Cas12a (Au-CRISPR) was tested for F. temperatum detection. The assay utilizes DNA-modified AuNPs as a colorimetric probe and the results can be readily observed by the naked eye. After optimization, the method can specifically detect as low as 100 copies/μL of target DNA or 10 −8 ng/μL of extracted DNA from F. temperatum . The detection platform allows high-throughput testing by integrating microtiter plates and microplate reader. Furthermore, a portable smartphone-based device was developed to realize point of care (POC) detection of F. temperatum in resource-limited settings. This simple, inexpensive and sensitive detection platform has great potential for the applications in field detection.
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