npj Science of Food
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2026
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IF 9.7
Wang Juan,Li Xiang,Zhang Jing,Zhao Chao,Shi Xuening
Microbial Contamination serves as an important issue in dairy safety. CRISPR/Cas12a-based methods possess tremendous potential, but are limited by nucleic acid pretreatment and specialized operations. Herein, a photothermal nanomaterial-assisted enhanced One-Tube CRISPR/Cas12a system was established to detect S. typhimurium in milk . S. typhimurium was lysed through the photothermal effect to obtain genomic DNA. A multi-crRNA strategy enables rapid signal accumulation, and the whole system was integrated into a single tube via trehalose-based aqueous biphasic systems. The proposed method requires only one step of sample addition to achieve sensitive detection within 40 min, with an LOD of 10 CFU/mL, showing potential in application.
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MICROCHIMICA ACTA
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2024
·Vol. 191
·期 8
·1-12
·
IF 5.3
He Qian,Chen Qun,Lian Lijin,Qu Jiuxin,Yuan Xi,Wang Chuhui,Xu Lidan,Wei Jiazhang,Zeng Shaoling,Yu Dongmei,Dong Yuhan,Zhang Yongbing,Deng Lin,Du Ke,Zhang Canyang,Pandey Vijay,Gul Ijaz,Qin Peiwu
The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13’s reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg 2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform. Graphical Abstract
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研究领域:
生物传感
·
分子生物学
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纳米技术
·
病毒学
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ANALYTICAL CHEMISTRY
·
2026
·
IF 7.3
Hui Liu,Hang Yin,Tao Yang,Jia Xiang,Tianhui Xue,Baiqi Lu,Yang Wu,Yongmei Liu,Tiantian Deng,Tingting Sun
Circular RNAs (circRNAs) are emerging as promising biomarkers in liquid biopsy, yet their accurate detection in circulating tumor cells remains challenging due to low abundance and interference from linear RNA counterparts. Here, we develop a dCas13a/crRNA-Linked Immunosorbent Mimic Assay (dCLIMA) for sensitive and specific colorimetric detection of circRNAs. The dCas13a/crRNA complex immobilized on a microplate specifically captures target circRNAs via their back-splicing junction (BSJ) region, eliminating linear RNA interference. Upon capture, the circRNA unlocks a hairpin probe and facilitates padlock circularization, triggering rolling circle amplification (RCA) to produce tandem G-rich repeats. Notably, this design enables RCA to recognize and probe dual functional domains on the same circRNA, allowing more comprehensive functional characterization. The G-rich repeats fold into G-quadruplex/hemin DNAzyme that catalyzes TMB oxidation to generate a blue product for label-free final readout. The dCLIMA assay achieves a detection limit of 0.63 fM for circRNA_100876, discriminates single-base mismatches, and exhibits excellent anti-interference capability. We successfully quantify circRNA_100876 in lung cancer-derived cells, cell lysates, and clinical serum samples, with results correlating well with qRT-PCR. This method holds great promise for advancing circRNA-based liquid biopsy and precision oncology.
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ANALYTICAL CHEMISTRY
·
2026
·
IF 6.7
Miao He,Jinhua Chen,MuRilege Chao,Feifan Yin,Han Pan,Qiufeng Wang,Wenting Cheng,Zhongyun Wang,Yang Xiang
mRNA vaccines, as a new form of vaccine, are gradually revealing their potential in the field of tumor treatment. With the development of mRNA vaccines, monitoring their levels has become particularly important. In this work, a novel and innovative strategy was proposed for the first time, which directly used the target as crRNA and simultaneously triggered an entropy-driven split Cas12a system for triple signal amplification for the high-sensitivity detection of mRNA vaccines. This study broke the traditional full-length crRNA and innovatively designed a split crRNA structure, directly using the target mRNA as the variable sequence of the crRNA. This design avoided the need for additional introduction of variable sequences, thereby eliminating nonspecific signal leakage and multicomponent interference from the source. At the same time, the target molecule served as the initiating chain for the EDC reaction, allowing the split Cas12a system to be coupled with the entire entropy-driven reaction process, achieving efficient collaboration and triple signal amplification and significantly improving the detection sensitivity and specificity. Moreover, the entire detection process required only one reaction step, which was simple, fast, and efficient. The aim was to provide strategies for screening and pharmacokinetic studies of mRNA vaccines.
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Analytical Methods
·
2026
·
IF 3
Jing Chen,Yanna Le,Luoxian Yang,Hua Qin,Wanli Yang
Chronic exposure to lead ions (Pb2+) disrupts intestinal flora homeostasis, underscoring the need for sensitive and practical detection methods. Herein, we report a dual-mode analytical strategy combining split-DNA re-assembly-driven colorimetric reaction with CRISPR/Cas12a trans-cleavage activity for Pb2+ analysis. A bifunctional magnetic probe is constructed, where Pb2+-specific DNAzyme recognition triggers cleavage and release of crRNA, which subsequently activates Cas12a to generate fluorescence by cleaving a fluorophore-quencher reporter. Concurrently, the remaining magnetic bead-anchored trigger initiates split G-quadruplex re-assembly, catalyzing a visible colorimetric reaction. Under optimized conditions, the fluorescence mode achieves a detection limit of 1.03 fM with a linear range of 5 fM to 100 pM, while the colorimetric mode offers a limit of 62.1 fM from 100 fM to 500 pM. Both modes exhibit excellent specificity against competing metal ions. The platform's clinical feasibility is validated using human serum samples, showing strong correlation with ICP-MS, satisfactory recoveries (97.1–103.4%), and good repeatability (CV = 4.1%). Magnetic separation effectively minimizes matrix interference, making the assay suitable for complex biological specimens such as intestinal contents and fecal samples. This dual-mode design integrates fluorescence for precise quantification and colorimetry for equipment-free visual detection, holding great promise for point-of-care testing, early risk assessment of lead-induced gut microbiota dysbiosis, and environmental monitoring.
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NUCLEIC ACIDS RESEARCH
·
2024
·
IF 16.6
Wu Yaokang,Li Yang,Liu Yanfeng,Xiu Xiang,Liu Jiaheng,Zhang Linpei,Li Jianghua,Du Guocheng,Lv Xueqin,Chen Jian,Ledesma-Amaro Rodrigo,Liu Long
Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.
研究领域:
分子生物学
·
基因工程
·
微生物学
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