TALANTA
·
2025
·
IF 6.1
Qiyu Zhang,Zhaoying Liu,Enze Sheng,Zhenjiang Liu,Rujin Zhao,Ming Li
To address the challenge of readily available and cost-effective monitoring, a novel aptasensor platform was proposed relying on the clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a)-driven and glucosemeter-assisted system for zearalenone (ZEN). The magnetic bead (MB)-based probes of MB@Apt-cDNA and MB@invertase were prepared to provide specific recognition and efficient conversion. When ZEN presented, the released cDNA activated the cutting function of CRISPR/Cas12a for ssDNA, releasing numerous invertase to convert the environmentally friendly sucrose. Then, the generated glucose signals were positively related to ZEN levels, which quantified by a glucosemeter. The proposed aptasensor displayed an excellent sensitivity, achieving the limit of detection (LOD) and detection range of 0.218 ng/mL and 0.218−109.89 ng/mL, respectively. The platform also exhibited high specificity (with cross-reactivity values below 6.81 % for other analytes) and high accuracy for ZEN detection. The satisfactory reliability and practicability had been demonstrated through its application in real agricultural byproduct samples. The area under the curve was 0.979, with an optimal operating point being (0.125, 1.000). This study can provide an alternative pathway for improving the detection performance and accessibility to public in limited situation, suggesting the efficient CRISPR/Cas12a and green glucosemeter detection strategies for other pollutant monitoring.
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Biomolecules
·
2025
·Vol. 15
·期 3
·315
·
IF 4.8
Dongxia Pan,Mukaddas Mijit,Hui Wang,Chaoqun Sun,Bantan Pingcuo,Zhixue Yu,Benhai Xiong,Xiangfang Tang
The A-to-G mutation (FecB) in theBMPR1Bgene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of theFecBgene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting theFecBmutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detectingFecBmutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.
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Nature Communications
·
2025
·Vol. 16
·期 1
·1-14
·
IF 15.7
Hu Hao,Guo Songcheng,Li Yiyuan,Dong Kejun,Lu Yan,Ye Keyi,Li Longjie,Zhou Xiaoyu,Cheng Liming,Xiao Xianjin
Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system’s robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.
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ACS Nano
·
2025
·
IF 16
Ye Zhang,Wenbin Li,Siting Chen,Yu Zhang,Yitong Zhu,Fei Lan,Huijun Du,Rui Fan,Junfang Zhu,Weilun Pan,Bo Situ,Lei Zheng,Shihua Luo,Xiaohui Yan
The lack of selective tumor targeting and the high toxicity of conventional chemotherapy treatments remain major challenges in cancer therapy. Here, we develop a self-controlled DNA nanostructure-CRISPR-12a system, a triple-locked cascade tumor therapy nanocapsule (Tatna), for efficient and targeted tumor treatment. Tatna integrates structural DNA tetrahedrons (DTs) with high drug-loading capacity, Cas12a/crRNA ribonucleoprotein (Cas12a RNP), and doxorubicin (DOX) to enable multisite response for precise drug delivery and augmented tumor treatment. By incorporation of a nucleolin-targeting aptamer, Tatna achieves selective targeting and efficient tumor cell internalization. Encapsulation in pH-responsive poly l-lactic-co-glycolic acid (PLGA) nanocapsule ensures stable circulation and controlled release of both DOX and Cas12a until tumor-specific activation in the acidic microenvironment. The Cas12a RNP, triggered by APE1 mRNA overexpression in tumor cells, induces trans-cleavage of DTs, releasing DOX and Cas12a to transport into the nucleus and induce enhanced cell apoptosis. This self-regulating and multifunctional approach enhances the efficacy of chemotherapy while reducing off-target effects. Tatna’s programmable, tumor-specific delivery system represents a powerful strategy for advancing precision medicine and personalized cancer treatment.
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BIOELECTROCHEMISTRY
·
2026
·
IF 4.9
Mingzhu Yang,Dujuan Li,Ao Wang,Yifan Zhang,Jiale Ouyang,Xinyi Wang,Liang Hong,Dingyi Tong,Minghua Wang,Jianhan Lin,Qiong Luo,Gaofeng Wang
Breast cancer, a globally prevalent malignancy in women, requires early diagnosis to improve patient outcomes, where ctDNA serves as a key biomarker. In the present study, an electrochemical biosensor based on CRISPR/Cas12a and Au@UiO-66 nanozyme synergistic dual amplification was developed to achieve ultrasensitive detection of breast cancer marker ctDNA. Au@UiO-66-modified ssDNA probes were anchored to the gold electrode via thiol groups. The intact ssDNA probe anchors Au@UiO-66 nanozyme to the electrode, where its peroxidase-like activity efficiently catalyzes H 2 O 2 reduction to generate amplified reduction peak currents. Target ctDNA activates Cas12a's trans-cleavage capacity by binding specifically to crRNA. Under the presence of the target, Au@UiO-66 nanozymes are released, resulting in a discernible drop in the peak current linked to H 2 O 2 reduction. Benefiting from the optimized experimental conditions, the biosensor demonstrates a broad linear detection range (from 10 fM to 10 nM) for the target ctDNA, along with an exceptionally low detection limit of 6.14 fM. Successful detection in human serum samples demonstrates its practicality. The platform's high specificity is attributed to the programmable crRNA design, enabling detection of specified DNA sequences and showcasing significant adaptability for diagnosing multiple genetic targets, highlighting its potential in clinical cancer diagnosis.
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BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Yuanyuan Miao,Chuang Wang,Yue Peng,Xinyuan Sun,Zhaokun Zheng,Qianlei Zhang,Wenting Cheng,Jinlong Li
Prostate specific membrane antigen (PSMA)-positive exosomes hold significant potential for the diagnosis and risk assessment of prostate cancer. However, accurate detection is severely hindered by their low abundance in blood and interference from similarly sized particles. In this study, we have developed a protospacer adjacent motif (PAM)-assembled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for the highly sensitive detection of PSMA-positive exosomes. In this work, two split strands respectively containing CD63 and PSMA aptamers are utilized to bind CD63 and PSMA on the exosome surface, forming a template that induces the opening of a hairpin DNA (HP DNA). A PAM site forms via hairpin-to-double-stranded structure transition. CRISPR-Cas12a recognizes PAM, activates to cleave FAM-labeled probes for fluorescence, while cerium dioxide nanozyme (CeO 2 NZ) (with phosphatase-mimicking activity) hydrolyzes cleavage products. Hydroxyl radicals from hydrolysis oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to induce colorimetry. CRISPR-Cas12a-nanozyme dual recognition significantly improves prostate cancer exosome detection selectivity and sensitivity. Under optimized conditions, the limits of detection for the fluorescence and colorimetric modes reach 49 particles/μL and 63 particles/μL, respectively. By mutually validating dual detection modes, this biosensing technology accurately distinguishes prostate cancer patients from healthy individuals, holding great promise for early diagnosis.
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Shichao Peng,Hang Zhai,Yunyun Tan,Yang Liu,Junliang Chen,Fan'ge Kong
Pseudomonas tolaasii , a widely distributed pathogenic bacterium, causes brown blotch disease of edible mushrooms, limiting the advancement of the edible mushroom industry in China. A one-tube method platform that combined clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system and recombinase-aided amplification (RAA) for P. tolaasii detection was developed. The sub-optimal protospacer adjacent motif (PAM) sequence was selected for the recognition of target DNA to achieve the one-tube detection. Additionally, fluorescent DNA probe and lateral-flow dipsticks (LFD) were utilized for visualization. The established one-tube detection method enabled visual detection, achieving detection limits of 1.6 × 10 −6 ng/μL and 1.6 × 10 −5 ng/μL P. tolaasii recombined plasmid, respectively, by integrating LFD or fluorescent ssDNA probe. It exhibited good specificity, successfully recognizing the P. tolaasii from other 5 common bacteria, including P. aeruginosa , Staphylococcus aureus , Escherichia coli , Brevundimonas di min uta , and Bacillus thuringiensis. The detection time was approximately 25 min. Overall, a CRISPR-coupled RAA method employing suboptimal PAM sequences was developed for one-tube, visual detection of P. tolaasii with high sensitivity and specificity, enabling visual naked-eye readout. The developed method offers a convenient and user-friendly tool for the early diagnosis of P. tolaasii -related diseases in edible mushroom cultivation.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Yaqi Wang,Xinning Wang,Xin Ji,Yunxuan Weng,Xiaoying Zhao
Zearalenone (ZEN) is a mycotoxin that poses severe health risks to humans and livestock through contaminated cereals and feed. In this study, a hydrogel-based sensor was developed for sensitive visual detection of ZEN. The sensor integrates a DNA aptamer-crosslinked polyacrylamide hydrogel encapsulating gold nanoparticles (AuNPs) with a rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for signal enhancement. Upon ZEN binding, the released complementary DNA triggers RCA, generating repetitive DNA strands that activate Cas12a. The activated Cas12a efficiently cleaves numerous single-stranded DNA crosslinkers within the hydrogel, leading to substantial dissociation and release of AuNPs for colorimetric readout. This strategy achieves high specificity and a significantly improved detection limit of 1.5 nM for ZEN, which is over 3000-fold lower than that of the aptamer-hydrogel system without amplification. The sensor enables rapid, simple, and cost-effective on-site quantification of ZEN, with the detection sensitivity and linear range well suited for food safety monitoring applications.
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MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 7
·1-7
·
IF 5.3
Wang Shirong,Wei Zaiwa,Li Liangxian,Luo Yu,Huang Zhimei,Yang Xing,Tang Yafang
CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu 2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis. Graphical Abstract
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BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Jienan Shen,Zhi Chen,Ruibin Xie,Jingfeng Li,Chunyan Liu,Yaqing He,Xiaopeng Ma,Hui Yang,Zhongjian Xie
Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H 1 N 1 , H 3 N 2 , IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.
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FOOD CHEMISTRY
·
2023
·
IF 8.8
Xuening Shi,Hang Li,Shuo Yao,Yukun Ding,Xiuzhu Lin,Hui Xu,Yi Liu,Chao Zhao,Tong Zhang,Juan Wang
The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans -cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.
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ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Guobin Mao,Xing Luo,Silu Ye,Xun Wang,Jin He,Jilie Kong,Junbiao Dai,Wen Yin,Yingxin Ma
It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.
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