ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Junzhuo Shan,Yixiao Sheng,Lun Luo,Wenhai Wang,Xinjiang Liu,Yi Ma,Jufang Wang
Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10–4 U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid–based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2024
·
IF 8.3
Runlin Yang,Siying Xie,Bin Zhou,Mingming Guo,Jun Fan,Fengli Su,Zhirun Ji,Yue Chen,Bingzhi Li
CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
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.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2023
·Vol. 190
·期 11
·1-9
·
IF 5.7
Wang Jiaying,Wang Xianliang,Li Bin,Zhang Kai,Mao Jingyuan
An electrochemical biosensor is reported for controlling CRISPR/Cas12a activity through the utilization of entropy-driven reactions, alongside the construction of a highly sensitive biosensor for B-type natriuretic peptide (BNP) detection. In the biosensor, entropy-driven reactions are employed to regulate the activity of CRISPR/Cas12a - a gene editing tool - capable of nonspecific cleavage of single-stranded DNA (ssDNA). The biosensor architecture encompasses an electrode that is modified with ssDNA probes designed to hybridize with target BNP aptamers. These aptamers, furnished with labeled ssDNA triggers, facilitate the activation of CRISPR/Cas12a through interaction with its guide RNA. Upon the presence of BNP, it associates with the aptamers, subsequently liberating the triggers that instigate the entropy-driven reactions. As a consequence of these reactions, more stable duplexes emerge between the triggers and guide RNA, thereby activating CRISPR/Cas12a. The activated CRISPR/Cas12a subsequently executes cleavage of ssDNA probes residing on the electrode surface, culminating in the generation of an electrochemical signal directly (the calibration plots of differential pulse voltammetric detection were acquired at a working potential of 0.2 V (vs. ref. electrode)) proportional to the BNP concentration. Validation of the biosensor’s performance is undertaken, wherein BNP detection is demonstrated in both buffer and human serum samples. Evident in the findings is the biosensor’s discernible sensitivity and specificity for BNP detection, exemplified by a detection limit of 13.53 fM and a lack of interference originating from other cardiac biomarkers, respectively. Furthermore, the biosensor’s potential to discriminate between healthy individuals and those afflicted by heart failure, predicated on distinctive BNP levels, is illustrated. Graphical Abstract
✓ 已验证
DOI
PubMed
高影响因子
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.
✓ 已验证
DOI
PubMed
高影响因子
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.
✓ 已验证
产品关联
DOI
高影响因子
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.
✓ 已验证
产品关联
DOI
高影响因子
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.
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）