METHODS
·
2025
·
IF 4.3
Wensi Li,Zhichao Tang,Xiang Zhu,Beijuan Wang,Xiuqin Zhang,Ze Ji,Suhang Cheng
In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor’s surface was constructed using a AuNPs/Ti 3 C 2 Tx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1 . The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring.
研究领域:
生物传感器
·
分子诊断
·
CRISPR技术
·
纳米技术
·
慢性阻塞性肺疾病
·
酶检测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ADVANCED FUNCTIONAL MATERIALS
·
2025
·
IF 19
Shuofeng Li,Fangfang Wang,Chenhuan Wang,Lin Hao,Ningzhao Shang,Pengbo Zhang,Yawen Zhang,Nuo Xiao,Yaai Kang,Jin Liu,Shutao Gao,Chun Wang,Zhi Wang,Qiuhua Wu
Single-atom nanozyme (SAZ) with peroxidase-like activity has attracted great attention in point-of-care (POC) diagnostic, but the use of unstable H 2 O 2 in peroxidase catalytic reactions often leads to low detection accuracy. Thus, developing SAZ with high oxidase (OD)-like activity to construct RNA sensing systems independent of H 2 O 2 is imperative for improving accuracy. Herein, a novel strategy to fabricate boron-nitrogen co-doped zinc SAZ (ZnBNC-SAZ) with excellent OD-like activity by carbonizing Zn based zeolitic boron imidazolate frameworks is reported. The electron deficient B in imidazolate ligand can form Zn─N─B bond in situ during high temperature pyrolysis, which can regulate the electronic structure of Zn and further upshift the d-band center of Zn to improve the OD-like activity. With ZnBNC-SAZ as signal generator, a new method for sensitive RNA detection is developed by combining CRISPR/ cas13 system with terminal deoxynucleotidyl transferase-induced DNA extension reaction. The limits of detection for RNAs are as low as 20 aM. The proposed biosensor is adaptable to a lateral-flow-based readout and is universally applicable for sensing various RNAs by programming the guide RNA. Importantly, the proposed biosensor can monitor the cellular differentiation and identify patients with cervical carcinoma, showing great potential for application in facile POC diagnosis.
研究领域:
纳米技术
·
生物化学
·
分子生物学
·
医学诊断
·
生物医学工程
✓ 已验证
产品关联
DOI
高影响因子
TALANTA
·
2024
·
IF 5.6
Yuanxun Gong,Danlei Tong,Ping Qiu,Rilian Song,Zhidan Li,Yanlin He,Qisheng Luo,Qianli Tang,Kai Zhang,Xianjiu Liao
B-type natriuretic peptide (BNP) is a biomarker for heart failure, a serious and prevalent disease that requires rapid and accurate diagnosis. In this study, we developed a novel electrochemical biosensor for BNP detection based on CRISPR/Cas13a and chain substitution reaction. The biosensor consists of a DNA aptamer that specifically binds to BNP, a T7 RNA polymerase that amplifies the signal, a CRISPR/Cas13a system that cleaves the target RNA, and a two-dimensional DNA nanoprobe that generates an electrochemical signal. The biosensor exhibits high sensitivity, specificity, and stability, with a detection limit of 0.74 aM. The biosensor can also detect BNP in human serum samples with negligible interference, demonstrating its potential for clinical and point-of-care applications. This study presents a novel strategy for integrating CRISPR/Cas13a and chain substitution reaction into biosensor design, offering a versatile and effective platform for biomolecule detection.
研究领域:
生物技术
·
生物化学
·
分子生物学
·
电化学
·
医学诊断
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Zhaleh Asadi Fakhr,Wei Xie,Su Zeng,Sheng Cai
Background CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis–Menten kinetic parameters. Results Three ciRS-7–specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (ΔS = –862, –813, and –615 cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA–target pair. Michaelis–Menten analysis showed k cat values of 1.39, 1.31, and 1.16 s -1 for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity. Significance Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.
研究领域:
分子诊断
·
CRISPR技术
·
RNA生物学
·
生物化学工程
·
即时检测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Nano
·
2023
·
IF 17.1
Dou Wang,Xuedong Wang,Feidi Ye,Jin Zou,Jiuxin Qu,Xingyu Jiang
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
研究领域:
生物技术
·
分子生物学
·
诊断方法
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Dan Zhao,Jiutang Tang,Qin Tan,Xiaohong Xie,Xin Zhao,Dingpei Xing
Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application.
研究领域:
分子生物学
·
医学诊断
·
生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2026
·
IF 6.1
Zhenhua Zeng,Ayang Wu,Haimin Gao,Yanping Zheng,Huicong Yang,Xiaojie Yao,Zhongjie Yang
MicroRNAs (miRNAs) are important liquid biopsy biomarkers, and their multiplex detection is highly important for the early screening of hepatocellular carcinoma. This work developed a CRISPR/Cas13a biosensing platform based on near-infrared (NIR) light and glutathione (GSH) dual activation for highly sensitive and specific determination of miRNA-21, miRNA-155, and miRNA-224. The advantages of this platform lie in the introduction of GSH-responsive mesoporous silica carriers and photolytic linkers (pc-linkers), which transform the sensing process from “passive triggering” to “controllable activation”, effectively suppressing nonspecific signals. Specifically, GSH can degrade mSiO 2 with tetrasulfide bonds, thereby degrading the carrier. It will release the CRISPR/Cas13a system loaded on the carrier, thereby achieving specific recognition and cleavage of miRNA-21/155; at the same time, 808 nm NIR light excites upconversion nanoparticles (UCNPs) to generate ultraviolet light, which cleaves the pc-linker to activate the detection of miRNA-224. The detection limits of the developed assay for detecting miRNA-21, miRNA-155, and miRNA-224 were 4.2 pM, 5.7 pM, and 0.205 nM, respectively. The recoveries in the serum spiked experiments were 98.97–109.24%, with a relative standard deviation of less than 4.613%, indicating that the method has good accuracy and reliability. This work proposes a novel and reliable strategy for detecting multiple miRNAs in complex biological samples. MicroRNAs (miRNAs) are important liquid biopsy biomarkers, and their multiplex detection is highly important for the early screening of hepatocellular carcinoma.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2025
·Vol. 15
·期 43
·36514-36524
·
IF 4.6
Xiwei Zhuang,Zhihao Duan,Xue Ge,Yanling Wang,Yuan Gao,Jianlei Liu,Shaoxiong Zhou,Chijia Zeng,Feiyun Cui,Qin Zhou
Dysregulation of PEAK1 (pseudopodium-enriched atypical kinase 1) plays a critical role in various cellular processes, including cell migration, proliferation and survival. Its aberrant expression or activity has been implicated in the pathogenesis of several diseases, particularly cancer. Early detection allows for the development and application of targeted therapies that can inhibit PEAK1 activity, potentially improving treatment outcomes. This study provides a new method for early diagnosis of PEAK1 by utilizing the specific RNA cleavage ability of Cas13a combined with electrochemical sensing technology. CRISPR/Cas13 has cis cleavage activity and can specifically recognize and cleave target RNA. Subsequently, its trans cleavage activity is activated to non-specifically cleave other single stranded RNAs, resulting in detectable signal changes. In the experiments conducted, high sensitivity for detecting PEAK1 mRNA was achieved by optimizing the interface cleavage of the hairpin reporter probe (ReRNA) molecule. The linear detection range is from 1 pg μL−1 to 10 ng μL−1, with a detection limit of 0.45 pg μL−1. In addition, the results showed that the developed biosensor has good repeatability, reproducibility, and stability, which provides a novel method for the early screening of PEAK1.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Junlan Liu,Yuan Gao
The exceptionally sensitive and specific CRISPR-Cas systems have revolutionized molecular diagnostics, but their application remains largely confined to nucleic acid targets. To overcome this limitation and enable one-pot, mix-and-read detection of non-nucleic acid analytes, we present a generalizable CRISPR-Cas13a sensing platform that utilizes a hybrid DNA:RNA strand displacement-based switch to conditionally activate Cas13a. The switch leverages Cas13a's strict requirement for single-stranded RNA activators to mediate non-RNA target-triggered activation while protecting functional DNA elements from collateral cleavage. By integrating the modular Cas13a switch with various functional DNAs, including DNAzyme, aptazyme, and duplexed aptamer, we constructed a suite of one-pot sensors capable of detecting non-RNA targets such as lead ion, ATP, and thrombin. These CRISPR-Cas13a sensors demonstrate enhanced sensitivity, high selectivity, and robust performance in complex biological matrices. The high modularity of the CRISPR-Cas13a sensor allows rapid reconfiguration for new targets by simply altering the output sequence of functional DNA without extensive re-engineering. This versatile and robust sensing strategy expands the application landscape of CRISPR-Cas systems and lays the foundation for flexible, sensitive, and one-pot detection of a wide array of clinically and environmentally relevant targets.
研究领域:
分子诊断学
·
合成生物学
·
生物传感器
·
CRISPR技术
·
核酸化学
·
生物分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Sensors
·
2022
·
IF 9.618
Haiyang Yu,Huibin Zhang,Jinhua Li,Zheng Zhao,Minhua Deng,Zhanpeng Ren,Ziqin Li,Chenglong Xue,Mitch Guijun Li,Zhaowei Chen
The disease caused by severe acute respiratory syndrome coronavirus, SARS-CoV-2, is termed COVID-19. Even though COVID-19 has been out for more than two years, it is still causing a global pandemic. Due to the limitations of sample collection, transportation, and kit performance, the traditional reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method has a long detection period and high testing costs. An increased risk of infection is inevitable, since many patients may not be diagnosed in time. The CRISPR-Cas13a system can be designed for RNA identification and knockdown, as a promising platform for nucleic acid detection. Here, we designed a solution-gated graphene transistor (SGGT) biosensor based on the CRISPR-Cas13a system. Using the gene-targeting capacity of CRISPR-Cas13a and gate functionalization via multilayer modification, SARS-CoV-2 nucleic acid sequences can be quickly and precisely identified without the need for amplification or fluorescence tagging. The limit of detection (LOD) in both buffer and serum reached the aM level, and the reaction time was about 10 min. The results of the detection of COVID-19 clinical samples from throat swabs agree with RT-PCR. In addition, the interchangeable gates significantly minimize the cost and time of device fabrication. In a nutshell, our biosensor technology is broadly applicable and will be suitable for point-of-care (POC) testing.
研究领域:
生物技术
·
纳米技术
·
分子生物学
·
医学诊断
✓ 已验证
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Jihua Wei,Jiahui Wang,Runze Wu,Jiayi Zhang,Haozhen Ren,Qianli Tang,Longjian Huang,Kai Zhang,Xianjiu Liao
Sensitive and reliable detection of DNA repair enzymes is critical for early cancer diagnosis and therapeutic monitoring. Herein, we report a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Flap Endonuclease 1 (FEN1), integrating an aptakiss-assisted CRISPR/Cas13a signal amplification strategy with a perovskite-based nanocomposite sensing interface. Specifically, a hybrid nanomaterial composed of CsPbBr 3 nanocrystals coated with polydopamine (PDA) and decorated with gold nanoparticles (AuNPs) was constructed to form a core–shell–satellite structure (CsPbBr 3 @PDA@AuNPs). The PDA coating enhanced the aqueous stability of CsPbBr 3 and introduced functional groups for probe attachment, while the AuNPs facilitated electron transfer and signal amplification. Upon recognition and cleavage of a flap-structured DNA substrate by FEN1, a downstream transcription reaction was initiated to generate RNA triggers, which in turn activated the Cas13a system. Cas13a cleaved a surface-tethered RNA strand required for forming the aptakiss complex, thereby removing ferrocene-based ECL quenching and restoring strong luminescence. The proposed biosensor exhibited an excellent detection limit of 1.73 fM, with high selectivity against non-specific nucleases, and demonstrated remarkable reproducibility and long-term stability. This study not only presents a powerful biosensing platform for FEN1 but also highlights the potential of CsPbBr 3 @PDA@AuNPs as a versatile ECL-active material. The modular design and tunable functionality of the nanocomposite enable broad applicability in detecting various biomolecules, paving the way for the development of advanced perovskite-based ECL biosensors for clinical and environmental diagnostics.
✓ 已验证
DOI
PubMed
高影响因子
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
·
传染病监测
·
生物传感器
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）