BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Qiang Tang,Jiayi Zhang,Jialuo Pang,Yingying Huang,Ying Guan,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
This study introduces an innovative electrochemiluminescence (ECL) biosensor for the highly sensitive and specific detection of Argonaute 2 (Ago2) activity. Ago2, a key enzyme in the RNA interference (RNAi) pathway, plays a crucial role in gene regulation, and its dysregulation is associated with diseases such as cancer and viral infections. The biosensor integrates hybridization chain reaction (HCR) amplification and the CRISPR/Cas12a system, leveraging a multi-stage signal amplification strategy. The detection mechanism begins with Ago2-mediated cleavage of a designed hairpin RNA (HP-RNA), releasing single-stranded RNA (ssRNA) that triggers HCR. This amplification step generates long DNA polymers, which serve as activators for the CRISPR/Cas12a system. Cas12a's collateral cleavage activity amplifies the signal further by cleaving a DNA reporter labeled with a ruthenium-based luminophore, enhancing the ECL output. This dual amplification strategy achieves exceptional sensitivity, with a detection limit of 0.126 aM. The biosensor demonstrates excellent specificity, distinguishing Ago2 from other Argonaute proteins, and maintains high reproducibility and stability, retaining 94 % of its signal after two weeks of storage. Real-world applicability was confirmed by accurately detecting Ago2 in spiked cell lysates, with recovery rates exceeding 100 %. The combination of HCR, CRISPR/Cas12a, and ECL establishes a robust platform for biomarker detection, offering superior sensitivity and adaptability for clinical diagnostics, disease monitoring, and therapeutic evaluation. This biosensor represents a significant advancement in the development of next-generation diagnostic tools.
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高影响因子
TALANTA
·
2024
·
IF 5.6
Lifeng Zhang,Shihua Luo,Rui Fan,Ruixi Li,Wenbin Li,Siting Chen,Fei Lan,Yitong Zhu,Tingting Ji,Ye Zhang,Ling Li
APE1, an essential enzyme for DNA repair, is overexpressed in various cancers and has been identified as a potential biomarker for cancer diagnosis. However, detecting APE1 at low expression levels in the early stage of cancer presents a significant obstacle. Here, we introduced a novel localized Cas12a-based cascade amplification (LCas12a-CA) method. This method confined both the terminal deoxynucleotidyl transferase and the crRNA/Cas12a complex onto the surfaces of gold nanoparticles (AuNPs). This confinement not only boosts the stability of the multiple enzymes but also induces a substrate channeling effect. As a result, it significantly accelerates the reaction rate and enhances the sensitivity of APE1 detection. Upon the addition of APE1, the AP sites within the APE1 primer can be recognized and cleaved by APE1, exposing the 3′-OH ends. In the presence of LCas12a-CA, polyA sequences are generated at 3′-OH ends with the help of TdT and dATP. The sequences directly enter the Cas12a system, activating the trans -cleavage activity of Cas12a, thereby cutting the reporters on the surface of AuNPs and releasing fluorescence. Our platform demonstrates a detection limit (LOD) as low as 2.51 × 10 −6 U/mL, which is more than 60 times lower than that of free Cas12a-CA. Furthermore, the LCas12a-CA exhibits enhanced resistance ability in extreme environments and has been proven effective for the detection of APE1 in clinical samples. Overall, this work offers a promising platform for robust biosensing in cancer diagnosis and prognosis.
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BIOSENSORS & BIOELECTRONICS
·
2024
·
IF 10.7
Xueying Lei,Lihua Ding,Xiaonan Yang,Feng Xu,Yongjun Wu,Songcheng Yu
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a p adlock activator which could i nhibit the t rans -cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca 2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca 2+ dependent. In the presence of Ca 2+ , DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans -cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca 2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Na + , Al 3+ , K + , Fe 2+ , and Fe 3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSD s from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca 2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
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VIEW
·
2024
·
IF 9.7
Jie Luo,Binpan Wang,Xiaoqi Tang,Ping Huang,Sha Yang,Shuang Zhao,Shuang Xie,Qiaofeng Li,Kai Chang,Ming Chen
Tumor-derived exosomes are crucial for early non-invasive and accurate tumor diagnosis in clinical diagnostics. The development of highly sensitive, simple, and intuitive exosome assays has sparked a research upsurge in clinical diagnostics. Here, we develop a bio-responsive intelligent DNA hydrogel loaded with CRISPR/Cas12a for universal and ultrasensitive detection of the exosomes. The aptamer serves as the target response unit and switch, competitively disintegrating the region of the DNA linkers and then Cas12a/crRNA was released and activated, resulting in a high fluorescent intensity for exosome detection at the detection limit of 119 particles/μL. Moreover, a constructed colorimetric tube is made by loading a colorimetric filter membrane on the tube lid and intelligent DNA hydrogel on the tube bottom, which enables one-pot portable colorimetric detection. Without the need for laboratory instruments and professionals, this strategy allows for naked eye detection with limit of detection as low as 10 4 particles/μL, and shows great applicability in distinguishing between healthy individuals, pretreatment patients, and post-treatment patients after obtaining a testable analyte. In this study, an ultrasensitive detection platform for exosomes that enables one-step sensing and dual signal output was introduced. The findings here suggest that this platform is a promising tool for the application of liquid biopsy based on exosomes in clinical diagnosis.
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MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Yang Song,Yiwen Xu,Ruirui Wang,Jianping Wang,Xiaoping Yu,Xuping Shentu
Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 × 10°CFU/mL, 1 × 10 4 CFU/mL, 1 × 10 2 CFU/mL, 1 × 10 3 CFU/mL, 1 × 10 1 CFU/mL for Escherichia coli , Salmonella typhimurium , Vibrio parahaemolyticus , Staphylococcus aureus , and Listeria monocytogenes , which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.
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ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Luyu Wei,Zhilong Wang,Yongzhen Dong,Deyang Yu,Yiping Chen
CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.
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Biosensors-Basel
·
2025
·Vol. 15
·期 12
·821
·
IF 5.6
Jie Chen,Shutao Liu,Sunyi Chen,Jingwen Mai,Maiwula Abudukadi,Yao Chen,Jie Lu,Guanglei Li,Chenchen Ge
Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.
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BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Mengqing Cheng,Xinyue Chen,Hongrui Cheng,Xianglin Gao,Haoyue Ao,Xinyang Bao,Xiaojun Song,Yulei Tai,Dazhi Jin,Lei Zhang
Clostridioides difficile ( C. difficile ) infection (CDI) represents a formidable global healthcare challenge, necessitating the development of rapid, accurate, and cost-effective diagnostic platforms to mitigate nosocomial transmission and improve patient outcomes. Compared with the conventional methods, CRISPR/Cas systems featured by specific target reorganization by a single chain RNA, coupled with electrochemical technology enables highly sensitive detection of various biomarkers. However, their application to CDI has remained unexplored due to the lack of tailored crRNAs. Herein, we present the integration of CRISPR/Cas12a with electrochemical transduction for the direct detection of C. difficile . A novel crRNA was engineered to specifically recognize the toxin B gene ( tcdB ), activating the trans-cleavage activity of Cas12a upon target binding. This cascade triggers the cleavage of immobilized ssDNA reporters on the electrode surface, generating measurable amperometric signal changes. The developed biosensor demonstrates exceptional performance, achieving a detection limit of pM level for tcdB DNA within 40 min, while exhibiting high specificity against non-target pathogens and robust stability over 7 days. This work establishes a rapid and reliable CRISPR-electrochemical diagnostic platform, offering significant potential for point-of-care CDI management.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Feifei Zhou,Yingzhan Fan,Qinrui Cao,Yingying Yuan,Yingxiao Chen,Lingling Liu,Jingwen Mai,Nandi Chen,Tianxing Ji,Dou Wang,Chenchen Ge
Dual-function DNA probe (dfDNA) enables a split crRNA/Cas12a (SCas12a) assay. • The dfDNA acts as both a Cas12a activator and a fluorescent reporter. • The dfDNA activates both the cis - and trans -cleavage activities of Cas12a. • The dfDNA/SCas12a assay detects microRNA-21 (miR-21) without amplification. • Bead-assisted dfDNA format improves the sensitivity for miR-21 detection. MicroRNA-21 (miR-21) is a promising non-invasive biomarker for tumor diagnosis, but its short length and low abundance make amplification-free detection challenging. Here, we developed a dual-function DNA probe (dfDNA)-enabled split crRNA-Cas12a (SCas12a) system for miR-21 detection. This design separates crRNA into a constant scaffold fragment (sc-crRNA) and a variable spacer fragment (sp-crRNA), and miR-21 functionally replaces the spacer fragment to activate Cas12a in the presence of dfDNA. The dfDNA acts as both a target-recognition probe and a fluorescent reporter: it hybridizes with miR-21, activates Cas12a, and presents a single-stranded fluorescent overhang that can be cleaved after Cas12a activation. Because target recognition and signal reporting are integrated within a single probe, dfDNA positions the activated Cas12a complex close to the cleavable overhang, improving signal conversion compared with the conventional single-stranded DNA reporter and fully complementary double-stranded DNA probe format. Under optimized conditions, the limit of detection (LOD) with the homogeneous ssDNA reporter-based assay was 347.9 pM for miR-21, whereas the homogeneous dfDNA-based assay reduced the LOD to 19.8 pM. When transferred to a magnetic bead (MB)-assisted format, the dfDNA-based system achieved an LOD of 1.1 pM, supporting that the localized reaction accelerates the trans -cleavage kinetics of Cas12a. The MB-assisted dfDNA assay achieves comparable or even superior sensitivity for miR-21 detection compared with previously reported split crRNA-based fluorescence methods. Further optimization, including target enrichment or signal amplification strategies, is needed before this method can be reliably used for low-abundance miRNAs in clinical serum samples. Download: Download high-res image (88KB) Download: Download full-size image
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ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Ling Deng,Xiaoyi He,Ke Liu,Yuanli Li,Han Xia,Hang Qian,Xiaoxue Lu,Xuhu Mao,Yang Xiang
Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance , and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a O ne- P ot C RISPR-integrated assay for I nstant and V isual D etection (termed OPC-IVD) of B. pseudomallei . The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans -cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei , and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.
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ACS Applied Materials & Interfaces
·
2022
·
IF 10.383
Lina Wu,Xinjie Wang,Xiangchuan Wu,Shiqi Xu,Ming Liu,Xizhong Cao,Taishan Tang,Xingxu Huang,He Huang
The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3′,5,5′-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.
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ACS Applied Materials & Interfaces
·
2023
·
IF 9.5
Shaoqiong Feng,Hanjun Chen,Ziao Hu,Tingting Wu,Zhihong Liu
MicroRNA (miRNA) is a promising biomarker for the diagnosis, monitoring, and prognostic evaluation of diseases, especially cancer. The existing miRNA detection methods usually need external instruments for quantitative signal output, limiting their practical applications in point-of-care (POC) settings. Here, we propose a distance-based biosensor through a responsive hydrogel, in combination with a CRISPR/Cas12a system and target-triggered strand displacement amplification (SDA) reaction for visual quantitative and sensitive measurement of miRNA. The target miRNA is first converted into plenty of double-stranded DNA (dsDNA) via target-triggered SDA reaction. Then, the dsDNA products trigger the collateral cleavage activity of CRISPR/Cas12a, leading to the release of trypsin from magnetic beads (MBs). The released trypsin can hydrolyze gelatin, and hence the permeability of gelatin-treated filter paper is increased, resulting in a visible distance signal on a cotton thread. Using this system, the concentration of the target miRNA can be quantified visually without any assistance of instruments, and a detection limit of 6.28 pM is obtained. In addition, the target miRNA in human serum samples and cell lysates can also be detected accurately. Owing to the characteristics of simplicity, sensitivity, specificity, and portability, the proposed biosensor provides a new tool for miRNA detection and holds great promise in POC applications.
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