MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Yi Duan,Meilin Liu,Dianhui Men,Yiyao Luo,Xuhui Xia,Li Wang,Fei Deng,Danqun Huo,Changjun Hou
CA15–3 is a vital biomarker for monitoring and evaluating treatment efficacy in breast cancer patients, especially those with advanced-stage disease. Conventional immunoassays face limitations, including high cost, limited sensitivity, and dependence on potentially error-prone single-fluorescence signal outputs. To address these challenges, we designed a dual-signal-emitting sensor based on CdTe quantum dots@ZIF-8 nanomaterials, incorporating both the CRISPR/Cas12a system and catalytic hairpin assembly (CHA) technology. The binding of CA15–3 to its specific aptamer inhibits the CHA cascade reaction, thereby preventing the generation of double-stranded DNA activators necessary to initiate Cas12a-mediated trans-cleavage activity. As a result, FAM-ssDNA is extensively adsorbed onto the CdTe QDs@ZIF-8 surface, where efficient electron transfer effectively quenches its fluorescence. The incorporation of the aptamer facilitates dual-signal amplification through both the CHA and CRISPR/Cas12a systems, achieving a detection limit of 0.131 U/mL. This dual-signal output mechanism provides the system with an internal reference function, thereby significantly improving the reliability of the results. Overall, this work establishes a novel tumor biomarker detection platform that combines high sensitivity with excellent robustness.
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ACS Sensors
·
2026
·
IF 10.9
Shuai Liu,Zishan Ding,Xing Lu,Zhiyong Liu,Wenrui Ma,Hanqing Xu,Hengjun Zhang,Xianling Dai,Man Shen,Yingchun Huang,Mingxuan Gao,Jing Bao,Ming Chen
Rapid and sensitive detection of antibiotic-resistant bacteria (ARB) remains a critical challenge in clinical and public health settings. This study describes the successful construction of a portable DNA activator-triggered entropy-driven catalysis-modulated CRISPR/Cas12a-based sensor (PSDA) for the ultrasensitive and rapid detection of multiple ARB. This PSDA platform utilizes a CRISPR/Cas12a-mediated signal transduction strategy, in which a target-specific DNA activator initiates an entropy-driven dynamic DNA network for signal amplification. To further enhance detection performance, a 3D-printed microfluidic chip device with a smartphone-based readout system has been integrated into the sensor, using green-emitting Zn2GeO4:Mn persistent luminescent nanoparticles as a novel molecular beacon for fluorescence enhancement. This platform enables the simultaneous detection of methicillin-resistant Staphylococcus aureus, carbapenem-resistant Pseudomonas aeruginosa, and Klebsiella pneumoniae carbapenemase 2 (KPC-2)-expressing Klebsiella pneumoniae (KPC-2 KP) with a broad dynamic range (1−107 CFU/mL), an ultralow detection limit (1 CFU/mL), and rapid analysis (∼45 min). The assay results are also highly consistent with those of conventional plate counting methods (95.48−115.15%). Overall, this study presents a cost-effective, rapid-response biosensing platform for the simultaneous detection of multiple ARB, with direct applications in clinical diagnostics, food safety monitoring, and environmental surveillance.
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International Journal of Nanomedicine
·
2026
·
IF 8.7
Jian Tao,Binbin Zeng,Caili Bi,Minyi Xu,Xiaxian Gu,Dingding Jing,Jun Sun
Purpose Ultra-sensitive detection of exosomal miR-21 is crucial for the early diagnosis of colorectal cancer. This study aims to develop a cascade amplification platform based on surface-enhanced Raman scattering (SERS), combining rolling circle amplification (RCA) and CRISPR/Cas12a, to enable ultra-sensitive quantitative detection of exosomal miR-21.Methods A SERS substrate was constructed using streptavidin-modified concave gold nanocubes (CGNs), and gold double-stranded probes loaded with Raman reporter molecules were used as SERS probes. The two components are controllably linked via a single-stranded DNA molecule with a thiol group at one end and biotin at the other, forming a stable detection system. Target-triggered RCA generates a repeat sequence, which, under the mediation of crRNA, activates Cas12a cleavage, causing the probe to be released from the substrate surface and resulting in a significant attenuation of the SERS signal.Results This platform achieves three-stage cascading signal amplification via RCA, CRISPR/Cas12a, and SERS, with a detection limit at the femto-mole (fM) level and a total detection time of approximately 80 minutes. The results were consistent with those of qRT-PCR, demonstrating high sensitivity, high specificity, and reliability.Conclusion This study proposes an RCA-CRISPR-SERS cascade amplification platform that provides a highly sensitive and specific method for detecting miR-21 in exosomes, offering potential clinical value for the early diagnosis and dynamic monitoring of colorectal cancer.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Cheng Qin,Xinlan Bian,Wei Lv,Bingzhi Li
CRISPR/Cas12a-based biosensors typically output fluorescent, colorimetric, and electrochemical signals, relying on reporters that suffer from low sensitivity, high cost, and low stability. Herein, we introduced a label-free chemiluminescent (CL) reporting system based on a DNAzyme known as “Supernova”. Supernova catalyzes the translocation of phosphate from a 1,2-dioxetane substrate (specifically, CDP-Star) to its 5′-OH ends, thereby triggering a CL reaction that produces light. Upon activation by the target, Cas12a cleaves Supernova, rendering it inactive in reacting with CDP-Star. Without pre-amplification, this biosensor can detect synthetic DNA corresponding to the SARS-CoV-2 N gene and monkeypox virus (MPXV) F3L gene with detection limits (LOD) of 1.5 and 2.4 pM, respectively. Integration with pre-amplification further enables the detection of SARS-CoV-2 and MPXV genes down to 1 copy/μL in real samples. Furthermore, the application of this CL system was expanded to the detection of non-nucleic acids with the integration of ATP aptamers, resulting in improved sensitivity in ATP biosensing compared to fluorescent assays. Overall, this work represents the first development of a label-free CL Cas12a reporting system, demonstrating its enhanced performance and broad applicability in synthetic biology-enabled sensing technology.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Ailing Su,Yuan Liu,Xiumian Cao,Weiqing Xu,Chongyang Liang,Shuping Xu
A new gene detection platform based on surface-enhanced Raman spectroscopy (SERS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system is developed, which is applied for virus gene detections. CRISPR/Cas12a activated by CRISPR ribonucleic acid (crRNA) can recognize and lock the complementary target double-stranded deoxyribonucleic acid (dsDNA), followed by the cis -cleavage on the dsDNA and the trans -cleavage on the nearby linker single-stranded DNA (ssDNA). Target-induced trans -ssDNA cleavage leads to a failure in an aggregation behavior of the designed SERS probe pair that was bridged by the linker ssDNA, which causes the SERS intensity reduced relative to the control trial that gives much stronger SERS due to the severe aggregation state of SERS probes caused by intact linker ssDNA. This sensing method achieves the highly sensitive detections of human papillomavirus (HPV) genes in serum and pseudovirus within 40 min. The detection sensitivity of this CRISPR/Cas-SERS platform can reach pM. Owing to the flexible designs of the DNA probe and crRNA, this CRISPR/Cas-SERS sensing system is a universal gene detection tool, which can be applied for the in-vitro diagnosis field and point-of-care testing.
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TALANTA
·
2022
·
IF 6.556
Siwenjie Qian,Yanju Chen,Cheng Peng,Xiaofu Wang,Hui Wu,Yang Che,Huanying Wang,Junfeng Xu,Jian Wu
African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/μL. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.
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ACS Synthetic Biology
·
2022
·
IF 5.249
Chunfeng Feng,Wenbin Liang,Fei Liu,Yu Xiong,Man Chen,Pan Feng,Mingjing Guo,Yunxia Wang,Zhongjun Li,Liqun Zhang
The mutation status of epidermal growth factor receptor (EGFR) exon 19 is of great importance for predicting sensitivity to tyrosine kinase inhibitors (TKIs) in the treatment of non-small-cell lung cancer (NSCLC). However, the development of simple, sensitive, and no-nonspecific amplification platforms for EGFR 19del detection in NSCLC remains a challenge. Herein, we developed a novel, simple, and highly sensitive naked-eye assay utilizing CRISPR/Cas12a-triggered no-nonspecific nucleic acid amplification (NAA) with rolling circle amplification (RCA) as a model for EGFR 19del detection. Typically, circular padlocks are designed to be the trans-cleavage substrate of Cas12a/crRNA and serve as templates for RCA. Since the target EGFR 19del induces robust trans-cleavage activity of the Cas12a/crRNA duplex, the surrounding circular padlocks are cleaved into random short linear fragments that are unable to initiate RCA, resulting in a colorless solution. However, in the absence of EGFR 19del, the inactivated Cas12a enzymes cannot cleave the circular padlocks, and they remain able to serve as templates to initiate RCA to generate long single-stranded DNA to further fold into G-quadruplex/hemin DNAzymes to catalyze the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS2–), generating a color response that is obvious to the naked eye. As expected, this strategy with a detection limit as low as 20 fM exhibited robust selectivity and anti-interference ability. Moreover, this method was applicable for detecting EGFR 19del in real serum samples and showed high consistency with real-time quantitative polymerase chain reaction (qPCR) and sequencing results, providing a promising strategy for the early noninvasive diagnosis and guidance of clinical treatment for cancer.
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BIOELECTROCHEMISTRY
·
2024
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IF 4.8
Longjian Huang,Wenzhao Zhang,Mingxuan Liu,Yuanxun Gong,Qianli Tang,Kaihua Wang,Xianjiu Liao,Kai Zhang,Jihua Wei
We present an innovative biosensor designed for the precise identification of Escherichia coli ( E.coli ), a predominant pathogen responsible for gastrointestinal infections. E.coli is prevalent in environments characterized by substandard water quality and can lead to severe diarrhea, especially in hospital settings. The device employs entropy-driven reactions to synthesize copious amounts of double-stranded DNA (dsDNA), which, upon binding with crRNA, triggers the CRISPR/Cas12a system’s cleavage mechanism. This process results in the separation of a ferrocene (Fc)-tagged DNA strand from the electrode, enhancing the electrochemical signal for E.coli ’s rapid and accurate detection. Our tests confirm the biosensor’s ability to quantify E.coli across a dynamic range from 100 to 10 million CFU/mL, achieving a detection threshold of just over 5 CFU/mL. The development of this electrochemical biosensor highlights its exceptional selectivity, high sensitivity, and user-friendly interface for E.coli detection. It stands as a significant step forward in pathogen detection technology, promising new directions for identifying various bacterial infections through the CRISPR/Cas mechanism.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2024
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IF 8
Kai Zhang,Yuwen Bao,Changjin Ou,Guoyin Zhu,Yizhou Zhang
This study presents a novel biosensor that integrates CRISPR-Cas12a technology with DNA tile-based architectures to enhance thrombin detection. Utilizing PAM-engineered toehold switches, the biosensor dynamically activates the CRISPR-Cas12a system upon thrombin recognition, significantly improving both sensitivity and specificity. The label-free electrochemical detection method offers rapid, cost-effective analysis, making it ideal for real-time clinical applications. The biosensor achieved an impressive detection limit of 103 aM, surpassing traditional methods and setting a new standard for thrombin assays. The biosensor's robust performance in complex biological samples, such as diluted human serum, demonstrates its reliability for clinical diagnostics. This is particularly important for the early diagnosis and management of thrombotic and coagulation-related disorders. By combining advanced genetic engineering with traditional electrochemical techniques, the biosensor not only optimizes detection but also expands its potential applications across various biomedical fields. Overall, this biosensor provides a powerful and innovative tool for thrombin detection, offering enhanced diagnostic accuracy, faster patient management, and potential cost savings in healthcare. Its superior performance and adaptability highlight its promise for broader adoption in clinical diagnostics and other biosensing applications.
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ANALYST
·
2024
·Vol. 149
·期 15
·3920-3927
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IF 3.6
Zhenhuan Jin,Wei Xiao,Lin Shen,Xiaoxue Shi,Jianping Li
Anti-cancer therapy is crucial in cancer prevention and anti-cancer, and thus, highly sensitive methods for detecting cancer biomarkers are essential for cancer early diagnosis. Herein, an electrochemical aptamer biosensor based on the CRISPR-Cas12a system was constructed for the detection of cancer tumor biomarker MUC1 mucin. The sensitivity was significantly prompted by enzyme-catalyzed signal amplification, and the selectivity was improved by the dual recognition of the aptamer to MUC1 and crRNA-Cas12a system to the aptamer. Glucose oxidase (GOD) was loaded on the surface of magnetic Fe3O4@Au (MGNP) via probe single-stranded DNA (pDNA) with the terminal modification of mercapto (–SH) to form GOD-pDNA/MGNP. The corresponding aptamer of MUC1 (MUC1 Apt) binds to its complementary ssDNA (cDNA) to form the activator Apt/cDNA, which is specifically recognized by crRNA-Cas12a and excites the trans-cleavage function of Cas12a, thus in turn trans-cleaves pDNA and detaches GOD from the magnetic particles. The magnetic beads were separated and transferred into a glucose solution, and the oxidation current of H2O2 produced by the catalytic reaction of GOD was measured on a Pt-modified magnetically-controlled glassy carbon electrode, resulting in an indirect determination of MUC1. The current change was linear with the logarithm of MUC1 concentration in the range from 1.0 × 10−17 g mL−1 to 1.0 × 10−10 g mL−1. The detection limit was as low as 7.01 × 10−18 g mL−1. The method was applied for the detection of MUC1 in medical samples.
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ANALYTICA CHIMICA ACTA
·
2024
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IF 5.7
Tianyi Zhang,Xiaoqin Wang,Yingtao Jiang,Yunyun Zhang,Shuhao Zhao,Jian Hu,Fei Hu,Niancai Peng
Background Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) Results In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 minutes with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) Significance This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52)
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MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Yafang Shen,Fei Jia,Yawen He,Yaping Peng,Yingchun Fu,Weihuan Fang,Yanbin Li
Rapid detection methods are urgently needed to help prevent the spread of Salmonella among food products. Herein, a CRISPR-Cas12a-powered, quantum dot-based and magnetic nanoparticle-assisted (QD-CRISPR-MNP) biosensor was proposed for the screening of Salmonella. The QD-CRISPR-MNP biosensor was designed and fabricated relying on the CRISPR-Cas12a system for better guidance of the coupling between magnetic nanoparticles (MNPs) and quantum dots (QDs). In this biosensor, the activity of the CRISPR-Cas12a system was unlocked by Salmonella and displayed collateral cleavage activity towards linker DNAs, which inhibited the coupling of the MNPs and QDs. Then, Salmonella could be quantitatively detected by simply measuring the fluorescence emitted by the QDs on the MNPs surface. This biosensor showed good detection performance for Salmonella with a detection limit of 86 CFU mL −1 , and exhibited a satisfactory specificity as well. Moreover, it overcomes the low fluorescence quantum yields and the possible high fluorescence background noise in conventional dye-based CRISPR detection platforms, acting as a powerful alternative for the detection of foodborne pathogens.
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危险品化学品经营许可证（带存储）