International Journal of Nanomedicine
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2026
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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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高影响因子
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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Food Bioscience
·
2026
·
IF 6.2
Lihua Fan,Mingying Hu,Zongyuan Ou,Fan Li,Yiheng Shi,Di Wu,Yongning Wu,Guoliang Li
Sensitive, rapid, and reliable detection of aflatoxin B1 (AFB1) in food is of great importance for ensuring food safety. In this study, a dual-mode paper-based biosensing platform was developed by integrating the CRISPR/Cas12a nucleic acid cleavage system with a nanozyme-responsive DNA hydrogel for highly sensitive and portable detection of AFB1. Upon the presence of the target, AFB1 binds specifically to the aptamer, thereby activating the CRISPR/Cas12a system and subsequently inducing the degradation of the hydrogel network, which leads to the release of the encapsulated Au@Pt nanozyme. The released nanozyme catalyzes the oxidation of TMB in the presence of H 2 O 2 to generate a blue-colored product, while simultaneously producing a quantifiable diffusion signal on the paper-based substrate, enabling dual-mode colorimetric and distance-based detection. The detection limit of naked-eye detection is 0.1 ng mL −1 , and it is 0.075 ng mL −1 in the distance detection mode. The proposed method can complete the signal output within 30 min and exhibits the advantages of operational simplicity, rapid response, and high sensitivity. In practical peanut samples, satisfactory recoveries and good stability were achieved, demonstrating its potential as a promising strategy for on-site detection of trace mycotoxins in food.
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npj Science of Food
·
2026
·
IF 9.7
Wang Juan,Li Xiang,Zhang Jing,Zhao Chao,Shi Xuening
Microbial Contamination serves as an important issue in dairy safety. CRISPR/Cas12a-based methods possess tremendous potential, but are limited by nucleic acid pretreatment and specialized operations. Herein, a photothermal nanomaterial-assisted enhanced One-Tube CRISPR/Cas12a system was established to detect S. typhimurium in milk . S. typhimurium was lysed through the photothermal effect to obtain genomic DNA. A multi-crRNA strategy enables rapid signal accumulation, and the whole system was integrated into a single tube via trehalose-based aqueous biphasic systems. The proposed method requires only one step of sample addition to achieve sensitive detection within 40 min, with an LOD of 10 CFU/mL, showing potential in application.
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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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PubMed
高影响因子
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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高影响因子
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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SENSORS AND ACTUATORS B-CHEMICAL
·
2023
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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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Analytical Methods
·
2024
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IF 2.7
Qiong Liu,Mei Yang,He Zhang,Wenjie Ma,Xin Fu,Huiqing Li,Sainan Gao
Tuberculosis caused by Mycobacterium bovis poses a global infectious threat to humans and animals. Therefore, there is an urgent need to develop a sensitive, precise, and easy-to-readout strategy. Here, a novel tandem combination of CRISPR/Cas12a system with dual HCR (denoted as CRISPR/Cas12a-D-HCR) was constructed for detecting Mycobacterium bovis. Based on the efficient trans-cleavage activity of the active CRISPR/Cas12a system, tandem-dsDNA with PAM sites was established using two flexible hairpins, providing multiple binding sites with CRISPR/Cas12a for further amplification. Furthermore, the activation of Cas12a initiated the second hybridization chain reaction (HCR), which integrated complete G-quadruplex sequences to assemble the hemin/G-quadruplex DNAzyme. With the addition of H2O2 and ABTS, a colorimetric signal readout strategy was achieved. Consequently, the CRISPR/Cas12a-D-HCR achieved a satisfactory detection linear range from 20 aM to 50 fM, and the limit of detection was as low as 2.75 aM with single mismatched recognition capability, demonstrating good discrimination from different bacterial species. Notably, the practical application performance via the standard addition method was verified, with the recovery ranging from 96.0% to 105.2% and the relative standard deviations (RSD) ranging from 0.95% to 6.45%. The proposed CRISPR/Cas12a-D-HCR sensing system served as a promising application for accurate detection in food safety and agricultural fields.
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高影响因子
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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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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MICROCHEMICAL JOURNAL
·
2024
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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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