BIOSENSORS & BIOELECTRONICS
·
2024
·
IF 10.7
Chuanyu Yang,Caiyi Du,Feiyu Yuan,Peitong Yu,Boxu Wang,Changshun Su,Ruiqi Zou,Junyang Wang,Xu Yan,Chunyan Sun,Hongxia Li
Benefiting from specific target recognition and trans-cleavage capabilities, the CRISPR/Cas12a system has great application prospects in the design of highly sensitive and rapid fluorescence biosensors. The CRISPR/Cas12a-based fluorophore-quencher molecular beacons exhibit single-color emission and are easily exposed to interference from environmental factors. Herein, we design a CRISPR/Cas12a-derived ratiometric fluorescence sensor for Pb 2+ detection based on embedded carbon dots@zeolitic imidazolate framework-8 (CDs@ZIF-8) composites and DNAzyme. The functions of ZIF-8 about encapsulating red emissive CDs in the inner cavity and adsorbing DNA on the outer surface are integrated to establish dual fluorescence signals, thereby reducing the possibility of interference and improving sensing accuracy. The presence of Pb 2+ is converted into the change of activator by the GR5 DNAzyme to activate the CRISPR/Cas12a system, which provides signal amplification through multiple turnovers of side branch cutting, achieving highly sensitive detection of Pb 2+ with a low detection limit of 18 pM. This method has the advantages of simplicity, universality, and excellent quantitative ability, and has broad prospects in sensing applications.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Honglian Wu,Ruolin Zhang,Huang Lai,Luogen Lai,Yi Liu,Yadi Fan,Yu Zhang,Jingyu Shi,Mo Yang
Endotoxins or lipopolysaccharides (LPS) are potent inflammatory agents that pose serious health risks even at low concentrations, thus requiring sensitive and reliable detection methods. Current methods for endotoxin detection, primarily the Limulus Amebocyte Lysate (LAL) assay and Enzyme-linked Immunosorbent Assays (ELISA) are limited by complex procedures, prolonged assay time, and susceptibility to interference. To address these challenges, we developed a single-reporter probe-based dual-mode biosensor integrating MXene-Au nanosheets with the CRISPR/Cas12a system for endotoxin detection. The sensing platform was constructed by conjugating ssDNA-Cy5 reporters onto MXene-Au nanosheets. MXene-Au nanosheets served dual functions, including efficient quenching of fluorescence and strong enhancement of SERS signals. In the presence of target endotoxins, CRISPR/Cas12a remains inactive, leaving the ssDNA-Cy5 reporters intact on the MXene-Au surface. The close proximity of Cy5 to the MXene-Au surface results in quenched fluorescence (“FL off”) but enhanced SERS signals (“SERS on”). Conversely, in the absence of endotoxins, activated CRISPR/Cas12a cleaves the ssDNA-Cy5, releasing Cy5 from the surface, thus leading to fluorescence recovery (“FL on”) but diminished SERS signals (“SERS off”). By measuring the changes in SERS and FL signals and analysing the ratiometric I SERS /I FL signals, the biosensor achieved a lower limit of detection (LOD) of 15.9 pg/mL within 30 minutes compared to single detection modes. Validation studies in complex matrices, including spiked water and milk samples, showed performance comparable to commercial endotoxin detection kits. Overall, our CRISPR-Cas12a-mediated fluorescent-SERS dual-mode biosensor not only improves detection sensitivity but also provides built-in quality control, improving the reliability of results through cross-validation.
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Sensing and Bio-Sensing Research
·
2025
·
IF 4.9
Baolin Li,Yaxin Huang,Zixin Zhu,Min Zhong,Guangrong Li,Jinbo Liu
Adenosine triphosphate (ATP) plays a critical role in cellular energy metabolism, acting as the energy source for many biological reactions. Abnormal fluctuations in ATP levels are directly linked to various diseases such as malignancies, bacterial infections, and cardiovascular disorders, making early-stage disease detection through rapid, highly sensitive ATP measurement essential. The current methods for ATP detection suffer from drawbacks such as complex procedures, reliance on expensive instruments, and prolonged reaction times. To this end, we have developed a novel biosensor named ATP Output Sensor Activated by CRISPR (AOSAC).This biosensor employs aptamer-based specific recognition combined with a dual-enzyme, amplification-free system involving Exo III and CRISPR-Cas12a for ATP detection. This sensor operates through an aptamer in a closed duplex structure, upon binding ATP, releases a trigger strand. This strand displaces an activation chain, which in turn interacts with Cas12a's crRNA, initiating trans-cleavage activity that cleaves a single-stranded DNA probe to produce a fluorescent signal. Concurrently, the activation chain's release sites are targeted by Exo III, enhancing signal amplification without the need for complex nucleic acid amplification, thus providing a highly selective, rapid detection method. This technique offers a linear detection range from 0 nM to 20 μM with a detection limit of 44.2 nM, presenting a novel strategy for signal amplification and high specificity in distinguishing ATP from its analogues.
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LWT-FOOD SCIENCE AND TECHNOLOGY
·
2025
·
IF 6.6
Xingyue Tang,Yafang Shen,Jiangqi Wang,Zhaohui Qiao,Fangkai Han,Yanna Cui,Qiuyue Hu,Yuxin Wu,Jiahua Wang,Xiaodan Liu,Huang Dai,Guijie Hao
Sensitive and accurate detection of enrofloxacin (ENR) residues is essential for food safety. Herein, a competitive immunosensor was developed for the detection of ENR, using a gold nanoparticle (AuNP)-enhanced CRISPR/Cas12a system. In this method, bovine serum albumin-ENR- and single-stranded DNA-modified AuNPs (AuNPs-BSA-ENR/ssDNA) was used to act as both a competitor and CRISPR/Cas12a activator. The use of AuNPs-BSA-ENR/ssDNA helped transduce the target recognition information to the cleavage activity of the CRISPR/Cas12a system. Moreover, benefiting from the high ssDNA loading capability of AuNPs, it enabled one-to-multiple CRISPR/Cas12a activation. The synergetic signal amplification from AuNPs and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a limit of detection of 1 ng/mL. Its feasibility for the detection of food and environmental samples was also investigated with recovery rates ranging from 91.0% to 115.2%. The developed immunosensor offered a promising alternative for ENR detection.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Yuqian Guo,Yuanqian Xiong,Weipeng Tong,Xiaolin Huang,Yonghua Xiong
Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.
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JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY
·
2026
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IF 4.7
Li Zhou,Xianwei Jiao,Yuting Zhang,Changling Yan
L-selectin, a leukocyte adhesion receptor, is essential in the early step of the adhesion cascade, facilitating the migration of leukocytes into surrounding tissues during inflammation and immune surveillance. Clinical studies have identified L-selectin as a prospective treatment option for many both acute and ongoing inflammatory conditions. Clarifying the cellular signaling pathways that function in the immediate vicinity of L-selectin may yield unique treatments tailored to certain cell types or illnesses. The study describes the development of a precise and sensitive fluorescence biosensor designed for the detection of L-selectin using a split aptamer specific to L-selectin. This biosensor utilizes nicking enzyme-assisted DNA walking together CRISPR-Cas12a for dual-signal multiplication. The calibration curve for L-selectin demonstrates a detection limit of 0.45 ng/mL, with an extensive linear range spanning from 10 to 1280 ng/mL. The fluorometric sensors effectively identified L-selectin in human blood samples, producing excellent outcomes. The fluorometric sensors effectively identified L-selectin in human blood samples, producing excellent outcomes. The original appearance not only validates the concept but also possesses substantial possibilities for identifying diverse targets.
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MICROCHEMICAL JOURNAL
·
2026
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IF 5.2
Yaru Zhang,Yongchang Sun,Jing Yuan,Lihua Ding,Leiliang He,Yongjun Wu,Songcheng Yu
MicroRNAs (miRNAs), as crucial regulators of gene expression, play significant roles in cancer development. Among them, miRNA-21 has been recognized as a promising biomarker for the diagnosis and prognosis evaluation of multiple cancers. However, existing CRISPR/Cas12a-based miRNA detection methods generally require reverse transcription and pre-amplification steps, which complicate the operational workflow and are prone to introducing cross contamination. Furthermore, the dependence on single-fluorescence signal detection makes them vulnerable to environmental and instrumental fluctuations, which results in risk of false-positive outcomes. Herein, we developed a ratiometric and sensitive CRISPR/Cas12a system with trans-cleavage-triggered feedback signal amplification for amplification-free detection of miRNA-21. The strategy employs a novel multifunctional switch probe that integrates target recognition, signal transduction, and cascade amplification within a unified system. A trans-cleavage-triggered feedback signal amplification circuit was constructed to enhance detection sensitivity without the need for additional enzyme systems. Moreover, a ratiometric fluorescence signal was implemented by calculating the fluorescence intensity ratio between CY5 and Auramine O, which effectively minimizes interference from environmental and instrumental variations and substantially improves detection accuracy and reliability. Experimental results demonstrate that the method achieves a detection limit of 1.28 nM, along with excellent specificity, accuracy, and reproducibility. In real sample analysis, differential expression levels of miRNA-21 in lung adenocarcinoma cells (A549) and non-cancerous adult bronchial epithelial cells (BEAS-2B) was observed, which highlights its potential value for clinical diagnostics.
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Food and Bioprocess Technology
·
2026
·Vol. 19
·期 5
·244
·
IF 5.6
Shen Jingqi,Zhao Yan,Duan Miaolin,Zhang Weijia,Wang Kaidi,Zhang Xiaowei,Dai Ruitong,Liu Zunying,Jia Fei
Foodborne pathogens have posed a major threat to food safety and public health, necessitating the development of accurate and sensitive methods for the detection of foodborne pathogens. In this study, a metal–organic framework (MOF) nanozyme-based CRISPR/Cas12a colorimetric biosensing system was developed and integrated with a smartphone-based portable device to meet the demand for on-site detection of Salmonella Typhimurium. Inspired by the catalytic characteristics of gold nanoparticles (AuNPs), the ZIF-8@AuNPs nanozyme with oxidase-like activity was synthesized by immobilizing AuNPs onto zeolitic imidazolate framework-8 (ZIF-8) via a self-assembly method. In this detection system, the target DNA is preamplified via the recombinase-aided amplification (RAA) procedure to achieve primary signal amplification. The amplified target DNA subsequently triggers colorimetric signal readout and secondary signal amplification through CRISPR/Cas12a-mediated specific recognition and cleavage, and the high catalytic performance of the ZIF-8@AuNPs nanozyme, thereby facilitating the biosensor to exhibit excellent detection sensitivity and specificity. The concentration of bacteria can be quantified and analyzed using a self-programmed “DeepFood” APP with the help of smartphones and 3D-printed devices. Consequently, this method allows the quantitative detection of Salmonella Typhimurium within a linear range of 10 1 to 10 7 CFU/mL, with detection limits of 11 CFU/mL for colorimetric detection and 65 CFU/mL for smartphone-based RGB detection in the range of 10 2 to 10⁷ CFU/mL. Additionally, this method facilitates sensitive, specific, and portable detection of Salmonella Typhimurium in milk samples, in comparison with traditional methods. The inherent programmability of CRISPR RNA also enables the detection of other bacterial pathogens, making this platform a valuable tool for food safety monitoring. Graphical
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SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 8.3
Wenji Bao,Ailing Su,Gerile Aodeng,Lu Ga,Lan Yu,Jun Ai
A highly sensitive colorimetric sensing platform was developed based on the trans-cleavage activity of CRISPR/Cas12a and the controllable aggregation of gold nanoparticles (AuNPs) for rapid and visual detection of the foodborne pathogen Staphylococcus aureus. Following PCR or RPA, the target DNA was specifically recognized by the Cas12a/crRNA complex, thereby activating its trans-cleavage activity. This process led to the degradation of linker single-stranded DNA (ssDNA) responsible for crosslinking AuNPs probes, resulting in significant changes in both UV–vis absorption signals and solution color. Three different crRNA sequences were systematically evaluated, revealing distinct efficiencies in target recognition and signal amplification. Among them, the crRNA-3 mediated system exhibited superior analytical performance, achieving an ultralow detection limit of 1.14 fM and a good linear response over a low concentration range. Furthermore, the proposed platform demonstrated excellent anti-interference capability in complex matrices, including serum, milk, bovine serum albumin (BSA), and phosphate-buffered saline (PBS), with recovery rates ranging from 83.2% to 125.9%. This work provides a robust and visual tool for the on-site detection of S. aureus and offers new insights into the design of portable CRISPR-based diagnostic platforms.
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ACS Sensors
·
2026
·
IF 10.9
Zhenghan Shi,Liya Ding,Li-ang Zhou,Ye Liu,Yuhang Wang,Yi Xu,Yunrui Cao,Zhinong Jiang,Qingjun Liu
Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine whose rapid and quantitative detection is essential for disease diagnosis and monitoring. However, conventional methods are often limited by bulky instrumentation and are not well suited for analysis in complex biological environments. Herein, a portable CRISPR/Cas12a-based aptasensor system was developed for detection of TNF-α by integrating aptamer recognition, Cas12a trans-cleavage signal amplification, and a miniaturized fluorescence detection device. An aptamer for TNF-α was designed to regulate the accessibility of the target sequence to the CRISPR RNA (crRNA), and the resulting change in Cas12a activity was converted into fluorescence signals. The portable device integrated light-emitting diode (LED) excitation and photodiode-based detection, with modulated signals wirelessly transmitted to a smartphone for readout. The aptasensor showed concentration-dependent responses, and the inhibition rate exhibited a linear relationship with TNF-α concentration. The applicability of the system was validated in cell culture supernatants and cervical tissue samples, including tumor and adjacent normal tissues. Distinct TNF-α levels were observed between these samples, showing good agreement with enzyme-linked immunosorbent assay (ELISA) results. This portable CRISPR/Cas12a aptasensor system provides a practical approach for point-of-care TNF-α detection and extends CRISPR-based sensing to protein biomarkers.
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BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Licong Jia,Kai Kang,Wen Yan,Yan Jin,Kejia Xu,Jingyi Wang,Yipeng Wang,Meng Jiang,Wei Yuan,Shipeng Liu,Lingmei Niu
17β-Estradiol (E2) serves as both a reproductive accelerator and growth promoter, yet its uncontrolled application induces severe endocrine dysfunction. Point-of-care testing (POCT) has emerged as a promising analytical platform for on-site E2 quantification, due to its inherent advantages in terms of sensitive, specific, cost-effective, and instrument-free. We developed a novel strategy combining the high collateral cleavage activity of CRISPR/Cas12a with the strong amplification power of dual-cycle reaction, translating target presence into a colorimetric signal on pregnancy test strip (PTS). In the presence of E2, the dual-cycle amplification was initiated to produce HP2–HP3 (Hairpin DNA 2–Hairpin DNA 3) duplexes. These duplexes then bound to the Cas12a-crRNA complex, activating its trans-cleavage activity. The activated Cas12a subsequently cleaved the ssDNA linkers in the MBs (magnetic beads)-ssDNA (single-stranded DNA)-hCG (human chorionic gonadotropin) conjugates, releasing hCG for quantitative E2 detection through strip color intensity measurement. By integrating CRISPR/Cas12a's high cleavage efficiency with dual-cycle amplification, a visible biosensor was developed, with a linear range of 1.0 × 10 −1 –200.0 pM and a detection limit of 0.0403 pM for E2. In conclusion, an E2 detection platform characterized by prominent sensitivity and convenience was established for monitoring spiked E2 in milk and urine, providing a significant reference for the POCT detection of other targets.
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TALANTA
·
2026
·
IF 6.7
Mingqi Yang,Jiali Hu,Zhenlin Zhang,Liting Huang,Yahan Yu,Dexing Qiu,Yexin Zu,Yanyan Liu,Zhenyu Lin
Infection with hepatitis B virus (HBV) represents a major challenge to global health, especially in areas of inadequate healthcare infrastructure, rapid and on-site detection plays a crucial role in effective disease management and control. Consequently, it is necessary to develop some simple but sensitive HBV screening techniques. In this work, a homogeneous electrochemical biosensor was constructed for sensitive and specific HBV DNA detection through the integration of CRISPR/Cas12a target recognition and magnetic bead-based separation. The sensor exploits the collateral cleavage activity of CRISPR/Cas12a protein upon recognition of target double-stranded DNA, enabling the degradation of methylene blue-labeled single-stranded DNA captured on magnetic beads (MBs). Then the MBs are separated and redissolved in the solution and the electrochemical response of the system can be tested in homogeneous solution. The electrochemical signal has relationship with the concentration of HBV DNA within a range of 10 fM - 10 nM, and the LOD is 3.74 fM. The sensor demonstrates excellent selectivity and biological stability, which has potential application in clinical diagnosis, especially in resource-limited environments.
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