SENSORS AND ACTUATORS B-CHEMICAL
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2023
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IF 8.4
Qing Han,Xinlan Bian,Yue Chen,Bingzhi Li
Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans -cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.
研究领域:
分子生物学
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合成生物学
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癌症诊断
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生物技术
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2025
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IF 6
Zhongheng Wei,Huini Luo,Danlu Huang,Peixin Wei,Jiayi Zhang,Jihua Wei,Qianli Tang,Longjian Huang,Kai Zhang,Xianjiu Liao
Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5′-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti 3 C 2 /AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10 -9 U μL -1 and a wide dynamic range (1 × 10 -8 to 1 × 10 -5 U μL -1 ), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.
研究领域:
生物分析化学
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分子诊断
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癌症研究
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酶检测
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生物传感器与生物电子学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2021
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IF 6.558
Bingzhi Li,Peng Zhang,Bin Zhou,Siying Xie,Anqi Xia,Tiying Suo,Shuang Feng,Xing Zhang
Flap endonuclease 1 (FEN1), a ubiquitous enzyme involved in DNA repair and replication, is overexpressed in highly proliferative cancer cells. FEN1 has been recognized as a promising diagnostic marker of cancers; however, very few analytical techniques have been developed for the convenient detection of FEN1. To realize the simplified quantification of FEN1, we developed a FEN1-responsive fluorescent nanoprobe based on DNA-silver nanoclusters (DNA-AgNCs). The nanoprobe was rationally designed with a double-flapped dumbbell conformation, where its 5′ flap was produced with DNA-AgNCs, and the 3′ flap was elongated by a guanine-rich enhancer sequence (GRS). Rigidified by the DNA scaffold, DNA-AgNCs and the GRS are in close proximity, resulting in high fluorescence because of the GRS-induced activation of DNA-AgNCs. Upon the addition of FEN1, the 5’ flap of the nanoprobe is cleaved due to the structure-specific endonuclease activity of FEN1. This cleavage released the DNA-AgNCs from the nanoprobe, broke the proximity between DNA-AgNCs and the GRS, and caused decreased fluorescence. This nanoprobe can be applied in the sensitive detection of FEN1 with a detection limit of 40 fM, and it showed high specificity for the monitoring of FEN1 in clinical samples. As the first attempt to develop biosensors targeting FEN1 based on DNA-AgNCs, this work provided a potent platform for monitoring FEN1 and screening FEN1 inhibitors.
研究领域:
癌症生物学
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分子生物学
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纳米技术
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分析化学
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生物医学工程
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2021
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IF 7.46
Bin Zhou,Lei Lin,Bingzhi Li
Flap endonuclease 1 (FEN1) is a nuclease overexpressed in cells with a high replicate rate, which has been recognized as a potential biomarker in the molecular diagnosis of cancers. In this work, we developed a fluorescent biosensor based on exponential amplification reaction (EXPAR) to realize the rapid and sensitive detection of FEN1. A DNA probe specific for FEN1 is used for the generation of reporter DNA (rDNA), and then the rDNA hybridizes with a template, triggering EXPAR to produce substantial rDNA exponentially. However, when no FEN1 is in the system, the EXPAR will not be initiated because of the lack of rDNA. Derived from the high amplification efficiency of EXPAR, the rapid quantification of FEN1 with a limit of detection (LOD) of 0.5 fM was realized. It is also revealed that this biosensor exhibits good selectivity towards FEN1 under the challenge from real samples including extracts of cells and solid tumors. Furthermore, this sensor was reengineered for the analysis of FEN1 inhibitors, which shows promise in the screening and evaluation of potential drugs targeting FEN1. This work integrates EXPAR into the biosensing of FEN1 for the first time, which provides a promising tool for FEN1-related biomedical investigations.
研究领域:
癌症诊断
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分子生物学
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生物医学工程
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分析化学
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DOI
高影响因子
ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Junqiu Zhai,Han Zhang,Wenzhi Zhu,Jiewei Deng,Xinyan Li,Tiangang Luan
Simultaneous in situ monitoring of base excision repair (BER) correlated enzymes like apurinic/apyrimidinic endonuclease 1 (APE1) and flap endonuclease 1 (FEN1) in living cells offers valuable insights into their roles in disease development and cytotoxicity caused by pollutants, but comprehensive analysis is currently hindered by diverse enzyme functions and limited methods. In this study, we developed a dual-activatable DNA fluorescent probe (AP-FLAP) to simultaneously visualize APE1 and FEN1 activities, revealing the BER-related DNA damage caused by various environmental pollutants within living cells. The AP-FLAP probe was designed by ingeniously integrating a dumbbell structure containing a 5′ flap and a hairpin structure containing AP sites into a single oligonucleotide probe. APE1 specifically hydrolyzed the AP sites, releasing a 5-carboxy-X-rhodamine (ROX) signal, while FEN1 recognized and cleaved the 5′ flap, releasing a 6-carboxyfluorescein (FAM) signal. The probe allowed for independent determination of APE1 and FEN1 activities with good specificity and sensitivity. Subsequently, we applied the AP-FLAP probe to investigate base damage induced by 1-methylphenanthrene (1-MP) and 6-chlorobenzo[a]pyrene (6-Cl-BaP) in human umbilical vein endothelial cells (HUVECs). Significant base damage by 1-MP and 6-Cl-BaP exposure was revealed, with a positive correlation of damage degree with different exposure concentrations from 0.1 to 100 μM. Notably, 6-Cl-BaP caused significant damage even at 0.1 μM, in a concentration-dependent manner. Our work provides a powerful tool for elucidating BER molecular mechanisms and DNA damage repair under environmental exposure and opens new avenues for developing multifunctional nucleic acid probes for a wide range of applications in chemical biology and biomedical research.
研究领域:
分子生物学
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细胞生物学
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环境毒理学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Liyan Zhang,Xiaoting Liu,Nan Zhang,Xianqiang Liu,Wei Jiang
Sensitive and accurate detection of flap endonuclease 1 (FEN1) is essential to understand its roles in DNA replication and repair and explore its functions in tumor diagnosis and prognosis. Here, we designed a magnetic separation-assisted cascade hybridization chain reaction (HCR) amplification strategy for enzyme-free and sensitive detection of FEN1. Firstly, the forked dsDNAs contained the recognition site (5′ overhanging DNA flap) of FEN1 were modified on magbeads surface to form magnetic recognition probes. By the recognition and catalytic cleavage of FEN1, the 5′ flaps were removed into solution and then purified by magnetic separation. Then, the obtained flaps served as triggers to activate the linear-assembly of hairpin probes H1 and H2 (primary HCR), forming nicked double helices with split fragments. Finally, the fragments acted as new triggers to activate the branched-assembly of hairpin probes H3 and H4 (secondary HCR), causing fluorophore/quencher labeled on H3 far away and fluorescence recovery. Benefiting from the purification of triggers by magnetic separation and the signal amplification effect of cascade HCR, this strategy achieved a low detection limit of 3.6 × 10 −5 U μL −1 for FEN1, and enabled the assay of it in actual samples. The results demonstrate that our strategy will provide a potent approach for sensitive detection of FEN1 in early tumor diagnosis and prognosis.
研究领域:
分子生物学
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肿瘤诊断
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生物化学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2026
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IF 10.5
Han Pan,Murilege Chao,Miao He,Jinhua Chen,Feifan Yin,Yongliang Cai,Wenting Cheng,Zhongyun Wang,Yang Xiang
A single-base mutation in the CHCHD10 gene can impair thermogenic responses, and inhibit the efficient conversion of white adipose tissue to beige adipose tissue. However, achieving high-fidelity detection of such low-abundance single nucleotide polymorphisms (SNPs) within complex biological matrices remains a critical technological challenge. This study developed a novel method for detecting a single-base mutation in CHCHD1 0 by integrating the specific recognition of flap endonuclease 1 (FEN1) with the cascade amplification of an entropy-driven circuit reaction (EDCR), supplemented with magnetic separation to suppress background interference. In this process, the mutant target (MT) hybridizes with the upstream probe (UP) and a hairpin downstream probe (DP) to form a specific three-base overlapping structure. FEN1 recognizes and cleaves this structure, releasing a cleavage sequence (CS). Following magnetic purification, the CS activates the EDCR, enabling dual-signal amplification. The hairpin DP design effectively conceals the CS, significantly reducing background noise. The method achieves a detection limit of 0.23 pM, discriminates mutation abundances as low as 0.01% in mixed DNA samples, and demonstrates robust performance in complex matrices such as human serum and high-background genomic DNA. This work provides an innovative and reliable methodological tool for the detection of low-abundance point mutations in complex samples.
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产品关联
DOI
PubMed
高影响因子
TALANTA
·
2024
·
IF 5.6
Lei-Xin Jin,Qi-Meng Wang,Shu-Jiao Zhu,Cai-Lian Zhu,Jian-Jun Sun,Shao-Hua Wu
The flap endonuclease 1 (FEN1) plays a key role in DNA replication and repair, its aberrant expression is associated with tumor development, so it has been recognized as a promising biomarker for a variety of cancers. Here, a novel “turn on” mode gold nanocube-enhanced surface-enhanced Raman scattering (SERS) biosensor was constructed by combining a heated Au electrode (HAuE), exonuclease III (Exo III)-assisted cycle amplification, and gold nanocube (AuNC)-based SERS enhancement to achieve highly sensitive detection of FEN1 activity. The SERS tag was prepared using the Raman reporter modified on the AuNC surface, and the high electromagnetic field provided by the sharp geometric feature of AuNC greatly enhanced the SERS signal. At the same time, HAuE was used to increase the electrode surface temperature and enhance the FEN1 activity, leading to more trigger DNA being cleaved, which was used to initiate the Exo III-assisted cycle amplification. Taking all these advantages, the proposed method possessed high sensitivity and good selectivity, with a low limit of detection (LOD) of 3.19 × 10 −7 U μL −1 . In addition, this method was successfully applied to detect FEN1 activity in real cellular extracts.
研究领域:
生物传感技术
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分子诊断
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癌症研究
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产品关联
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.
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DOI
PubMed
高影响因子
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