SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2024
·
IF 4.3
Bowen Xiong,Xiang Li,Yuting Zhang,Changling Yan,Gongke Wang
The overexpression of vascular endothelial growth factor 165 (VEGF 165 ) in cancer cells plays a pivotal role in promoting tumor metastasis by facilitating their excessively rapid proliferation and division. Hence, the development of analytical methods possessing high sensitivity and resistance to interference is imperative for the detection of VEGF 165 . Various types of aptasensors have been devised for VEGF 165 detection; however, the performance of these biosensors can be influenced by non-target signals caused by conformational changes in unbound aptamers. The paper shows the creation of a precise and sensitive fluorescence biosensor designed to detect VEGF 165 by using a VEGF 165 -specific split aptamer. Additionally, this biosensor employs nicking enzyme-assisted DNA walker coupling with CRISPR-Cas12a to achieve dual-signal amplification. The VEGF 165 calibration curve shows a detection limit of 268 fM and has a broad linear range from 5 to 4000 nM. The fluorometric biosensor was utilized to detect VEGF 165 in human serum and cellular homogenate samples, yielding good outcomes. The innovative design serves as proof of concept and demonstrates significant potential in detecting various targets.
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Advanced Science
·
2025
·
IF 14.1
Shuai Wu,Yincheng Liu,Tianyu Zeng,Tianci Zhou,Yanting Sun,Ying Deng,Juan Zhang,Genxi Li,Yongmei Yin
Continued development of clustered regularly interspaced short palindromic repeats (CRISPR)-powered biosensing system on the electrochemical interface is vital for accurate and timely diagnosis in clinical practice. Herein, an electrochemical biosensor based on manganese metal-organic frameworks (MOFs)-enhanced CRISPR (MME-CRISPR) is proposed that enables the efficient detection of circulating tumor DNA (ctDNA). In this design, customized enzyme stimulants (Mn 2+ ) are co-assembled with Cas12a/crRNA to form enzyme-MOF composites, which can be released quickly under mild conditions. The MOFs-induced proximity effect can continuously provide adequate Mn 2+ to sufficiently interact with Cas12a/crRNA during the release process, enhancing the trans-cleavage activity of complex available for biosensor construction. The MOFs-based enzyme biocomposites also afford efficient protection against various external stimulus. It is demonstrated that the developed biosensor can achieve ultrasensitive detection of epidermal growth factor receptor L858R mutation in ctDNA with a low detection limit of 0.28 f m without pre-amplification. Furthermore, the engineered mismatch crRNA enables the biosensor based on MME-CRISPR to detect single nucleotide variant with a high signal-to-noise ratio. More importantly, it has been successfully used to detect the targets in clinical practice, requiring low-dose samples and a short time. This strategy is believed to shed new light on the applications of cancer diagnosis, treatment, and surveillance.
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Journal of Future Foods
·
2026
·
IF 7.2
Xiaobo Zhang,Zekun Long,Wei Wang,Yafeng Jiang,Qiuyue Zheng,Jijuan Cao
Aflatoxin B1 (AFB1), the most toxic mycotoxin among aflatoxin family, which presents a risk to food safety and human health at a trace level. Herein, the self-enhanced electrochemiluminescence (ECL) emitters of Au nanoclusters-anchored g-C 3 N 4 nanosheet (Au NCs@g-C 3 N 4 ) were synthesized, in which the Au NCs as a co-reaction accelerator enhanced the ECL stability and intensity. Integrating with CRISPR/Cas12a system, the Au NCs@g-C 3 N 4 behaved as sensing substrate to construct a “off-on” switch-type ECL sensor for AFB1 detection based on ECL resonance energy transfer (ECL-RET). The presence of targets AFB1 can specifically bind its aptamer to trigger the release of the activator. Using dual aptamer-regulated activation of CRISPR-Cas12a, AFB1 can be quantified via the ECL sensor, displaying a wide detection range from 0.2 pg/mL-100 ng/mL and a detection limit of 0.08 pg/mL. Furthermore, good practicability was verified in real samples assay, providing an efficient tool for the early warning of AFB1 pollution.
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FOOD CHEMISTRY
·
2026
·
IF 10.4
Yipeng Zhang,Shijia Wu,Yisheng Chen
A portable biosensor for the peanut allergen Ara h 1 was developed by integrating a polydopamine-functionalized pipette tip, CRISPR/Cas12a signal amplification, and a personal glucose meter. Ara h 1 binding induced partial displacement of a cDNA activator, which activated Cas12a trans-cleavage and released glucose oxidase from GOx–DNA conjugates. The released enzyme catalyzed glucose oxidation, producing a quantitative decrease in glucose concentration. Under optimized conditions, the assay showed a linear range of 50–1000 ng/mL and a solution-based limit of detection of 22.19 ng/mL. Post-extraction matrix-spike tests in commercial peanut-biscuit and peanut-beverage extracts yielded recoveries of 101.58–104.68% with relative standard deviations below 3.44%. These results demonstrate the analytical feasibility and preliminary matrix compatibility of the platform, although validation using pre-extraction fortified, incurred, and naturally contaminated foods is still required.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Chunlei Yu,Keyun Ren,Xuezhen Xu,Jinmiao Ma,Falan Li,Yanyan Zhang,Qingqing Yang
Most applications of CRISPR-Cas12a technology for mycotoxin detection rely on aptamers as recognition elements. However, although antibodies exhibit high affinity and specificity, they require converting immune signals into nucleic acid signals and cannot directly activate the CRISPR-Cas12a system, thereby limiting their utility for highly sensitive small-molecule target detection. In this work, we conjugated CRISPR-Cas12a activator DNA (aDNA) to antibodies via strain-promoted azide-alkyne cycloaddition (SPAAC) to achieve dual functionality: transducing small-molecule target signals into nucleic acid signals and partially immobilizing the Cas12a protein. Magnetic spherical nucleic acids (MSNAs) served as trans-cleavage substrates for the CRISPR-Cas12a system. Terminal deoxynucleotidyl transferase (TdT) was used to extend polyA sequences by adding dATP to the 3’-OH ends of numerous cleaved DNA fragments. This process, combined with high-concentration NaCl-induced aggregation and color change of gold nanoparticles (AuNPs), established a cascade signal amplification system. We developed a novel ultrasensitive immunosensor, termed Cas12a-TdT Cascade Amplification for Antigen-Toxin Immunoassay (CTCA-ATI), for mycotoxin quantification. The limits of detection for aflatoxin B1 and deoxynivalenol (DON) were 2.63 × 10 −5 ng/mL and 6.23 × 10 −4 ng/mL, respectively, with linear ranges of 1 × 10 −4 –10 ng/mL for aflatoxin B1 and 1 × 10 −3 –10 ng/mL for DON. This study provides new insights into the development of cascade-amplified biosensors using CRISPR-Cas12a and demonstrates substantial potential for integrating antibodies with CRISPR-Cas technology to detect other critical targets.
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CHEMICAL ENGINEERING JOURNAL
·
2021
·
IF 13.273
Kai Zhang,Zhenqiang Fan,Yuedi Ding,Minhao Xie
In this work, we constructed an exonuclease III cleavage reaction-based isothermal amplification of nucleic acids with CRISPR/Cas12a-mediated pH-induced regenerative Electrochemiluminescence (ECL) biosensor for ultrasensitive and specific detection of SARS-CoV-2 nucleic acids for SARS-CoV-2 diagnosis. The triple-stranded nucleic acid in this biosensor has an extreme dependence on pH, which makes our constructed biosensor reproducible. This is essential for effective large-scale screening of SARS-CoV-2 in areas where resources are currently relatively scarce. Using this pH-induced regenerative biosensor, we detected the SARS-CoV-2 RdRp gene with a detection limit of 43.70 aM. In addition, the detection system has good stability and reproducibility, and we expect that this method may provide a potential platform for the diagnosis of COVID-19.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2021
·
IF 7.46
Kai-Ren Zhao,Li Wang,Peng-Fei Liu,Xiao-Min Hang,Hui-Yi Wang,Shu-Ying Ye,Zhi-Jun Liu,Guo-Xi Liang
Herein, we report a “on-off” signal-switchable electrochemiluminescence (ECL) biosensor for simple, sensitive and quantitative detection of two kinds of virus genes, human immunodeficiency virus (HIV) and human papilloma virus (HPV-16) DNAs, by the integration of spherical nucleic acid (SNA) with CRISPR/Cas12a. The concept behind this approach is “on” signal based on a sandwich type assay after ECL luminophores of carbon dots (CDs) labeled SNA incubated with the biosensor in the presence of HIV DNA; and “off” signal upon the biosensor incubation with assistant DNA (HIV DNA sequence), CDs-labeled SNA and Cas12a/crRNA/HPV-16 DNA ternary complex relying on the collaterally cleavage of Cas12a. The cross-interference between HIV and HPV-16 DNAs can be avoided by adding Cas12a effector to the system or not. Using this strategy, HIV and HPV-16 DNAs are successfully quantitatively detection with detection limits of 30 fM and 0.32 pM (S/N = 3), respectively. Importantly, this system does not rely on multiplex signal reporting molecules, thus simplifying the detection system and avoiding the likelihood of cross interferences presented in the assay. Furthermore, multiplex detection of HIV/HPV-16 DNAs in human serum samples are successfully achieved within 2 h without additional nucleic acid amplification step. As demonstrated, the proposed biosensor has the potential for multiplex detection in clinical diagnosis.
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BIOSENSORS & BIOELECTRONICS
·
2021
·
IF 10.618
Kai Zhang,Zhenqiang Fan,Bo Yao,Yuedi Ding,Jing Zhao,Minhao Xie,Jianbin Pan
Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a′s side-cutting feature to build a 2D ultra-thin Ti 3 C 2 T x (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Zhengfang Liang,Shuwei Xiao,Junhong Li,Guoli Zuo,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision repair pathway, is frequently overexpressed in multiple cancers and has emerged as an important biomarker for early diagnosis and therapeutic response evaluation. In this work, a highly sensitive electrochemiluminescence (ECL) biosensor was developed for APE1 detection by integrating a PAM-regulated CRISPR-Cas12a activation mechanism with a multifunctional nanocomposite-modified electrode. A Ti₃C₂/AuNPs/CsPbBr₃ ternary nanocomposite was fabricated through ultrasonic assembly to provide enhanced conductivity, water stability, and probe-loading capacity. Meanwhile, a DNA duplex containing an AP site within the PAM region was engineered to regulate Cas12a activation through APE1-mediated cleavage and polymerase-assisted repair, thereby minimizing background interference and enabling highly specific signal generation. Ferrocene-labeled DNA tetrahedrons (TDNs) were further introduced as a spatially ordered probe scaffold to improve hybridization efficiency and facilitate Cas12a trans-cleavage–mediated signal release. This work presents three key innovations: (1) Construction of a Ti₃C₂/AuNPs/CsPbBr₃ nanocomposite that simultaneously enhances ECL stability, conductivity, and electron-transfer efficiency, addressing the inherent water-instability of perovskite emitters. (2) Coupling of a PAM-regulated APE1–KF repair mechanism with Cas12a activation to achieve highly specific AP-site recognition and selective signal generation. (3) Introduction of a TDN-based probe architecture that provides rigid spatial orientation and high-density probe loading, markedly improving hybridization efficiency and Cas12a trans-cleavage performance. Benefiting from these synergistic innovations, the biosensor achieves a detection limit of 6.28 × 10⁻⁸ U μL⁻¹ and a wide linear range (1 × 10⁻⁷ to 1 × 10⁻ 1 U μL⁻¹). The platform also demonstrates excellent specificity, reproducibility, and reliable performance in diluted serum, highlighting its promise for clinical and biochemical applications.
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Jiaying Wang,Yu Tian,Kai Zhang,Jiantong Jiao,Yi Zhao
NF-κB p50 is a critical transcription factor that plays an essential role in regulating immune responses, inflammatory pathways, and tumor progression. Its aberrant expression is closely linked to various pathological conditions, making it a valuable biomarker for early disease diagnosis and therapeutic monitoring. In this work, we developed a highly sensitive and reusable electrochemical biosensor capable of detecting NF-κB p50 at ultralow concentrations. The biosensor integrates a pH-responsive regeneration mechanism and a multi-stage signal amplification cascade involving Exonuclease III (Exo III)-assisted recycling, CRISPR/Cas12a-mediated trans -cleavage, and DNA tetrahedron nanostructure-functionalized electrodes. This synergistic design significantly enhances detection sensitivity and stability. The platform offers a broad dynamic detection range from 5 aM to 100 fM with a remarkably low detection limit of 2.38 aM. More importantly, the biosensor demonstrates excellent reusability through efficient regeneration under alkaline conditions (pH 10.0), with minimal loss in signal output. Validation in tenfold-diluted human serum samples yielded high recovery rates (99.61 %–106.23 %) and low relative standard deviations, confirming its practical applicability. This study not only presents a robust approach for ultrasensitive protein detection but also introduces a generalizable strategy for the regenerative electrochemical analysis of protein-based biological macromolecules in clinical diagnostics.
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Journal of Materials Chemistry B
·
2022
·Vol. 10
·期 32
·6107-6117
·
IF 7.571
Siying Xie,Cheng Qin,Furong Zhao,Zhaoyang Shang,Pei Wang,Muhammad Sohail,Xing Zhang,Bingzhi Li
CRISPR-driven biosensing is developing rapidly, but current studies mostly adopt dye-labeled ssDNA as the signal reporter, which is costly and unstable. Herein, we developed a label-free and low-background reporter for CRISPR/Cas12a signaling by integrating DNA-templated copper nanoclusters (DNA–CuNCs) and exonuclease I (EXO I). The template of the DNA–CuNCs was rationally designed as a ds-/ss-DNA hybrid, ensuring that after a quick and nonpersistent cut of Cas12a, a majority of the template can be digested by EXO I. Based on this novel reporter, a biosensor termed CRISPR-CNS (cost-effective, nimble, and sensitive copper nanocluster sensor integrating CRISPR) was developed. Due to the high signal-to-background ratio of our proposed reporter, CRISPR-CNS shows excellent performances for nucleic acid detection, yielding a detection limit of 20 copies for SARS-CoV-2 RNA. Considering its facile synthesis, robust fluorescence, effective cost, and good sensitivity, this combination shall serve as a highly potential output for CRISPR-based point-of-care testing.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2023
·
IF 6.1
Xiaobo Zhang,Zhiru Li,Lili Yang,Bing Hu,Qiuyue Zheng,Jiang Man,Jijuan Cao
The sensitive and accurate detection of ochratoxin A (OTA) is crucial for public health due to its high toxicity. Herein, using Au nanoparticle (NP)-attached CdS/UiO-66-NH2 heterostructures as photoactive materials, a photoelectrochemical (PEC) aptasensor was presented for the ultrasensitive assay of OTA based on a competitive displacement reaction triggering the trans-cleavage ability of CRISPR/Cas12a. In this sensing strategy, methylene blue-labeled single-stranded DNA (MB-ssDNA) was immobilized on the Au NPs/CdS/UiO-66-NH2 electrode to accelerate the separation of the photogenerated carrier, thus producing a significantly increased PEC response. In the presence of OTA, it specifically bound with the aptamer (Apt) and resulted in the release of the activation chain, triggering the trans-cleavage characteristics of CRISPR/Cas12a. MB-ssDNA was cut randomly on the electrode surface to convert the PEC signal from the “on” to the “off” state, thereby achieving a quantitative and accurate detection of OTA. The CRISPR/Cas12a-derived PEC aptasensor exhibited excellent sensitivity and specificity, with a linear range from 100 to 50 ng/mL and a detection limit of 38 fg/mL. Overall, the proposed aptasensor could provide a rapid, accurate, and sensitive method for the determination of OTA in actual samples.
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