ANALYTICAL CHEMISTRY
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2023
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IF 7.4
Yin Zhao,Dongjuan Chen,Zhichen Xu,Tao Li,Jiang Zhu,Rui Hu,Guoyong Xu,Ying Li,Yunhuang Yang,Maili Liu
Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10–18 M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.
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ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
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2023
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IF 6.8
Hangyu Guo,Yaqin Zhang,Fange Kong,Chunxia Wang,Shanshan Chen,Jiasi Wang,Di Wang
Alternaria solani ( A. solani ), the causal agent of early blight in potatoes, poses a serious and persistent threat to potato production worldwide. Therefore, developing a method that can accurately detect A. solani in the early stage to avoid further spread is urgent. However, the conventional PCR-based method is not appropriate for application in the fields. Recently, the CRISPR-Cas system has been developed for nucleic acids analysis at point-of-care. Here, we propose a gold nanoparticles-based visual assay combining loop-mediated isothermal amplification with CRISPR-Cas12a to detect A. solani . After optimization, the method could detect 10 −3 ng/μL genomic gene of A. solani . The specificity of the method was confirmed by discriminating A. solani from other three highly homologous pathogens. We also developed a portable device that could be used in the fields. By integrating with the smartphone readout, this platform holds significant potential in high-throughput detection of multiple pathogens in the fields.
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ANALYTICA CHIMICA ACTA
·
2023
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IF 6.2
Jiuying Cui,Qisheng Luo,Cheng Wei,Xiandong Deng,Hongqu Liang,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
Background Escherichia coli ( E.coli ) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract , posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. Results We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli . Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. Significance With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 10 2 - 1 × 10 7 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.
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BIOSENSORS & BIOELECTRONICS
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2023
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IF 12.6
Yu Li,Miaomiao Cai,Wenwen Zhang,Ying Liu,Xiaoqing Yuan,Na Han,Jing Li,Shengnan Jin,Chunming Ding
The CRISPR-Cas12a RNA-guided complexes hold immense promise for nucleic acid detection. However, limitations arise from their specificity in detecting off-targets and the stability of the signal molecules. Here, we have developed a platform that integrates multiplex amplification and nanomolecular-reporting signals, allowing us to detect various clinically relevant nucleic acid targets with enhanced stability, sensitivity, and visual interpretation. Through the electrostatic co-assembly of the Oligo reporter with oppositely charged nanoparticles, we observed a significant enhancement in its stability in low-pollution environments, reaching up to a threefold increase compared to the original version. Additionally, the fluorescence efficiency was expanded by three orders of magnitude, broadening the detection range considerably. Utilizing a multiplex strategy, this assay can accomplish simultaneous detection of multiple targets and single-point indication detection of nine specific targets. This significant advancement heightened the sensitivity of disease screening and improved the accuracy of diagnosing disease-related changes. We tested this assay in a colorectal cancer model, demonstrating that it can identify DNA methylation features at the aM-level within 40–60 min. Validation using clinical samples yielded consistent results with qPCR and bisulfite sequencing, affirming the assay's reliability and potential for clinical applications.
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MICROCHEMICAL JOURNAL
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2023
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IF 4.8
Xianfeng Wang,Chengxiang Chu,Yuanyi Deng,Yi Ma,Mei Yang,Huibo Luo,Danqun Huo,Changjun Hou
Accurate identification of Staphylococcus aureus ( S. aureus ) is crucial for food safety and clinical diagnosis. Particularly, identifying drug-resistant strains guides effective treatment and prevention of infectious diseases. Herein, we presented a highly efficient programmable CRISPR/Cas12a system for identification of methicillin-resistant S. aureus (MRSA). Genus-specific gene (spec gene) and methicillin-resistant gene (mecA gene) of S. aureus were amplified by PCR. According to the sequences of spec gene and mecA gene, corresponding programmable crRNAs were designed and prepared to construct CRISPR/Cas12a system, which recognized and bound to the PCR products through complementary interactions to activate the trans -cleavage activity of Cas12a. This led to the indiscriminate digestion of surrounding FQ-reporter, releasing a distinct fluorescence signal for MRSA identification. In addition, we devised a pair of AuNPs-DNA probes for colorimetric recognition of S. aureus . Target-triggered trans -cleavage for linker probe led to an non-aggregation of AuNPs-DNA probes pair. This led to a noticeable appearance of red color, enabling naked-eye detection. Thus, MRSA identification was realized with remarkable sensitivity and exceptional selectivity. Successful identification of MRSA in clinical samples validated the practical applications of the proposed CRISPR/Cas12a assay, establishing it as a universal platform for diagnosing pathogenic microorganisms with versatility and compatibility.
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BIOELECTROCHEMISTRY
·
2025
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IF 4.5
Yun Zhang,Liang Gao,Zhe Shi,Qiong Wu,Xiangmin Miao
Sensitive telomerase activity detection becomes particularly significance since the important value of it in early cancer diagnosis as a potential biomarker. Herein, we developed a paper-based analytical devices (PADs) for telomerase activity detection, using positively charged Au@luminol nanoparticles ((+)Au@luminol NPs) as electrochemiluminescence (ECL) signal probe coupling with hybridization chain reaction (HCR) and CRISPR/Cas12a dual signal amplification. Firstly, the initial strong ECL signal was obtained based on the electrostatic adsorption of (+)Au@luminol NPs onto the surface of HCR double-stranded hybrid aggregates. In the presence of telomerase, the primer was efficiently elongated with telomeric repeats of (TTAGGG) n to release activator DNA and trigger the CRISPR/Cas12a, which can prevent the happen of HCR and the adsorption of (+)Au@luminol NPs through cleaving the capture probe on the electrode surface, such results directly inducing the decrease of the ECL signal that was proportional to telomerase concentration, due to the efficient signal amplification of HCR and CRISPR/Cas12a, a low detection limit of 2.3 cells/mL for telomerase could be detected. Moreover, the sensor realized the effective application for telomerase extracts analysis in human serum samples, making it possess potential application value for telomerase activity assays in cancer diagnostics.
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MICROCHEMICAL JOURNAL
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2024
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IF 4.9
Maolin Li,Yuting Zhang,Xiuyuan Jiang,Jiao Jiang,Xina Gu,Hua Zhang
Transcription factors play crucial roles in governing gene expression and are implicated in various physiological and pathological processes. Here, we propose a novel electrochemiluminescence (ECL) biosensor for the sensitive and specific detection of the transcription factor NF-kB p50, utilizing CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (HRCA) technology. The biosensor achieves a remarkable limit of detection (LOD) of 3.6 fM and demonstrates excellent specificity against interfering proteins. Optimization of experimental parameters further enhances the biosensor’s performance, ensuring its reliability and accuracy. In addition, validation in real biological samples confirms the feasibility of biosensor for practical applications, offering a promising tool for clinical trials and disease diagnosis.
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ANALYST
·
2025
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IF 3.3
Shaoying He,Wei Lin,Xin Liu,Fei Li,Hong Liang,Huo Xu,Chunhua Lu,Chao Xing
Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb2+ based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb2+, the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb2+ concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb2+, the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb2+ in milk and lake water samples.
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ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Weihong Yin,Kai Hu,Yunxing Yang,Jianjian Zhuang,Zheyu Zou,Yuanjie Suo,Liping Xia,Jiale Li,Yehong Gui,Haohua Mei,Juxin Yin,Tao Zhang,Ying Mu
Escherichia coli O157:H7 (E. coli O157:H7) is a prominent pathogenic bacterium that poses serious risks to food safety and public health. Rapid and accurate detection of live E. coli O157:H7 is of great importance in food quality monitoring and clinical diagnosis. Here, we report a propidium monoazide-assisted nonamplification digital CRISPR/Cas12a assay for sensitive and rapid detection of live E. coli O157:H7. The incorporation of propidium monoazide into the method enables the selective detection of live bacteria by eliminating 98% of interference from the dead bacterial nucleic acid. Implemented on microfluidic digital chips, this method can achieve absolute quantification of nonamplified nucleic acid. The entire detection process of live bacteria can be completed within 120 min without the need for establishing a standard curve, and the sensitivity of the method reaches 1.2 × 103 CFU/mL. The method was validated using various samples, yielding results consistent with the plate counting method (Pearson’s r = 0.9490). Consequently, this method holds significant potential for applications in fields requiring live bacterial detection.
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ACS Applied Materials & Interfaces
·
2024
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IF 8.3
Runlin Yang,Siying Xie,Bin Zhou,Mingming Guo,Jun Fan,Fengli Su,Zhirun Ji,Yue Chen,Bingzhi Li
CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.
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ANALYTICAL CHEMISTRY
·
2025
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IF 6.7
Junzhuo Shan,Yixiao Sheng,Lun Luo,Wenhai Wang,Xinjiang Liu,Yi Ma,Jufang Wang
Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10–4 U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid–based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Wang Guo,Yiqing Guo,Hong Xu,Chen Li,Xinai Zhang,Xiaobo Zou,Zongbao Sun
Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal–organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 100 to 7.9 × 108 CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.
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