FOOD CONTROL
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2021
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IF 5.548
Shujuan Wang,Yiling Fan,Zhen Feng,Minghui Song,Qiongqiong Li,Bo Jiang,Feng Qin,Hao Liu,Lefu Lan,Meicheng Yang
Escherichia coli O157:H7 ( E. coli O157:H7) is one of the most dangerous foodborne pathogens causing severe illnesses in the elderly, children and immunocompromised people. Traditional methods could not meet the detection requirements of high speed, sensitivity, and specificity. The clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic is a novel high sensitivity and specificity detection method. In this study, a rapid nucleic acid of Escherichia coli O157:H7 was detected using rfbE gene based on CRISPR/Cas12a system. The limit of detection for DNA and bacterial concentrations was 0.9 pg/μL and 6.5 × 10 4 CFU/mL, respectively. Furthermore, when the initial bacterial inoculum was 14 CFU/mL in the ground beef sample, the target was detected after 4 h of culture through Metal Organic Framework immunomagnetic beads (Fe 3 O 4 @ [email protected] 2 ) enrichment.
研究领域:
微生物学
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分子生物学
·
食品科学
·
生物技术
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高影响因子
Current Research in Food Science
·
2026
·
IF 7
Kan Yang,Changan Cheng,Youjia Zhang,Zhaoying Liu,Enze Sheng,Zhen Zhang,Ming Li
Multi-mode biosensors are promising rapid detection methods for mycotoxins, while these methods are still challenging in practice due to accessibility and efficiency. A tri-mode aptasensor platform based on CRISPR/Cas12a-driven cascade strategy is presented for ultrasensitive quantification of ochratoxin A (OTA) in this study. The probes of Apt@cDNA, fluorophore-quencher (FQ-reporter) and glucose oxidase (GOx)-reporter were prepared in advance to provide specific recognition and efficient signaling conversion. When target of OTA presented, the released cDNA aroused CHA reaction and then activated CRISPR/Cas12a, which not only used to turn-on the FQ-reporter but also release the GOx. To further improve the performance, the glucose was selected to generate numerous H 2 O 2 molecules. The ultrasensitivity had been exhibited with limit of detection (LOD) values of 23.7 pg mL −1 , 9.10 pg mL −1 and 16.9 pg mL −1 for fluorescence, colorimetric and strip mode, respectively. The tri-mode aptasensor demonstrated satisfactory specificity, accuracy, and practicability by its application in spiked and real samples. Furthermore, the highlights, including quantitative capability, homogeneous reaction, and simplified operation, had been integrated. This proposed aptasensor has provided an ultrasensitive tri-mode rapid detection platform through CRISPR/Cas12a-driven generalduty optical signals, which might have promising prospects in the harmful substance monitoring in various scenarios.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
研究领域:
分子生物学
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生物工程
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诊断技术
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合成生物学
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核酸扩增
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 8.3
Xiaolong Li,Daxiu Li,Yanni Wang,Caiyue Xiang,Shuang Tan,Rou Fan,Kai Shi,Wenjiao Zhou
A novel logic-gated D -CHA/Cas12a biosensor enables accurate dual-miRNA detection. • PS-G-triplex mediates label-free fluorescence, colorimetric, and RGB outputs. • Ultra-low femtomolar sensitivity was realized through D -CHA and Cas12a amplification. • Dynamic AIH progression and prednisone efficacy in mice were tracked successfully. An innovative trimodal optical biosensor incorporating dual catalytic hairpin assembly ( D -CHA) and CRISPR/Cas12a was fabricated to specifically and sensitively detect miR-21 and miR-122, two core biomarkers of autoimmune hepatitis (AIH). The platform employs a phosphorothioate (PS)-modified G-rich hairpin (SHG3) as the reporter, enabling fluorescence, colorimetric, and smartphone-based Red Green Blue (RGB) signal readouts. Target-driven AIH produces extended double-stranded DNAs that trigger Cas12a trans-cleavage, thereby cleaving SHG3 to release G-rich strands for PS G-triplex (SG3) formation. Benefiting from this synergistic amplification, the platform achieved ultra-low limits of detection (LOD) of 153 fM (fluorescence), 516 fM (colorimetric), and 6.17 pM (smartphone) for the dual targets, coupled with exceptional specificity capable of discriminating single-base mismatches. The platform was successfully applied to detect miR-21 and miR-122 in mouse serum from a concanavalin A (ConA)‑induced AIH model, showing significantly elevated signals in model mice that were notably reduced after prednisone treatment, consistent with liver function indices and inflammatory cytokine profiles. This sensing strategy features simple operation, low cost, and no need for sophisticated instruments, providing a reliable point-of-care tool for AIH progression monitoring and therapeutic efficacy evaluation. It also offers a new methodological reference for multiplex biomarker detection in autoimmune diseases, showing promising potential for clinical translation. Download: Download high-res image (160KB) Download: Download full-size image This work presents a trimodal biosensor integrating D -CHA with CRISPR/Cas12a for sensitive detection of autoimmune hepatitis biomarkers miR-21 and miR-122. Target-triggered D -CHA activates Cas12a to cleave a G-rich reporter probe, enabling fluorescence, colorimetric, and smartphone-based RGB detection. The platform achieves fM-level sensitivity, single-base mismatch discrimination, and successful application in a mouse AIH model, offering a simple, low-cost POC tool for disease monitoring.
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ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Tian Jin Xie,Jia Li Xie,Yu Jie Luo,Kai Mao,Cheng Zhi Huang,Yuan Fang Li,Shu Jun Zhen
DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.
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MICROCHEMICAL JOURNAL
·
2023
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IF 4.8
Lilin Qin,Fangxu Lou,Yan Wang,Yinhao Zhang,Shishi Liu,Xu Hun
Molecular switches formed by enzyme-DNA hybrid compounds combined with CRISPR-Cas12a cis -cleavage used for photoelectrochemical nucleic acid assay was fabricated. A compound, molecular switch, consisting of Taq DNA polymerase, aptamer, block DNA and methylene blue labeled primer (primer-Mb) is formed. Block DNA is assembled on the surface of MBs (block DNA-MBs), and hybridized with the primer-Mb. In the presence of Human Immunodeficiency Virus (HIV) target, molecular switches are turned on by hybridization of the target with block DNA. The Taq DNA polymerase is released and activated. The activated Taq DNA polymerase elongates the primer via the template, block DNA, which attached onto Magnetic beads (MBs). A dsDNA with Mb (dsDNA-Mb) was formed. With the assistance of Cas12a, a amplified photoelectrochemical signal was obtained and the HIV target DNA was qualified. Under the best conditions, the limit of detection (LOD) can be as low as 0.3 fM. This photoelectrochemical assay extends the tool kit of molecular nanotechnology.
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高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Shiqi Xu,Songtao Wang,Ling Guo,Yuqin Tong,Lina Wu,Xingxu Huang
CRISPR-based detection was often based on the target preamplification to realize the high sensitivity. Here, we prepared a CRISPR-Cas12a system for the colorimetric detection of lead ion (Pb 2+ ) based on the assistance of DNAzyme and nanozyme instead of preamplification. The recognition between GR-5 DNAzyme and Pb 2+ could trigger the CRISPR-Cas12a system. MnO 2 nanozymes connected with magnetic beads through single stranded DNA were prepared as the colorimetric signal probes and catalyst of CRISPR-Cas12a system for the strong oxidase-like activity inducing the color change of 3,3′,5,5′-tetramethylbenzidine. The nanozyme-catalysed CRISPR-Cas12a system could be used to detect Pb 2+ through the color change with high specificity and sensitivity. The linear range of this approach was 0.8 nM–2500 nM, with a limit of detection of 0.54 nM. This method was applied for the detection of the Pb 2+ in food samples indicating good accuracy and anti-interference ability.
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2021
·
IF 7.46
Lijuan Yin,Ninghui Duan,Si Chen,Yuan Yao,Jifeng Liu,Long Ma
Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans -cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K + . This DNAzyme could catalyze the TMB-H 2 O 2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R 2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.
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Journal of Analysis and Testing
·
2025
·
IF 7
Yan Jieyu,Wang Zhen,Li Jingwen,Li Yifei,Wu Zhengjiang,Zhang Yirui,Han Kun
Accurate and sensitive detection of protein biomarkers is critical for early disease diagnosis and therapeutic monitoring. This study introduces a novel isothermal proximity CRISPR-Cas12a assay for the detection of C-reactive protein (CRP), a key biomarker for inflammation and disease progression. By employing a DNA-antibody bioconjugate strategy, this method converts protein recognition into DNA signals, enabling multiple signal amplification cascades. The proximity hybridization of antibody probes triggers the Cas12a trans-cleavage activity, releasing detectable signals in fluorescence and commercial test strips. Dual-mode detection was realized, with detection limits of 0.57 ng/mL for fluorescence and 2.63 ng/mL for test strips. Linearity detection ranges were found to be 0.1–100 µg/mL. Furthermore, it exhibited high specificity against interfering substances in blood. Clinical serum samples were analyzed, demonstrating the assay’s robustness and practicality under physiological conditions. This approach enhances the detection of CRP and provides a versatile framework for the quantitative analysis of various protein biomarkers, offering an alternative solution for clinical protein diagnosis for point-of-care tests.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Fan Li,Ruiting Yao,Xinxin Jiang,Haorui Zhu,Yiheng Shi,Jianpeng Wang,Yutao Luo,Guoliang Li
Rapid and sensitive nucleic acid detection is of immense value and broad application prospects in fields such as food safety monitoring and disease diagnosis. Conventional fluorescently labeled probes, while widely used in fluorescent biosensors for nucleic acid detection, suffer from inherent limitations including high background signal, photobleaching, and cost inefficiency. Meanwhile, G-quadruplex (G4) structures present inherent issues of insufficient design flexibility and weak output fluorescent signal strength. Herein, we innovatively developed a fluorescent biosensor integrating split G4 with the recombinase polymerase amplification (RPA)-CRISPR system for the label-free, rapid, and highly sensitive detection of Listeria monocytogenes . Through RPA of target DNA, exponential amplification of the target DNA sequence was achieved, which subsequently activated the trans -cleavage activity of Cas12a. This non-specifically cleaved the linker strand, inhibiting the assembly of split G4 and resulting in proportional fluorescence attenuation corresponding to the input DNA target. Under the optimized conditions, the detection sensitivity of this method for L. monocytogenes reached 1.9 × 10 1 CFU/mL, which is two orders of magnitude higher than that of the conventional fluorescent probe methods. Furthermore, this method exhibited excellent specificity in distinguishing L. monocytogenes from other common pathogens, with successful application in natural sample analyses. Therefore, we have established a split G4-based RPA-CRISPR/Cas12a method, which provides a label-free, highly sensitive and specific analytical tool for the detection of foodborne pathogens, offering significant reference value for practical applications.
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高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Yiyang Fan,Xiaoyi Yang,Xin Xu,Tong Shen,Ze Zhang,Fan Wang,Hongzhi Pan,Dong Chang
A CRISPR-responsive DNA hydrogel gates δ-MnO 2 nanozyme release. • RPA–Cas12a recognition enables detection of five common uropathogens. • The assay achieved LODs of 2.44–45.47 copies/μL across targets. • Parallel channels showed specificity, reproducibility, and 20-day stability. • KernelRidge-RBF provided the lowest prediction error among nine evaluated regression models. Urinary tract infection (UTI) requires rapid and accurate pathogen identification, yet conventional culture-based methods are time-consuming and many molecular assays remain dependent on sophisticated instrumentation. Here, we developed a multiplex visual biosensing platform for uropathogen detection by integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a recognition, a CRISPR-responsive DNA hydrogel gate, and a δ-MnO 2 nanozyme-mediated colorimetric module. In this system, target-derived amplicons activate pathogen-specific Cas12a, which degrades the DNA hydrogel and releases encapsulated δ-MnO 2 , thereby catalyzing the TMB/H 2 O 2 reaction to produce a visible and spectrophotometrically measurable signal. The platform was applied to five representative uropathogens, including Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa , Enterococcus faecium , and Enterococcus faecalis . Under optimized conditions, the assay showed good linearity for all five targets, with R 2 values of 0.9955, 0.9958, 0.9948, 0.9974, and 0.9956, respectively, and limits of detection of 2.60, 3.15, 2.44, 20.40, and 45.47 copies/μL, respectively. The platform also exhibited satisfactory specificity, multiplexing capability, reproducibility, and storage stability. To improve quantitative interpretation, an image-recognition- and machine-learning-assisted workflow was further introduced, in which KernelRidge-RBF showed the best overall performance among the tested models. Clinical urine sample testing further supported the applicability of the platform, with AUC values of 1.000, 0.976, 0.994, 0.947, and 0.939 for the five target pathogens, respectively. Collectively, the RPA-Cas12a-DNA hydrogel-δ-MnO 2 platform enables visual and parallel multi-target detection of uropathogens within approximately 3 h and provides a proof-of-concept framework for the further development of portable nucleic acid biosensors. Download: Download high-res image (194KB) Download: Download full-size image
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Biosensors-Basel
·
2026
·Vol. 16
·期 1
·32
·
IF 5.6
Zhiheng Zhang,Qiyu Fu,Tiantai Wen,Youmin Zheng,Yincong Ma,Shixian Liu,Guozhen Liu
Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a’s trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV.
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