Food Quality and Safety
·
2025
·
IF 4.4
Hu Jinyan,Liu Xiaogang,Liu Tiantian,Zhao Ting,Li Mingliang,Yang Qilin,Jiang Xiaofeng,Xiong Yanfei,Shen Caihong,Liu Shuangping,Mao Jian,Tu Rongkun
ObjectivesYeast plays a crucial role in fermented foods, yet the application of the CRISPR/Cas system for yeast detection has been rarely reported. The objective of this study is to develop a rapid and quantitative detection method for brewing yeast based on the CRISPR-Cas12a system, aiming to achieve higher sensitivity than the quantitative real-time polymerase chain reaction (qPCR) assay and fill the gap in the application of CRISPR for yeast detection in fermented foods.Materials and Methods: This work established one-step RPA-CRISPR/Cas12a (ORCC) detection for quantifying yeasts in fermented foods by integrating target amplification and the CRISPR system into a one-pot reaction.ResultsA numerical standardized definition was established for the quantifiable state in ORCC detection of yeasts. Using ORCC detection, which occurs at 37 °C, absolute quantification of Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Zygosaccharomyces bailii can be completed in less than 45 min. Under optimal conditions, the limits of quantification and the limits of detection for three yeasts were 100 copies/μL and 1 copy/μL, with no cross-reaction observed among non-target yeasts. In pure yeast samples, ORCC detection of the target yeasts achieved a detection rate of 91.60% - 116.57%. In simulated fermentation samples, the recovery rate for spiked target yeasts using ORCC detection ranged from 92.78% to 106.90%. In original naturally fermented samples, target yeasts can be quantified by ORCC detection but not by qPCR assay. In spiked naturally fermented samples, the recovery rates of the qPCR assay were generally lower than those of ORCC detection.ConclusionORCC detection is simple, rapid, accurate, and highly sensitive for detecting three yeasts in fermented foods. It has significant potential for precisely monitoring yeast growth and fermentation process in various fermentation systems.
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BIOSENSORS & BIOELECTRONICS
·
2025
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IF 10.5
Yining Zhao,Ziye Liu,Ziting Liu,Juan Zhang,Jinghui Peng,Genxi Li
Prosthetic joint infection (PJI), which is still difficult to be diagnosing, is caused by bacterial infection. Rapid detection for trace amounts of bacteria should be a solution; however, the existing assays cannot serve for PJI diagnosis due to the inherent limitations. Herein, a strategy based on supramolecular chemistry mediated Cas12a catalytic aggregation (CCA) is explored in this work for ultrasensitive detection of universal bacteria. In the design, supramolecular chemistry is introduced to impede the cis-cleavage of activated Cas12a, resulting in its aggregation in the surrounding of bacteria with the linkage of CCA probe. Since Cas12a aggregation owns excellently enhanced catalytic efficiency, the CCA-based strategy can be used for bacterial analysis with a limit of detection as low as 1 CFU/mL. With high sensitivity, reproducibility, generalizability, and stability, the CCA-based strategy can be applied for the detection of clinical effusion samples. The remarkable performance of CCA-based strategy has also demonstrated its robust potential to be a simple, rapid, and highly sensitive bacteria diagnostic tool, paving the way for prompt diagnosis and management of PJI.
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MICROCHEMICAL JOURNAL
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2026
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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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Journal of Analysis and Testing
·
2025
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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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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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MICROCHEMICAL JOURNAL
·
2026
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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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ANALYTICA CHIMICA ACTA
·
2023
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IF 6.2
Yuwan Du,Shuaifeng Ji,Qingyang Dong,Jiang Wang,Dianpeng Han,Zhixian Gao
Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans -activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy . This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.
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ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Xiaofen Zhao,Xiaoxiao Tian,Yuwei Wang,Linbin Li,Yan Yu,Shiqiao Zhao,Juan Zhang
Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA . After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment , direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.
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BIOSENSORS & BIOELECTRONICS
·
2022
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IF 12.545
Hai-Yan Jia,Hong-Li Zhao,Ting Wang,Pin-Ru Chen,Bin-Cheng Yin,Bang-Ce Ye
MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans -cleavage activity of Cas12a for further signal amplification . More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.
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TALANTA
·
2024
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IF 5.6
Jiayue Shi,Sijia Li,Rongguang Shao,Yang Jiang,Yanxia Qiao,Jin Liu,Yaqian Zhou,Yan Li
In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy) 3 2+ ) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy) 3 2+ /AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy) 3 2+ /AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy) 3 2+ /AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy) 3 2+ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.
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TALANTA
·
2025
·
IF 6.1
Xiuyu Chen,Dan Zhao,Changmin Yu,Jifu Wei,Guoren Zhou
The detection of specific protein biomarkers holds significant potential for the early diagnosis of colorectal cancer (CRC). However, the accurate quantification of low-abundance proteins in serum presents a major challenge due to factors such as limited sensitivity and the complexity of the required methodologies. In this work, we established a universal CRISPR/Cas biosensing platform by integrating novel photosensitive nanoprobes (DA/PL@Cu NPs) and CRISPR/Cas12 system (DPC-Cas) for the highly sensitive, specific and user-friendly detection of angiopoietin-like protein 2 (ANGPTL2). The DA/PL@Cu NPs serve as a critical component in the transduction of protein recognition information into nucleic acid amplification events to produce Cas12a activators. The DPC-Cas biosensor integrates DA/PL@Cu NPs-assisted amplification with Cas12a self-amplification, enabling ultrasensitive detection of ANGPTL2 at concentrations as low as 20.00 pg/mL. The proposed DPC-Cas biosensor successfully detected ANGPTL2 in serum, demonstrating significant potential for the early diagnosis of CRC.
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NUCLEIC ACIDS RESEARCH
·
2024
·
IF 16.6
Huang Shen,Lou Yongliang,Zheng Laibao
CRISPR-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a–RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.
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