Food Analytical Methods
·
2026
·Vol. 19
·期 9
·259
·
IF 3
Yang Yue,Liu Jianghua,Liu Fulu,Chen Jianing,Li Qianyu,Xu Bufan,Xu Qinfeng,Li Guoliang
The development of a detection technique with superior accuracy, high sensitivity, and visual applicability simultaneously for economically motivated adulteration (EMA) of fruit juice remains a formidable challenge. Herein, we developed a CRISPR/Cas12a-regulated fluorescence detection platform capable of converting trace level of target into detectable fluorescence signals. This strategy was inspired by the extraordinary trans- cleavage capacity of CRISPR/Cas12a and significant fluorescence response of the fluorescence probe [Ru(phen) 2 (dppz)] 2+ . After the target DNA had been identified, activated CRISPR/Cas12a cleaved the block ssDNA (bDNA), preventing the formation of short dsDNA and initiating a hybridization chain reaction (HCR). Consequently, large amounts of [Ru(phen) 2 (dppz)] 2+ intercalated into the resulting long dsDNA nanowires, enabling cascade signal amplification and a highly sensitive signal output. The output could be observed as a strong orange fluorescence only using a blue transilluminator. Then, we used the strategy to detect adulteration in blueberry juice, which displayed excellent specificity and satisfactory sensitivity with the limit of detection (LOD) as low as 19.5 fM. Our study demonstrated a novel method utilizing the fluorescence switch regulated by CRISPR/Cas12a, showing extremely high specificity, excellent sensitivity, and capability for visual detection of trace target substances in complex food matrices. Graphical
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Dandan Chen,Bin Yin,Zhirun Ji,Zhiqi Liu,Jiahao Li,Fei Liu,Xuping Wu
Amplification-free digital detection of SARS-CoV-2 via CRISPR/Cas12a. • Pneumatically actuated droplet microfluidics enables uniform ∼30 μm droplets. • Rapid detection of SARS-CoV-2 with demonstrated performance in clinical samples. • Offers a simple, portable method for rapid virus detection. Outbreaks of respiratory viruses, including severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), Influenza A (IAV), and Influenza B (IBV) viruses, present a formidable challenge to global public health, driving the urgent need for rapid, sensitive, and deployable diagnostic tools. Here, we present an amplification-free digital nucleic acid detection platform that synergistically integrates a pneumatically actuated droplet microfluidics platform (PADMP) with CRISPR/Cas12a. PADMP enables the precise generation of highly uniform, monodisperse microdroplets (∼30 μm), within which an optimized Cas12a reaction is compartmentalized. This digital partitioning strategy exponentially increases the local target concentration and facilitates single-molecule detection without pre-amplification. Through systematic optimization of droplet stability, surfactant formulation, and Cas12a assay kinetics, the PADMP-Cas12a platform achieves a detection limit (LOD) of 100 pM for synthetic SARS-CoV-2 N gene targets within 40 min. It demonstrates exceptional specificity, showing no cross-reactivity with non-target respiratory viruses (IAV, IBV, SARS-CoV, MERS-CoV, HRSV). The platform successfully detected SARS-CoV-2 in clinical positive samples, with mean droplet fluorescence intensity negatively correlated with Ct values. By combining the advantages of droplet microfluidics with the programmable recognition and trans -cleavage activity of CRISPR/Cas12a, this platform provides a rapid, amplification-free, and highly specific digital detection pathway for RNA viruses, marking an important advance toward high-throughput and point-of-care testing (POCT) for molecular diagnostics. Download: Download high-res image (127KB) Download: Download full-size image
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ANALYTICA CHIMICA ACTA
·
2026
·
IF 6.1
Lingjun Cheng,Qingmei Yang,Yuanyuan Yang,Yufeng Wang,Qingfei He,Zhiyan Zou,Canping Su,Jiaming Chen,Yinhuan Liu,Guolin Hong
Background Tuberculosis (TB) nucleic acid diagnosis urgently requires rapid, amplification-free methods to overcome limitations of quantitative real-time polymerase chain reaction (qPCR), including instrument dependency, prolonged time, and contamination risks from nucleic acid amplification. Results Here, an amplification-free electrochemiluminescence (ECL) biosensor based on peptide-templated gold nanoclusters and the clustered regularly interspaced short palindromic repeats-Cas12a system (CRISPR-Cas12a) has been constructed for the detection of Mycobacterium tuberculosis (MTB)-specific IS6110 sequences. Peptide-templated gold nanoclusters with low background and high ECL response can be used as sensitive signal probes. When CRISPR-Cas12a recognizes the target IS6110 DNA, it exhibits non-specific cleavage activity, repeatedly cleaving ferrocene-labelled DNA. This results in the restoration of ECL signals quenched by ferrocene, thereby achieving signal amplification. There is a linear relationship between the signal and the target concentration range from 10 CFU/mL to 10 4 CFU/mL, with a detection limit of 7 CFU/mL (S/N = 3). Clinical validation ( n = 40) showed strong agreement with qPCR ( κ = 0.90). Significance Critically, this amplification-free strategy eliminates the need for temperature cycling equipment, reduces detection time to 1 h, and completely avoids the risk of amplicon contamination, providing an ideal solution for rapid screening of highly infectious diseases such as TB.
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Analytical Methods
·
2025
·Vol. 17
·期 25
·5298-5307
·
IF 2.6
Yang Cao,Chun Huang,Kui Li,Xi Zhang,Mengfei Tong,Jiale Tu,Kun Yang,Shuai Yuan,Hong Zhang
To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2′-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)32+/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL−1 in a linear detection range of 10−5–100 ng mL−1. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.
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高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2026
·
IF 4.8
Wenjie Liao,Shiqiang Li,Shaoyong Wu,Xiaosong Yang,Bo Liu,Liansheng Ling,Qiuyao Zeng,Ji Zhang
Prostate Cancer Antigen 3, a type of long non-coding RNA, exhibits outstanding specificity as a biomarker for the diagnosis of prostate cancer, offering a highly effective diagnostic indicator, whereas the currently used prostate specific antigen exhibits low specificity, leading to reduced accuracy in prostate cancer diagnosis. Herein, we designed a novel fluorescent sensing platform for targeted detection of PCA3, which integrates the non-specific trans-cleavage activity of the CRISPR/Cas12a with the remarkable fluorescence quenching effect of Gold Nanorods. The Cas12a recognizes and binds to specific sequences of PCA3, thereby activating nonspecific cleavage activity, which cleaves fluorescent reporter probes adsorbed on AuNRs, thus leading to the recovery of fluorescence signals and enabling sensitive detection. The proposed fluorescent sensor exhibits excellent accuracy and convenience for the detection of PCA3 in urine, and a detection limit as low as 1.65 pM was obtained. This sensing system has achieved effective detection of clinical samples of prostate cancer and is expected to provide significant assistance in the screening and therapeutic feedback of prostate cancer in clinical diagnosis.
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高影响因子
ANALYST
·
2022
·Vol. 147
·期 24
·5687-5693
·
IF 5.227
Yuyuan Zhang,Jing Lei,Wei Liu,Yan Jin,Baoxin Li
Various CRISPR/Cas12a-based biosensing systems have been developed in the past few years, and most of these systems used Taqman probes to report signals through fluorescence resonance energy transfer (FRET). In this study, we explored chemiluminescence resonance energy transfer (CRET) as the readout mode for CRISPR/Cas12a-based biosensing. The chemiluminescence (CL) reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) and H2O2 was used to excite the fluorophore dye of the Taqman probe. Different from FRET, CRET does not need external excitation light, which can effectively avoid autofluorescence and photobleaching. The detection limit of this CRET readout mode was estimated to be 10 pM for target DNA, which was about 8 times lower than that of the widely used FRET readout mode. These results suggest that CRET can serve as a rapid, sensitive and simple readout mode of CRISPR/Cas12-based biosensing, and can further enrich the toolbox of CRISPR/Cas12-based biosensing.
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FOOD CONTROL
·
2022
·
IF 6.652
Baoqing Zhou,Qinghua Ye,Moutong Chen,Chufang Wang,Xinran Xiang,Ying Li,Jumei Zhang,Youxiong Zhang,Juan Wang,Shi Wu,Qihui Gu,Yu Ding,Qingping Wu
Rapid and accurate screening of foodborne pathogens in contaminated food is with great significance for food safety. AuNPs-based colorimetric assay is an attractive strategy in the field of food safety inspection, but is limited by tedious probe preparation, low color rendering resolution, and narrow color gamut. Herein, we proposed an e nhanced c olorimetric p latform based on the C RISPR/Cas12a system and label-free D NA-AuNP p robe (CDP-ECP) for high-throughput detection of Staphylococcus aureus ST398. In this strategy, a novel and specific gene targeting S. aureus ST398 was identified and efficient AuNP-bioprobe was successfully prepared. Through a PCR/RAA amplification, the trans -ssDNA cleavage ability induced by target DNA product triggered an aggregation performance for the prepared AuNP-bioprobe pairs, which could realize target colorimetric detection within 50 min. Under optimal conditions, the colorimetric assay could detect as low as 6.4 × 10 2 cfu/mL of S. aureus ST398 in pure culture. Specific gene mining and target-induced trans -cleavage character gave this method a superior specificity and anti-interference. More importantly, this colorimetric method can be extended to a high-throughput platform for rapid detection of spiked and natural sample by using a common microplate reader. Since it is simple to operate, low cost, high sensitivity and specificity, this new platform is fully qualified for clinical diagnosis, environmental monitoring onsite testing requirements.
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BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Feng Qiu,Xiaoyan Gan,Jianglong Yao,Bingying Jiang,Ruo Yuan,Yun Xiang
Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.
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LWT-FOOD SCIENCE AND TECHNOLOGY
·
2023
·
IF 6
Yingchun Li,Yaqin Zhang,Fange Kong,Chunxia Wang,Shanshan Chen,Jiasi Wang,Di Wang
Fusarium temperatum ( F. temperatum ) causes maize stalk rot disease, reducing grain yield and producing multiple harmful mycotoxins that threaten food safety and quality. However, current detection methods are limited by the requirement of well-trained personnel, complicated operations and expensive instruments. To address this challenge, gold nanoparticles (AuNPs)-based colorimetric assay using loop-mediated isothermal amplification (LAMP) coupled with CRISPR-Cas12a (Au-CRISPR) was tested for F. temperatum detection. The assay utilizes DNA-modified AuNPs as a colorimetric probe and the results can be readily observed by the naked eye. After optimization, the method can specifically detect as low as 100 copies/μL of target DNA or 10 −8 ng/μL of extracted DNA from F. temperatum . The detection platform allows high-throughput testing by integrating microtiter plates and microplate reader. Furthermore, a portable smartphone-based device was developed to realize point of care (POC) detection of F. temperatum in resource-limited settings. This simple, inexpensive and sensitive detection platform has great potential for the applications in field detection.
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TALANTA
·
2024
·
IF 5.6
Qiang Tang,Xiaoling Huang,Guangxing Liu,Hongran Huang,Jiayi Zhang,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Sirui Liu,Xinlan Bian,Furong Zhao,Min Cao,Zhaoyang Shang,Bingzhi Li,Xing Zhang
High mannose is overexpressed on the cell surface during carcinogenesis; however, there are currently no efficient techniques for its detection. Herein, we propose an assay called RADAR ( r emote high m a nnose d etection on cell surf a ce with C R ISPR) for the quantification of high mannose, leveraging the integration of high mannose-binding aptamer and CRISPR technique. The high mannose-binding aptamer is hybridized with a locker, which is released upon specific binding to high mannose. The released locker then activates CRISPR/Cas12a to produce a fluorescence signal. The "remote" concept in RADAR refers to the recognition of high mannose occurring on the cell surface, while signal generation takes place in a solution separate from the target. This feature makes RADAR suitable for the detection of suspended bio-samples without the need for sophisticated imaging. RADAR can detect high mannose Man 9 with a limit of detection of 2.5 nM, distinguish cancer cells from non-cancerous cells, and profile high mannose level in human serum for potential non-small cell lung cancer (NSCLC) diagnosis. This work represents the first attempt to integrate CRISPR technology into the aptasensing of high mannose, which may serve as a synthetic biology-enabled tool in glycochemistry and glycobiology.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Yan Zhao,Qianqian Yu,Miaolin Duan,Qi Zhang,Zhishang Li,Yan Zhang,Yana Liu,Han Wang,Xingmin Li,Ruitong Dai,Fei Jia
The efficient, sensitive, and rapid detection of Salmonella typhimurium (S. typhimurium) in food and food products is important to ensure food safety and health. This study developed a fluorescence biosensing assay that integrated recombinase-aided amplification (RAA) and CRISPR/Cas12a with a zeolitic imidazolate framework-8@fluorescein sodium (ZIF-8@FLS) nanocomposite for the sensitive detection of S. typhimurium. In this approach, using RAA as a preamplification module, CRISPR/Cas12a-AChE as a target recognition and dual-enzyme cascade amplification module, and the prepared ZIF-8@FLS with high porosity and rapid pH responsiveness as a fluorescence signal explosive amplification module, the RAA-CRISPR/Cas12a-ZIF-8@FLS biosensor was constructed. Under optimal conditions, it exhibited an excellent linear relationship for S. Typhimurium, with a sensitive detection limit as low as 1.3 × 102 CFU/mL and could complete sample detection within 2 h relying on the RAA and ZIF-8@FLS explosive fluorescence rapid response, demonstrating its significant advantages in specificity, sensitivity, and reliability in food-borne pathogens detection.
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