MICROCHEMICAL JOURNAL
·
2026
·
IF 5.1
Meilin Liu,Yi Duan,Yiyao Luo,Xuhui Xia,Mingyi Guo,Danqun Huo,Changjun Hou
Ochratoxin A (OTA), a prevalent cereal mycotoxin, exhibits carcinogenic and teratogenic properties that threaten public health. Consequently, sensitive and reliable OTA detection methods are critically needed. A CRISPR-Cas12a-mediated colorimetric biosensor was developed, integrating Cas12a regulation via modular hairpin structures with MOF@Pt@MOF nanozyme amplification. Hairpin structures incorporate target-specific sequences; target binding induces rearrangement, preventing Cas12a recognition while enabling dsDNA activators to restore Cas12a function. Magnetic beads (MBs) were coupled with MOF@Pt@MOF nanozymes via ssDNA to construct MBs-MOF@Pt@MOF probes. Following OTA-induced activation, Cas12a cleaves ssDNA, releasing nanozymes into the supernatant, catalyzing TMB oxidation to generate a blue chromophore. A detection limit of 0.055 ng/mL was achieved with excellent selectivity. This approach utilizes inhibitory aptamer conformational changes for precise Cas12a regulation and nanozyme signal amplification to enhance sensitivity and specificity. The modular design demonstrates versatility for diverse target detection in point-of-care testing (POCT) applications.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 7.7
Huikai Lin,Caiyi Du,Ningyi Xu,Yiru Wang,Chunyan Sun
CRISPR/Cas12a can activate trans-cleavage activity upon single-stranded DNA binding, making it widely applicable in highly sensitive fluorescent biosensors. Sensors utilizing monochromatic fluorescence output modes are susceptible to interference from multiple factors, resulting in unstable detection methods. To address this, we designed a ratio-based fluorescence sensor for detecting tetracyclines (TCs) based on the synergistic signal amplification of nucleic acid aptamer (42TET) and zirconium-based metal-organic framework (PCN-224) with CRISPR/Cas12a. PCN-224 adsorbs single-stranded DNA, quenching its FAM-labeled fluorescence while simultaneously emitting its own fluorescent signal. This dual-fluorescence output reduces background interference and enhances detection accuracy. By designing an activator as a complementary sequence (cDNA) to the nucleic acid aptamer, the system binds to 42TET in the presence of tetracycline, releasing the cDNA to activate the CRISPR/Cas12a system. Through this system's signal amplification capability, highly sensitive detection of tetracycline is achieved, with a detection limit reaching 8.7 nM. It possesses the same detection capabilities as traditional HPLC detection methods. This method is simple, rapid, efficient, and highly quantitative, offering broad prospects for future applications.
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ANALYTICAL CHEMISTRY
·
2026
·
IF 6.7
Xiang Chen,Chunxiang Mao,Yuanji Gao,Chaoting Shi,Yu Wang,Zhuolin Jin,Bing Xia,Yan Zhou
Direct quantitative analysis of low-abundance protein biomarkers by electrospray ionization mass spectrometry (ESI-MS) remains challenging due to poor ionization efficiency and matrix interferences. Herein, we report an ultrasensitive analytical platform, termed CRISPR/Cas12a-mediated liposomal amplification coupled with electrospray ionization mass spectrometry (CMLA-MS), that overcomes this limitation by integrating CRISPR/Cas12a-mediated dual-cascade signal amplification with an ESI-MS readout. The strategy converts the detection of poorly ionizable protein molecules into the quantification of numerous, highly ionizable small-molecule reporters: proteins trigger Cas12a trans-cleavage (first amplification), which subsequently cleaves single-stranded DNA (ssDNA) probes anchored to signal-loaded liposomes, causing the burst release of thousands of MS-detectable reporters (second, physical amplification). This dual-amplification strategy enabled an exceptionally low limit of detection (LOD) of 10.8 fg/mL, and the method successfully quantified cardiac troponin I (cTnI) in clinical serum samples with high recoveries (90.3–101.6%).
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ANALYTICAL CHEMISTRY
·
2026
·
IF 6.7
Kun Wang,Wanli Wang,Yufeng Hu,Xiaoyang Li,Haoyu Yu,Xin Xia,Tianhan Kai,Mei Wen
Staphylococcus aureus (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.
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ANALYTICAL CHEMISTRY
·
2026
·
IF 7.3
Liu Han,Liu Yin,Xu Yu,Wang Zhuoxuan,Yu Yanyan,Su Gaoxing,Qu Guangbo
Monitoring environmental drug-resistance genes (DRGs)
plays a pivotal
role in preventing the transmission of antimicrobial resistance, thereby
reducing public health risks. In this study, a one-pot recombinase
polymerase amplification (RPA)/clustered regularly interspaced short
palindromic repeat (CRISPR) assay was developed for monitoring DRGs
in river water. To overcome compatibility challenges between RPA and
CRISPR systems, four glycosaminoglycans (heparin sodium, nadroparin
calcium, dalteparin sodium, and chondroitin sulfate A sodium salt)
with different molecular weights or negative charge density were evaluated
as Cas-enzyme activity modulators. Among them, heparin sodium with
the high molecular weight and high strong negative charge density
exhibited the best performance in the one-pot DRG detection assay.
In the system, CRISPR-Cas12a activity was temporarily inhibited during
the RPA amplification phase. When sufficient amplicons were accumulated,
Cas12a was activated for signal readout, thereby achieving orderly
coupling and precise control of both reactions. To further simplify
and improve the reliability of environmental DRG monitoring, a pretreatment
method that can eliminate nucleic acid extraction was developed and
integrated with the inhibitor-controlled one-pot platform. This assay
achieved high sensitivity and specificity when it was applied to river
samples, matching the performance of qPCR. The developed assay is
simple to operate, has high sensitivity, and is widely adaptable,
providing a robust tool for rapid antimicrobial resistance surveillance
and exhibiting promise for public health management applications.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Zhenjie Zheng,Siqi Cai,Mengqi Ma,Xiaoyi Chen,Zhanglin Weng,Da Huang,Bin Qiu,Zhenyu Lin,Fang Luo
ATT-AuNCs provide intrinsically efficient solid-state AIE emission without additional material engineering. • CRISPR/Cas12a enables efficient target-triggered molecular signal amplification. • The biosensor achieves a 0.37 pg·mL −1 detection limit for NDMA in processed meat samples. N -Nitrosodimethylamine (NDMA) is a potent carcinogenic small-molecule contaminant, and its trace-level occurrence necessitates sensitive and reliable detection methods. Herein, we developed an electrochemiluminescence (ECL) aptasensor integrating a solid-state aggregation-induced emission (AIE)-active gold nanocluster (Au NCs) luminophore with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-mediated molecular signal amplification for ultrasensitive NDMA detection. 6-Aza-2-thiothymine (ATT)-protected Au NCs (ATT-AuNCs) served as the ECL luminophore with triethylamine as the coreactant, while ferrocene (Fc)-labeled single-stranded DNA (Fc-ssDNA) acted as the ECL quencher. Upon NDMA recognition, target-induced strand displacement released a DNA activator, which activated the Cas12a/crRNA complex and triggered trans-cleavage of electrode-bound Fc-ssDNA. Cleavage of the ssDNA backbone disrupted the linkage between the Fc moiety and the electrode-anchored terminus, allowing Fc-containing cleavage products to detach from the electrode interface and thereby reducing Fc-mediated quenching. As a result, the ECL emission of ATT-AuNCs was restored, translating target recognition into an enhanced ECL response. The proposed sensor exhibited a linear range of 5 pg·mL −1 to 500 ng·mL −1 with a detection limit of 0.37 pg·mL −1 . By exploiting the intrinsically efficient solid-state AIE emission of ATT-AuNCs and CRISPR/Cas12a-mediated signal amplification, this strategy enables ultrasensitive NDMA detection. Download: Download high-res image (118KB) Download: Download full-size image
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CHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 12.5
Shuang Guo,Jiashuo Xu,Huiquan Wang,Wen Li,Tao Meng,Zhe Zhao,Zhiguang Niu,Xiaoqun Gong
We reported a TdT combined with CRISPR/Cas12a driven cascade amplification platform for the sensitive detection of both heavy metal ions and viral targets. • Integrated with reverse fluorescence-enhanced lateral flow strip for visual readout, this platform achieves LODs of 2.5 × 10 −15 M for Pb 2+ and 3.6 × 10 −16 M for DENV-RNA. • The modular design allows flexible replacement of recognition modules, offering a universal solution for on-site rapid monitoring of diverse pollutants in complex environmental matrices. Currently, constructing flexible detection platforms capable of identifying diverse hazardous substances remains a significant challenge. Conventional detection methods are limited by target specificity and lack cross-class universality. Herein, we developed a general platform for sensitive detection of lead ions (Pb 2+ ) and dengue virus (DENV) as two distinct model targets. This platform specifically converted different types of targets into detectable nucleic acid molecules, which would be further recognized and signal amplified by the cascade cycling isothermal amplification system composed of terminal deoxynucleotidyl transferase (TdT) and CRISPR/Cas12a system. TdT has the property of extending the 3′ end of DNA molecule in a template-independent manner, providing long poly-T nucleic acid sequence for binding lots of crRNA, so as to activate the trans-cleavage activity of Cas12a to cleave the hairpin DNA reporters with exposing numerous novel 3′ ends for the same cycle. By combining this strategy with the reverse fluorescence-enhanced lateral flow test strip, the platform achieves a limit of detection (LOD) of 2.5 × 10 −15 M and 3.6 × 10 −16 M for Pb 2+ and DENV-RNA, respectively. Moreover, the detection platform for Pb 2+ in environmental water samples showed excellent agreement with standard methods. It also demonstrated high sensitivity for DENV-RNA detection in complex samples, confirming its reliability and broad applicability potential in practical scenarios. Download: Download high-res image (103KB) Download: Download full-size image
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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
The alternative text for this image may have been generated using AI.
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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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BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Tai Ye,Mei Xue,Bingjing Zhou,Shuwei Kang,Zhaobao Chen,Zilong Tang,Shihui Yang,Qianling Zhao,Min Yuan,Jinsong Yu,Hui Cao,Liling Hao,Xiuxiu Wu,Fengqin Yin,Fei Xu
One-pot autocatalytic CRISPR biosensing offers a promising route for signal amplification without nucleic acid pre-amplification, but its efficiency is limited by an intrinsic readout-amplification conflict: the ssDNA reporter required for signal output competes with the autocatalytic mediator required for feedback amplification. Here, we report a biphasic spatiotemporal regulation strategy (BS-Cas12a system) to resolve this substrate competition for one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets. Mechanistic studies revealed that Cas12a preferentially cleaved the ssDNA reporter over the autocatalytic circular mediator, leading to insufficient mediator linearization and impaired autocatalytic amplification. A glycerol/water biphasic system was therefore constructed to spatially delay reporter access to activated Cas12a, allowing preferential cleavage of the autocatalytic mediator and subsequent generation of additional Cas12a activators. This biphasic system achieved a 3-fold increase in the autocatalytic amplification efficiency. By integrating an aptamer-mediated target-to-activator conversion module, non-nucleic-acid recognition was programmably converted into Cas12a activation. Using sulfadimethoxine as a model target, the platform achieved a detection limit of 65 pM and showed good recoveries in fish samples. The system was further extended to Cd 2+ and thrombin detection with limits of detection of 0.65 nM and 11.63 pM, respectively. Moreover, by replacing the fluorescent reporter with a FAM/biotin-labeled reporter, a lateral-flow readout was achieved. This work provides a kinetic-regulation strategy for modular one-pot Cir DNA autocatalytic biosensing with an independent and exchangeable reporter and expands CRISPR-based detection toward diverse non-nucleic-acid targets.
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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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