Journal of Analysis and Testing
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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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Nature Communications
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2026
·
IF 18.1
Liu Kai,Li Xiang,Zhao Meng,Yang Zhuo,Wen Jingshuai,Liu Jinpeng,Zhang Haoli,Zhao Yuecan,Sun Yang,Li Zhengping
Nucleic acid testing is important for infectious-disease diagnosis, but combining high sensitivity with accessible testing remains challenging. Here we show a chip-based visual detection strategy for one or two viral RNA targets that involves only sample loading, without further instrumentation or manual actuation. Target-activated CRISPR-Cas13a alters the wettability of a molecular barrier, causing liquid to cross a preset threshold and generate a dam-break drainage signal. The chip detects a single target at 10 aM within 2 min and two targets at 100 aM within 5 min, with costs of $0.20 and $0.30 per test, respectively. We further establish a practical clinical-sample workflow combining room-temperature lysis with RNase inhibition and validate the platform across 40 nasal-swab samples tested for SARS-CoV-2, influenza A virus and influenza B virus, with results concordant with RT–PCR. This threshold-based fluidic strategy provides a route towards rapid, low-cost and instrument-free molecular testing outside centralized laboratories.
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ANALYTICAL CHEMISTRY
·
2026
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IF 7.3
Liang Zhu,Lejun Liao,Yan Huang,Zhou Nie,Chunyang Lei
The highly sensitive analysis of small-molecule targets holds profound significance across diverse fields, ranging from clinical diagnosis and environmental monitoring to food safety. Herein, we developed a label-free electrochemical CRISPR platform gated by allosteric transcription factors (aTFs) for the ultrasensitive detection of various small molecules. In this system, the specific binding of target analytes to their cognate aTFs induces the release of programmable DNA adaptors, which subsequently trigger Cas12a to trans-cleave DNA probes anchored to the electrode surface. Consequently, the truncated DNA probes serve as initiators to form electroactive G-quadruplex/hemin complexes in situ via terminal deoxynucleotidyl transferase (TdT)-mediated elongation, generating a robust electrochemical response signal. Using TetR as a model aTF, this integrated electrochemical CRISPR biosensor achieved tetracycline detection with picomolar sensitivity. Furthermore, the versatility of this platform was demonstrated by extending its application to p-hydroxybenzoic acid and copper ions through the simple substitution of the aTF modules. The practical utility of the assay was further demonstrated by the robust detection of tetracycline in complex matrices such as milk. Ultimately, this study not only provides a novel strategy for constructing universal, label-free electrochemical CRISPR platforms but also paves the way for the sensitive detection of low-abundance non-nucleic acid targets.
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Nature Communications
·
2026
·Vol. 17
·期 1
·2565
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IF 18.1
He Mingbin,Wang Weiwei,Zhou Haiwu,Liu Cong,Zhao Chunbei,Li Jian,Han Yuewen,Qin Yali,Chen Mingzhou
Pyroptosis plays a crucial role in immune defense against infections and endogenous threats by eliminating harmful cells and modulating the immune response through inflammation. However, the natural activation of pyroptosis involves intricate signaling pathways, posing significant challenges for its artificial manipulation in research and therapies. Here, we present DAMAGE ( D e a th Ma nipulation Ge ne), an innovative system that integrates gasdermins within the type III-E CRISPR framework, enabling the specific recognition of target RNA (tgRNA) and triggering pyroptosis. This mechanism allows DAMAGE to selectively identify and eliminate virus-infected, cancerous, and senescent cells, all of which exhibit altered RNA transcriptomes. Additionally, DAMAGE exhibits considerable promise as a platform for mRNA-LNP therapy. Our study highlights the potential of this CRISPR-based system in the controllable induction of pyroptosis, offering an innovative therapeutic strategy for treating RNA-heterogeneous diseases.
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MICROCHEMICAL JOURNAL
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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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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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TRENDS IN BIOTECHNOLOGY
·
2026
·
IF 16.6
Xiaoya Ren,Xu Cui,Ying Zeng,Yimei Guo,Junhua Chen,Jiafeng Pan
In this research article, an intelligent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas14a-based logic system was developed for microplastic monitoring on the basis of catalytic hairpin assembly (CHA) and DNA walkers on magnetic beads. Polyvinyl chloride (PVC) and polystyrene (PS) can activate the CHA-DNA walker sensing system to generate numerous single-stranded DNA fragments that can be recognized by Cas14a. After the cleavage reaction targeting the fluorophore-quencher reporter, a high Cy5 signal can be observed. The fluorescent biosensor is ultrasensitive, with a limit of detection of 3.4 ng/ml for PVC and 4.1 ng/ml for PS. Using PVC and PS as the two inputs, an OR logic gate was further developed. The input combinations of (10), (01), and (11) can give an output of 1. The sensing system is robust and can work in real water samples with good accuracy. Our proposed biosensor can provide an ultrasensitive and intelligent tool for microplastic monitoring.
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ACS Nano
·
2025
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IF 16
Wenxian Zhang,Zhi-Yuan Feng,Zhe Feng,Rui Lian,Zheng Liu,Jingjing Zhang
Despite the promise of cuproptosis in antitumor therapy, developing strategies to enhance its therapeutic efficacy within the tumor microenvironment remains a challenge. Inspired by the chronotherapy that manipulate circadian rhythms to enhance drug effectiveness, herein we report for a CRISPR-customized copper-DNA nanoplatform (Cu-RNP) that synergistically induces multimodal cell death, including potentiated cuproptosis, by manipulating circadian and metabolic pathways. Cu-RNP integrates coordination-driven self-assembly of Cu2+-DNA nanospheres with Cas13d/crRNA ribonucleoproteins targeting BMAL1. Upon cellular internalization, the acidic and reducing endo/lysosomal environment triggers Cu-RNP disassembly, releasing RNP to silence BMAL1 and disrupt circadian oscillations, leading to WEE1 downregulation and p21 upregulation, thereby inducing apoptosis. Simultaneously, liberated Cu2+ generates cytotoxic hydroxyl radicals for chemodynamic therapy (CDT) and concurrently depletes GSH, promoting mitochondrial copper overload for cuproptosis. Importantly, we demonstrate that silencing BMAL1 disrupts circadian rhythms, inhibits glycolysis, enhances mitochondrial respiration, and redirects metabolic flux to the TCA cycle, thereby amplifying the cell’s vulnerability to copper-induced cuproptosis. In vitro and in vivo results demonstrate that Cu-RNP sensitizes cancer cells to cuproptosis and elicit strong antitumor response through the synergistic combination of cuproptosis, CDT, apoptosis, and circadian-metabolic modulation. This study demonstrates a mechanistic link between BMAL1-regulated circadian rhythms and cuproptosis sensitivity, suggesting a potential treatment strategy for multimodal, cuproptosis-potentiating cancer therapies.
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Advanced Healthcare Materials
·
2025
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IF 9.6
Liuxian Chen,Qin Huang,Yongjie Liu,Ke Chen,Qing Yang,Hua Tang,Dan Wang,Zhenrong Tang
Complete hepatitis B virus (HBV) cure is hindered primarily by the stable persistence of covalently closed circular DNA (cccDNA). Gene editing approaches to eradicate HBV by targeting cccDNA face challenges and limitations due to suboptimal editing efficiency and substantial off-target effects. Herein, a combinatorial therapeutic strategy is developed that integrates CRISPR/Cas9-mediated cccDNA disruption with an antisense oligonucleotide (ASO)-targeted degradation of pregenomic RNA (pgRNA). To overcome delivery challenges, a hepatocyte-targeting nanocarrier (UACPG) is engineered, featuring low immunogenicity, high payload capacity, and dual-stimuli responsiveness. The UACPG platform enabled liver-specific delivery through surface-conjugated targeting ligands, followed by on-demand release of Cas9 ribonucleoprotein complexes and ASO via RNase H-dependent degradation and near-infrared (NIR) light activation. The results demonstrated that UACPG can effectively reduce HBV replication and viral antigen levels, while significantly lowering cccDNA in hydrodynamic HBV-infected mouse models, with no significant off-target effects observed. This nanocarrier achieved the spatiotemporally controlled release of gene-editing systems in vitro and in vivo, significantly inhibiting the replication of HBV, thereby establishing an innovative technological platform for developing curative HBV therapies.
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MICROCHIMICA ACTA
·
2026
·Vol. 193
·期 3
·163
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IF 5.4
Yi Fei,Li Zhixin,Jiang Fan,Zhang Zhule,Chen Yingmei,Zhao Zhe,Deng Xiaoyu,Chen Hao,Xu Shaohua,Tao Yingzhou
Aflatoxin B 1 (AFB 1 ), a potent mycotoxin, poses a critical threat to global food safety, demanding analytical methods with exceptional sensitivity. Here, we introduce a homogeneous electrochemiluminescence (ECL) biosensor that operates on a novel CRISPR-actuated electrostatic gating mechanism. The core of our strategy relies on controlling the access of ECL reporters to a positively charged electrode surface (PAH-ITO). In the absence of AFB 1 , cationic Ru(phen) 3 2+ reporters are electrostatically repelled from the electrode, resulting in a low background signal. The presence of AFB 1 triggers a CRISPR/Cas12a enzymatic cascade, which activates its trans-cleavage activity to release a highly anionic hybridization chain reaction (HCR) scaffold from magnetic beads (MB). This scaffold serves as a nanocarrier, capturing the Ru(phen) 3 2+ reporters and, by virtue of its strong negative charge, shuttling them to the electrode through potent electrostatic attraction. This action effectively “opens” the electrostatic gate, switching on a robust ECL signal. By synergistically integrating the high specificity of the aptamer-CRISPR system with the immense signal amplification of the HCR scaffold, all under the control of a charge-dominant switch, our biosensor achieves an outstanding limit of detection of 0.121 fg/mL and a broad linear range from 1 fg/mL to 100 pg/mL. Its successful application in spiked food samples validates its practicality and robustness, presenting a powerful new paradigm for designing minimal-background, high-gain ECL sensors for mycotoxin determination.
Section
Graphical
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Scientific Reports
·
2017
·Vol. 7
·期 1
·1-14
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IF 4.259
Lu Jian,Shao Yanjiao,Qin Xuan,Liu Daozhi,Chen Ang,Li Dali,Liu Mingyao,Wang Xin
Cytochrome P450 (CYP) 3A accounts for nearly 30% of the total CYP enzymes in the human liver and participates in the metabolism of over 50% of clinical drugs. Moreover, CYP3A plays an important role in chemical metabolism, toxicity, and carcinogenicity. New animal models are needed to investigate CYP3A functions, especially for drug metabolism. In this report, Cyp3a1/2 double knockout (KO) rats were generated by CRISPR-Cas9 technology, and then were characterized for viability and physiological status. The Cyp3a1/2 double KO rats were viable and fertile, and had no obvious physiological abnormities. Compared with the wild-type (WT) rat, Cyp3a1/2 expression was completely absent in the liver of the KO rat. In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats. The Cyp3a1/2 double KO rat model was successfully generated and characterized. The Cyp3a1/2 KO rats are a novel rodent animal model that will be a powerful tool for the study of the physiological and pharmacological roles of CYP3A, especially in drug and chemical metabolism in vivo .
研究领域:
药理学
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遗传学
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毒理学
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Processes
·
2026
·Vol. 14
·期 9
·1347
·
IF 3.4
Zhuoling Ding,Kadireya Tuerxun,Xueqing Luo,Shishui Zhou
γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter, is known for its physiological functions in alleviating anxiety and improving sleep. Currently, high-yielding GABA food products are mainly obtained through screening wild-type high-producing strains (e.g.,Saccharomyces cerevisiaeisolated from Sichuan pickles yielding 0.67 g/L) or employing co-culture systems (e.g.,Enterococcus faeciumandLactiplantibacillus plantarumreaching 6.35 g/L). While effective, these methods often rely on natural screening strains or multi-microbial interactions. This study employed CRISPR-Cas9 technology to knockout theUGA1gene inSaccharomyces cerevisiae, a key gene responsible for GABA degradation. Starting from the low higher alcoholSaccharomyces cerevisiaeSY-LH, we successfully constructed the recombinant strain SY-LHU. Remarkably, this study discovered a significant upregulation ofGAD1gene expression followingUGA1knockout, which further enhanced GABA synthesis capacity. Under optimal fermentation conditions (inoculum size 4 × 107cells/mL, wort concentration 10 °P, sugar addition 60 g/L, 30 °C for 10 days, and mixing the malt broth every 48 h), the validation fermentation was performed and the GABA content in the wort beverage reached 280.36 mg/L, representing a 385.4% increase compared to the pre-optimization level. Furthermore, sensory evaluation by a trained panel yielded a mean score of 88, with no significant off-flavors detected, demonstrating the product’s high consumer acceptance. This pioneering work provides a novel and feasible technical pathway for developing functional alcoholic beverages with sleep-aiding properties.
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