MICROCHIMICA ACTA
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2022
·Vol. 189
·期 10
·1-8
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IF 6.408
Du Yuwan,Han Dianpeng,An Zhaoxia,Wang Jiang,Gao Zhixian
Antibiotics have brought many benefits to public health systems worldwide since their first use in the last century, yet with their overuse in clinical treatment and livestock farming, new public health issues have arisen. Previously, we found in our experiments that the levels of macB genes in bovine raw milk ranked among the top of many drug resistance genes. In this paper, we present an analysis of regularly interspaced clustered short palindromic repeats (CRISPR) combined with surface-enhanced Raman scattering (SERS) technology for the detection of the drug resistance gene macB. The analysis was accomplished through the collaboration of the CRISPR system's ability to specifically identify genes and the more sensitive performance of the SERS. The analysis detects the drug resistance gene macB and does not yet require complex steps such as nucleic acid amplification. This method may prove to be an effective method for accurate detection of the drug-resistant gene macB, thus enabling more effective prevention of contamination of drug-resistant genes in food hygiene. Graphical abstract
研究领域:
微生物学
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分析化学
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食品科学
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基因工程
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高影响因子
MICROCHEMICAL JOURNAL
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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.
研究领域:
分子生物学
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生物化学
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纳米技术
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病毒学
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高影响因子
Synthetic and Systems Biotechnology
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2024
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IF 4.4
Bing Fu,Meng Chen,Xianfeng Bao,Jiajie Lu,Zhiwen Zhu,Fuyao Guan,Chuyang Yan,Peize Wang,Linglin Fu,Ping Yu
Vitamin B 2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.
研究领域:
生物工程
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分子生物学
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PubMed
高影响因子
MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 4
·1-8
·
IF 5.479
Chang Weidan,Liu Weipeng,Liu Ying,Zhan Fangfang,Chen Huifang,Lei Hongtao,Liu Yingju
A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA.
Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.
研究领域:
分子生物学
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植物病理学
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纳米生物技术
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BIOSENSORS & BIOELECTRONICS
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2024
·
IF 10.7
Xueying Lei,Lihua Ding,Xiaonan Yang,Feng Xu,Yongjun Wu,Songcheng Yu
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a p adlock activator which could i nhibit the t rans -cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca 2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca 2+ dependent. In the presence of Ca 2+ , DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans -cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca 2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Na + , Al 3+ , K + , Fe 2+ , and Fe 3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSD s from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca 2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
研究领域:
生物技术
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生物化学
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分析化学
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分子生物学
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DOI
PubMed
高影响因子
Analytical Methods
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2025
·
IF 2.6
Chen Lu,Jiayue Chen,Shengsheng Zhang,Yaru Tian,Xinyu Ying,Dan Zhang,Zhidan Luo,Xinxin Si,Ming Li
Two single nucleotide polymorphisms (SNPs) in the human SLCO1B1 gene, c.388A>G (rs2306283) and c.521T>C (rs4149056), are independent determinants of the efficacy and side effects of statin drugs. Multinational clinical guidelines recommend testing for SLCO1B1 genotypes before the initial use of statins. Current SLCO1B1 SNP identification methods, primarily based on quantitative fluorescence PCR, rely on expensive equipment, are time-consuming, and require cold-chain storage for reagents, making them unsuitable for use in resource-limited healthcare settings. In this study, we developed a CRISPR/Cas12b-based amplification-free genotyping technique for SLCO1B1 SNPs. Within 30 minutes of the isothermal reaction, genotyping of the c.388A>G and c.521T>C SNPs in the SLCO1B1 gene can be observed by the naked eyes under blue light. Additionally, maltodextrin was identified as an effective vitrification stabilizer for the CRISPR/Cas12b premix. A low-cost vitrification process was optimized to prepare a glass like solid reagent via room-temperature vacuum drying. The vitrified CRISPR/Cas12b reagent retained approximately 88% of its activity after 30 days of storage at 37 °C, eliminating the need for cold-chain storage and allowing for long-term preservation at room temperature. This vitrified CRISPR/Cas12b based rapid SNP detection technique is especially suitable for genotyping drug metabolism genes in primary healthcare settings, providing effective guidance for precision medicine in clinical practice.
研究领域:
分子生物学
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医学遗传学
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精准医疗
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高影响因子
CHEMICAL ENGINEERING JOURNAL
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2025
·
IF 13.2
Hongkai Wang,Yinzheng Niu,Hui Liu,Ye Zhu
Nucleic acid detection is crucial for early disease diagnosis and related clinical applications. Exploring nucleic acid amplification-free yet highly sensitive detection strategies is of significance for the development of accurate and user-friendly point-of-care testing devices. Herein, a modularized universal electrochemical sensing strategy was proposed based on the synergistic effect of CRISPR/Cas12 system and nanoporous materials. The trans -cleavage activity of Cas12a, which was activated by HPV-16 target, was utilized to cut the gatekeepers of mesoporous silica nanoparticle nanocontainers. The released methylene blue was entrapped by a mercaptobenzoic acid functionalized nanoporous gold (NPG) sensing platform. By virtue of the excellent electrocatalytic ability of NPG, ultrasensitive detection of HPV-16 was achieved over a broad linear range of 7 orders of magnitude with a detection limit as low as 0.41 fM. Moreover, determination of HPV-18 was easily achieved by only altering crRNA, revealing the remarkable universality of this strategy and its superiority of modularized design. The practical applicability of the proposed sensing strategy was verified in terms of specificity, stability, and complex biological sample analysis, demonstrating its promising prospects in the development of reliable, universal, robust, and user-friendly point-of-care diagnostic devices.
研究领域:
生物技术
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医学诊断
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纳米科学
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Jienan Shen,Zhi Chen,Ruibin Xie,Jingfeng Li,Chunyan Liu,Yaqing He,Xiaopeng Ma,Hui Yang,Zhongjian Xie
Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H 1 N 1 , H 3 N 2 , IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.
研究领域:
分子生物学
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医学诊断
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病毒学
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DOI
PubMed
高影响因子
TALANTA
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2024
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IF 5.6
Hongmin Gao,Hehua Zhang,Xue Qi,Meng Miao,Longbin Que,Xin Gu,Dong Chang,Hongzhi Pan
Accurate and reliable detection of Staphylococcus aureus ( S. aureus ) is essential for preventing infections, particularly in healthcare and food safety contexts. This work presents a novel dual-mode biosensor that integrates the CRISPR/Cas12a system with an enzyme-free isothermal amplification method for detecting S. aureus . Hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) amplify the aptamer-triggered response, significantly enhancing sensitivity. CRISPR/Cas12a′s nuclease activity is utilized in two modes: cis cleavage generates a fluorescence signal, while trans cleavage produces an electrochemical signal, enabling dual-mode detection. The biosensor demonstrates outstanding performance, with a limit of detection (LOD) as low as 5.7 CFU mL −1 in electrochemical mode and 133.7 CFU mL −1 in fluorescence mode, showcasing excellent accuracy, stability, and sensitivity. It has been successfully applied to detecting actual samples, confirming its practical applicability. This innovative approach offers a powerful tool for the swift and precise identification of S. aureus and paves the way for developing next-generation dual-mode biosensors for various analytes. Future research will aim to simplify the detection process further, making it more accessible for use in resource-limited settings.
研究领域:
分子生物学
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病原体检测
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生物传感技术
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2024
·
IF 3.1
Yan Na,Hu Zhiwei,Zhang Lingling
The development of a bio-sensing strategy based on CRISPR/Cas that is exceptionally sensitive is crucial for the identification of trace molecules. Colorimetric miRNA detection utilizing CRISPR/Cas13a-triggered DNAzyme signal amplification was described in this article. The developed strategy was implemented for miRNA-21 detection as a proof of concept. The cleavage activity of Cas13a was triggered when the target molecule bonded to the Cas13a-crRNA complex and cleaved uracil ribonucleotides (rU) in the substrate probe. As a consequence, the S chain was liberated from the T chain that had been modified on magnetic beads (MB). The G-rich sections were then exposed when the catalytic hairpin assembly between the H1 and H2 probes was activated by the released T@MB. G-rich section can fold into G-quadruplex. By catalyzing the formation of green ABTS 3 – via HRP-mimicking G-quadruplex/hemin complexes, colorimetric measurements of miRNA can be achieved visually through DNAzyme-mediated signal amplification. The method demonstrated a low limit of detection of 27 fM and a high selectivity towards target miRNA eventually. As a result, the developed strategy provides a clinical application platform for the detection of miRNAs that is both ultrasensitive and extremely specific.
研究领域:
生物技术
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分子生物学
·
诊断方法
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DOI
PubMed
高影响因子
ANALYST
·
2025
·
IF 3.3
Yang Li,Yikan Zhao,Zhihao Yi,Shitong Han
Timely and accurate diagnosis of RNA viruses, represented by the SARS-CoV-2, is the foundation for protecting people from health threats. Currently, direct detection of viral RNA genes remains the most accurate method. Herein, a rapid, ultrasensitive, and no heating equipment required CRISPR/Cas13a based evanescent wave fluorescence biosensing platform for quantitative detection of the SARS-CoV-2 gene is reported. The collateral effect of CRISPR/Cas13a for RNA is combined with a self-driven enzyme-free hybridization chain reaction (HCR) as a signal amplification step. When the initiator RNA strand is cleaved by Cas13a, the downstream signal amplification induced by HCR is blocked, and a multiple crRNA strategy is used to enhance the cleavage efficiency. The newly designed HCR assemblies are captured by the cDNA-modified optical fiber and generate a higher-intensity fluorescence signal induced by the evanescent field. The CRISPR/Cas13a-HCR evanescent wave fluorescence biosensing platform is capable of detection of SARS-CoV-2 with a LOD of 0.47 copies per μL and the detection time is within 35 min. The spike recovery tests in saliva and high specificity have demonstrated the potential of this method for point-of-care diagnosis. This method is also suitable for the detection of other RNA viruses, without the need to alter any design of the HCR component, and only the corresponding crRNA needs to be replaced.
研究领域:
生物技术
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医学诊断
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病毒学
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DOI
PubMed
高影响因子
Acta Pharmaceutica Sinica B
·
2024
·
IF 14.7
Xiaoyu Wu,Zijun Bai,Hui Wang,Hanqing Wang,Dahai Hou,Yunzhu Xu,Guanqun Wo,Haibo Cheng,Dongdong Sun,Weiwei Tao
Copper-based nanomaterials demonstrate promising potential in cancer therapy. Cu + efficiently triggers a Fenton-like reaction and further consumes the high level of glutathione, initiating chemical dynamic therapy (CDT) and ferroptosis. Cuproptosis, a newly identified cell death modality that represents a great prospect in cancer therapy, is activated. However, active homeostatic systems rigorously keep copper levels within cells exceptionally low, which hinders the application of cooper nanomaterials-based therapy. Herein, a novel strategy of CRISPR-Cas9 RNP nanocarrier to deliver cuprous ions and suppress the expression of copper transporter protein ATP7A for maintaining a high level of copper in cytoplasmic fluid is developed. The Cu 2 O and organosilica shell would degrade under the high level of glutathione and weak acidic environment, further releasing RNP and Cu + . The liberated Cu + triggered a Fenton-like reaction for CDT and partially transformed to Cu 2+ , consuming intracellular GSH and initiating cuproptosis and ferroptosis efficiently. Meanwhile, the release of RNP effectively reduced the expression of copper transporter ATP7A, subsequently increasing the accumulation of cooper and enhancing the efficacy of CDT, cuproptosis, and ferroptosis. Such tumor microenvironment responsive multimodal nanoplatform opens an ingenious avenue for colorectal cancer therapy based on gene editing enhanced synergistic cuproptosis/CDT/ferroptosis.
研究领域:
生物技术
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纳米技术
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肿瘤学
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基因编辑
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）