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来源:生物磁珠专家 2023-12-23 18:03:02      点击:

Zheng, L., Jiang, Y., Huang, F. et al. A colorimetric, photothermal, and fluorescent triple-mode CRISPR/cas biosensor for drug-resistance bacteria detection. J Nanobiotechnol 21, 493 (2023). https://doi.org/10.1186/s12951-023-02262-x


A colorimetric, photothermal, and fluorescent triple-mode CRISPR/cas biosensor for drug-resistance bacteria detection

Laibao Zheng, Yayun Jiang, Fuyuan Huang, Qiaoli Wu & Yongliang Lou 
Journal of Nanobiotechnology volume 21, Article number: 493 (2023) Cite this article

Abstract

A multimodal analytical strategy utilizing different modalities to cross-validate each other, can effectively minimize false positives or negatives and ensure the accuracy of detection results. Herein, we establish a colorimetric, photothermal, and fluorescent triple modal CRISPR/Cas12a detection platform (CPF-CRISPR). An MNPs-ssDNA-HRP signal probe is designed to act as a substrate to trigger three signal outputs. In the presence of the DNA target, MNPs-ssDNA-HRP is cleaved by the activated CRISPR/Cas12a, resulting in the release of HRP and generating short DNA strands with 3-terminal hydroxyl on magnetic beads. The released HRP subsequently catalyzed TMB-H2O2 reaction and oxidized TMB is used for colorimetric and photothermal signal detection. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), the remaining short DNA strands are used as primers to form poly-T and function as scaffolds to form copper nanoclusters for fluorescent signal output. To verify the practical application of CPF-CRISPR, we employed MRSA as a model. The results demonstrate the platform’s high accuracy and sensitivity, with a limit of detection of 101 CFU/mL when combined with recombinase polymerase amplification. Therefore, by harnessing the programmability of CRISPR/Cas12a, the biosensor has the potential to detect various drug-resistant bacteria, demonstrating significant practical applicability.
利用不同模态相互交叉验证的多模态分析策略,可以有效减少误报或假阴性,保证检测结果的准确性。在此,我们建立了一个比色、光热和荧光三模态CRISPR/Cas12a检测平台(CPF-CRISPR)。MNPs-ssDNA-HRP信号探针被设计为触发三个信号输出的底物。在DNA靶标存在的情况下,MNPs-ssDNA-HRP被活化的CRISPR/Cas12a切割,导致HRP的释放,并在磁珠上产生具有3-末端羟基的短DNA链。释放的HRP随后催化TMB-H2O2反应,氧化的TMB用于比色和光热信号检测。在末端脱氧核苷酸转移酶(TdT)的催化下,剩余的短DNA链用作引物形成poly-T,并作为支架形成铜纳米团簇用于荧光信号输出。为了验证CPF-CRISPR的实际应用,我们采用MRSA作为模型。结果表明,该平台具有较高的准确性和灵敏度,与重组酶聚合酶扩增联合使用时,检测限为101 CFU/mL。因此,通过利用CRISPR/Cas12a的可编程性,该生物传感器具有检测各种耐药细菌的潜力,显示出重要的实际适用性。

Experimental section

Materials and reagents

Carboxyl-coated magnetic nanoparticles were purchased from PuriMag Biotechnology Co., Ltd. (Xiamen, China). Lysostaphin and oligonucleotides (Table S1) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). 2-(N- morpholino) ethane sulfonic acid (MES), TMB, 1-ethyl-3-[3-di-methylaminopropyl] carbodiimide hydrochloride (EDC), and 4-Morpholinepropanesulfonic acid (MOPS) were bought from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). LbCas12a was purchased from New England Biolabs Inc. (United States). Terminal Deoxynucleotidyl Transferase (TdT) was bought from Takara Biotech Co., Ltd. (Dalian, China). Horseradish Peroxidase labeled streptavidin (SA/HRP) and DNase/RNase-free H2O were bought from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Copper sulfate (CuSO4·5H2O), Ascorbic acid (AA), and Sodium chloride (NaCl) were provided by Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). RAAFAST, the recombinase polymerase amplification (RPA) nucleic acid amplification kits were obtained from Qitian Gene Biological Co., Ltd. (Jiangsu, China).

羧基包被的磁性纳米颗粒购自PuriMag Biotechnology Co., Ltd.(中国厦门)。溶葡萄球菌素和寡核苷酸(表S1)购自Sangon Biotech Co. Ltd.(中国上海)。2-(N-吗啉基)乙烷磺酸(MES)、TMB、1-乙基-3-[3-二甲氨基丙基]碳二亚胺盐酸盐(EDC)和4-吗啉丙磺酸(MOPS)购自北京索拉生物科技有限公司(中国北京)。LbCas12a购自New England Biolabs Inc.(美国)。末端脱氧核苷酸转移酶 (TdT) 购自 Takara Biotech Co., Ltd.(中国大连)。辣根过氧化物酶标记的链霉亲和素(SA/HRP)和不含DNase/RNase的H 2 O购自上海百优泰生物科技有限公司(中国上海)。硫酸铜(CuSO4·5H2O)、抗坏血酸(AA)和氯化钠(NaCl)由阿拉丁生化科技有限公司(中国上海)提供。重组酶聚合酶扩增(RPA)核酸扩增试剂盒RAAFAST购自启天基因生物有限公司(中国江苏)。

Preparation of MNPs-ssDNA-HRP

Forty µL of magnetic beads (10 mg/mL) and 8 µL of 100 µM NH2-ssDNA-Biotin were added in 80 µL of MES Buffer (50 mM, pH 6.0). The mixture was incubated with shaking at room temperature for 30 min. Then, the 40 µL of the newly prepared 50 mg/mL EDC solution was added and shaken at room temperature for 4 h. The magnetic beads were washed with MES Buffer three times and isolated by magnetic decantation. 400 µL of 0.35 µg/mL SA/HRP was added in MNPs-ssDNA-Biotin and shaken at room temperature for 20 min. The MNPs-ssDNA-HRP were collected by magnetic separation and were washed with MES Buffer five times. Finally, to minimize background values, 400 µL of 0.5% BSA was added in MNPs, shaken at room temperature for 30 min, and washed 3 times with MES Buffer. Thus, MNPs-ssDNA-HRP is prepared.
在 80 μL MES 缓冲液(50 mM,pH 6.0)中加入 40 μL 磁珠 (10 mg/mL) 和 8 μL 100 μM NH2-ssDNA-生物素。将混合物在室温下振荡孵育30分钟。然后,加入40 μL新制备的50 mg/mL EDC溶液,并在室温下振荡4 h。磁珠用MES缓冲液洗涤3次,磁倾析分离。在MNPs-ssDNA-生物素中加入400 μL 0.35 μg/mL SA/HRP,并在室温下振荡20 min。通过磁选收集MNPs-ssDNA-HRP,并用MES缓冲液洗涤5次。最后,为了尽量减少背景值,在MNP中加入400μL 0.5%BSA,在室温下振荡30分钟,并用MES缓冲液洗涤3次。因此,制备了MNPs-ssDNA-HRP。


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