客戶采用G-COOH磁珠偶聯(lián)核酸在一區(qū)期刊Journal of Nanobiotechnology發(fā)表論文
2023-12-23 18:03:02點(diǎn)擊：
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 LouJournal 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.利用不同模態(tài)相互交叉驗(yàn)證的多模態(tài)分析策略，可以有效減少誤報或假陰性，保證檢測結(jié)果的準(zhǔn)確性。在此，我們建立了一個比色、光熱和熒光三模態(tài)CRISPR/Cas12a檢測平臺（CPF-CRISPR）。MNPs-ssDNA-HRP信號探針被設(shè)計為觸發(fā)三個信號輸出的底物。在DNA靶標(biāo)存在的情況下，MNPs-ssDNA-HRP被活化的CRISPR/Cas12a切割，導(dǎo)致HRP的釋放，并在磁珠上產(chǎn)生具有3-末端羥基的短DNA鏈。釋放的HRP隨后催化TMB-H2O2反應(yīng)，氧化的TMB用于比色和光熱信號檢測。在末端脫氧核苷酸轉(zhuǎn)移酶（TdT）的催化下，剩余的短DNA鏈用作引物形成poly-T，并作為支架形成銅納米團(tuán)簇用于熒光信號輸出。為了驗(yàn)證CPF-CRISPR的實(shí)際應(yīng)用，我們采用MRSA作為模型。結(jié)果表明，該平臺具有較高的準(zhǔn)確性和靈敏度，與重組酶聚合酶擴(kuò)增聯(lián)合使用時，檢測限為101 CFU/mL。因此，通過利用CRISPR/Cas12a的可編程性，該生物傳感器具有檢測各種耐藥細(xì)菌的潛力，顯示出重要的實(shí)際適用性。
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).
羧基包被的磁性納米顆粒購自PuriMag Biotechnology Co.， Ltd.（中國廈門）。溶葡萄球菌素和寡核苷酸（表S1）購自Sangon Biotech Co. Ltd.（中國上海）。2-（N-嗎啉基）乙烷磺酸（MES）、TMB、1-乙基-3-[3-二甲氨基丙基]碳二亞胺鹽酸鹽（EDC）和4-嗎啉丙磺酸（MOPS）購自北京索拉生物科技有限公司（中國北京）。LbCas12a購自New England Biolabs Inc.（美國）。末端脫氧核苷酸轉(zhuǎn)移酶 （TdT） 購自 Takara Biotech Co.， Ltd.（中國大連）。辣根過氧化物酶標(biāo)記的鏈霉親和素（SA/HRP）和不含DNase/RNase的H 2 O購自上海百優(yōu)泰生物科技有限公司（中國上海）。硫酸銅（CuSO4·5H2O）、抗壞血酸（AA）和氯化鈉（NaCl）由阿拉丁生化科技有限公司（中國上海）提供。重組酶聚合酶擴(kuò)增（RPA）核酸擴(kuò)增試劑盒RAAFAST購自啟天基因生物有限公司（中國江蘇）。
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 緩沖液（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。將混合物在室溫下振蕩孵育30分鐘。然后，加入40 μL新制備的50 mg/mL EDC溶液，并在室溫下振蕩4 h。磁珠用MES緩沖液洗滌3次，磁傾析分離。在MNPs-ssDNA-生物素中加入400 μL 0.35 μg/mL SA/HRP，并在室溫下振蕩20 min。通過磁選收集MNPs-ssDNA-HRP，并用MES緩沖液洗滌5次。最后，為了盡量減少背景值，在MNP中加入400μL 0.5%BSA，在室溫下振蕩30分鐘，并用MES緩沖液洗滌3次。因此，制備了MNPs-ssDNA-HRP。
更多羧基磁珠請參考 羧基磁珠|高密度羧基磁珠|聚合物羧基磁珠PuriMag G-COOH|carboxyl magnetic bead-生物磁珠專家 (purimagbead.com)
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