Ultrasensitive detection of NDM-1 resistant bacteria based on signal amplification with sandwich-type LNA electrochemical biochips
Ultrasensitive detection of NDM-1 resistant bacteria based on signal amplification with sandwich-type LNA electrochemical biochips
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基于夹心型LNA电化学生物芯片信号放大的NDM-1耐药菌超灵敏检测
DOI：
10.1016/j.snb.2019.127556
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发表时间：
2020-03
影响因子：
8.4
通讯作者：
Wang Yunxia
中科院分区：
文献类型：
--
作者：
Zhang Liqun;Liang Wenbin;Ran Qiaosheng;Liu Fei;Chen Dong;Xiong Yu;Jiang Dongneng;Li Yi;Tu Dianji;Luo Xiaoge;Pu Xiaoyun;Wang Yunxia
新德里金属β-内酰胺酶-1（NDM-1）是一种已鉴定的金属β-内酰胺酶，可对碳青霉烯类和除氨曲南外的所有其他β-内酰胺类抗生素产生耐药性。本文基于氧化锌（ZnO）和还原的氧化石墨烯（rGO）复合物（ZnO@rGO），以Au纳米笼（Au NCs）为载体，构建了一种新型的电化学生物芯片，用于NDM-1的早期临床诊断的超灵敏检测。在生物芯片表面合成了一种Au NC和一种巯基修饰的锁核酸-1（Au NC@LNA-1）探针。在杂交溶液中，Au NC与修饰有SH基团的LNA-2探针形成稳定的Au NC@LNA-2复合物。Au NC@LNA-1和Au NC@LNA-2通过互补碱基配对与NDM-1连接以形成Au NC@LNA-1/NDM-1/Au NC@LNA-2“类”复合物，并且该复合物可以结合靶序列的额外拷贝以进一步放大其检测信号。在优化的条件下，该体系的线性范围为1 ~ 100 μ g L-1，检出限为0.042 pg L-1。生物芯片探针修饰时间为45 min，孵育时间为30 min，电化学扫描检测时间为1 min。此外，结果表明，特异性LNA探针可识别单碱基错配，并对靶DNA分子表现出亲和力，从而无需PCR扩增即可直接检测复杂临床细菌样品中的NDM-1。总之，这种新的电化学生物芯片方法允许快速，特异性和灵敏度的检测NDM-1，表明其在疾病诊断中的潜在应用。
New Delhi metallo-beta-lactamase-1 (NDM-1) is an identified metallo-beta-lactamase that confers resistance to carbapenems and all other beta-lactam antibiotics, with the exception of aztreonam. Herein, a novel electrochemical biochip was constructed based on a zinc oxide (ZnO) and reduced graphene oxide (rGO) complex (ZnO@rGO) supported by Au nanocages (Au NCs) for signal amplification for ultrasensitive detection of NDM-1 in early clinical diagnosis. A Au NC and a sulfhydryl (SH) modified locked nucleic acid-1 (Au NC@LNA-1) probe was synthesized with stable Au-S bonds to on the biochip surface. In hybridization solution, Au NCs and LNA-2 probes modified with SH groups formed stable Au NC@LNA-2 complexes. Au NC@LNA-1 and Au NC@LNA-2 were linked with the NDM-1 to form a Au NC@LNA-1/NDM-1/Au NC@LNA-2 "sandwich-like" complex through complementary base pairing, and this complex can bind additional copies of the target sequence to further amplify it's the detection signal. Under optimized conditions, the system showed a linear range from 1 pg L-1 to 100 mu g L-1, and the detection limit was 0.042 pg L-1. The time of biochip probe-modification was 45 min, incubation was 30 min, and electrochemical scanning detection was 1 min. In addition, the results indicate that the specific LNA probe identified single base mismatches and exhibited affinity for the target DNA molecule, resulting in the direct detection of the NDM-1 in complex clinical bacteria samples without PCR amplification. In conclusion, this novel electrochemical biochip method allows for the rapid, specific, and sensitive detection of the NDM-1, indicating its potential application in disease diagnostics.
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