Real-time enzyme-digesting identification of double-strand DNA in a resonance-cantilever embedded micro-chamber.
Real-time enzyme-digesting identification of double-strand DNA in a resonance-cantilever embedded micro-chamber.
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DOI：
10.1039/c3lc51294k
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发表时间：
2014-02
期刊：
影响因子：
6.1
通讯作者：
Tiegang Xu;Haitao Yu;P. Xu;Wangjie Xu;Wenqing Chen;Chuanzhao Chen;Xinxin Li
中科院分区：
文献类型：
--
作者：
Tiegang Xu;Haitao Yu;P. Xu;Wangjie Xu;Wenqing Chen;Chuanzhao Chen;Xinxin Li
提出了一种在谐振悬臂式嵌入式微流控芯片中实时酶消化直接鉴定双链DNA的新方法。这种新的基因水平检测方法有望取代传统的基于DNA杂交的基因检测方法，这种方法不仅存在非特异性吸附诱导假阳性的问题，而且还需要复杂的单链DNA制备和杂交。由于检测到的DNA链具有特定限制性内切酶的唯一切割位点，因此通过谐振式微悬臂梁传感器的频移信号可以在线准确地记录切割质量(代表消化片段的长度)。通过对大肠杆菌O157：H7的stx2基因的实验鉴定，证实了这种酶消化技术。利用自制的悬臂梁嵌入型微流控芯片对直接聚合酶链式反应样品进行了直接分析。通过生物素-链霉亲和素结合固定化3776bpDNA，在10min内记录到增加的质量下降15.9 kHz。然后，用EcoRV-酶切位点2635bp，在6min内以10.2 kHz的频率实时检测截断质量。检测到的频移率为15.9/10.2=64.2%，与截断片段与DNA全链的长度比(2635/3776=69.8%)一致。实验验证了该方法简单、准确的双链检测方法。
A novel direct identification of double-strand DNA is proposed by using real-time enzyme-digestion in a resonant-cantilever embedded microfluidic chip. The new gene-level detection method is expected to replace the conventional DNA-hybridization based gene-detection that suffers from not only nonspecific adsorption induced false-positives but also complicated single-strand DNA preparation and hybridization. Since a detected DNA chain features a unique cutting site for a certain restriction-enzyme, the accurately cut-off mass (representing the length of the digested segment) can be online recorded by the frequency-shift signal of the resonant micro-cantilever sensor. This enzyme-digestion technique is confirmed by experimental identification of the stx2 gene of E. coli O157:H7. The direct-PCR sample is directly analyzed by using our lab-made cantilever-embedded microfluidic-chip. The 3776 bp DNA is immobilized via biotin-streptavidin binding and the added mass is recorded by a frequency-decrease of 15.9 kHz within 10 min. Then, with EcoRV-enzyme digestion at the site of 2635 bp, the cut-off mass is real-time detected by a frequency-increase of 10.2 kHz within 6 min. The detected frequency-shift ratio of 15.9/10.2 = 64.2% is consistent with the length ratio between the cut-off fragment and the whole DNA chain (2635/3776 = 69.8%). Hence, the simple and accurate double-strand detection method is verified experimentally.