Real-time enzyme-digesting identification of double-strand DNA in a resonance-cantilever embedded micro-chamber.
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.
State Key Lab of Transducer Technology and Science and Technology on Microsystem Lab, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai 200050, China. xxli@mail.sim.ac.cn.
Tiegang
T
Xu
Haitao
H
Yu
Pengcheng
P
Xu
Wangjie
W
Xu
Wenqing
W
Chen
Chuanzhao
C
Chen
Xinxin
X
Li
D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D019453:Escherichia coli O157 / Q000737:chemistry* / Q000235:genetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods
D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases