Real-time enzyme-digesting identification of double-strand DNA in a resonance-cantilever embedded micro-chamber.

Abstract

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.