As one of most widespread toxic heavy metal ion,mercury ion (Hg2+) causes so many adverse health effects that highly selective Hg2+ assay in aqueous media are urgently needed.It is reported that Hg2+ is able to interact with thymine in DNA aptamer [1].Not only specific G-rich DNA aptamer with thymine/thymine mismatches can complex with Hg2+,but can further bond hemin to form peroxidase-like DNAzymes as well,which enhances the luminol-H2O2 chemiluminescence reaction[2].In this work,a Hg2+ microassay methodology based on G-quadruplex DNAzyme chemiluminescence microfluidic biosensor has been proposed.Two G-rich ssDNAs,5-TCGCTTGTGGAGGG-3and 5-GGGACGGGTTGCG A-3,were employed and both of the molecules can be divided into three parts.Part Ⅰ(in italic) is G-rich sequence that can fold into G-quadruplex [3]; part Ⅱ (with underline) is able to form T-Hg2+-T mismatch and stabilize G-quadruplex structure; part Ⅲ of the four base pair used to form DNA duplex between the two ssDNAs.The experimental result showed that,in the present of mercury ion,thymine -Hg2+- thymine interaction were favorable for DNA duplex formation from the above ssDNA,which further promoted G-rich segment approaching closer to fold into a bi-molecular G-quadruplex,G4.ds(T-T),as it was called.Once hemin was added and coordinated with G-quadruplex,the complex catalyzed H2O2 mediated oxidation of luminol to produce chemiluminescence.The chemiluminescence intensity increase with the increase of the Hg2+ concentration.In the study,the effect of relevant parameters on chemiluminescence intensity,such as reaction temperature,pH of the solution,incubation time and chemiluminescent reagent concentration,had been investigated.Under the optimized conditions,the G4.ds(T-T)/hemin DNAzyme showed relative good catalytic properties that peroxidase-like activity increased about 8 fold in the presence of 400 nM of Hg2+.Compared with previous colorimetric reports,sample consumption of the present microfluidic chemiluminescence biosensor is only 3 μL.Moreover,this chemiluminometric microflow injection procedure,further developed from our previous work [4],is simple,automatic with a high throughput of 65 h-1.