Preparation Of Micrometer-Sized Fluorescent Hybridization Probe And Its Application In MicroRNA Detection

Fluorescent hybridization probes have been widely used in clinical diagnosis,food safety and environmental testing due to their good biocompatibility,easy synthesis and modification.However,most of the existing fluorescent hybridization probes have problems such as low sensitivity,complicated detection operation,slow reaction speed and need enzymatic amplification,which limit their application in real samples.Herein we synthesized a micron-size fluorescent hybridization probe,and explored its potential application in microRNA detection.The details are as follows:1.A micron-size DNA fluorescent hybridization probe(?SDat)was made.Using circular DNA as a template,a long-chain sDP1 was synthesized by rolling circle amplification(RCA)using phi29 DNA polymerase.The resultant sDP1 was then used as a template to derive sDP2 by strand displacement amplification.The final probes were obtained by the self-assembly of FAM-labeled DNA onto purified sDP2.2.The characteristics of the ?SDat probe was examined.sDP2 could correctly hybridize with DNA modified with FAM,making this probe have strong fluorescence itself.This probe was about 13.5 ± 3.2 ?m in length and contained two regions,including one target binding region at the 3' end and about 1,000 fluorescent reporter regions at the 5' end.3.A paper chip device was designed based on this novel probeThe presence of a miR21 target leads to the formation of a bDCP/miR21/?SDat complex on paper,producing a strong fluorescence.A limit of detection of 0.1 pM was achieved within 30 min.Furthermore,this paper device could be used for the detection of endogenous mature miR21 in various cell lines.The obtained results were comparable to that of qRT-PCR.This study first reported the hybridization probe with micrometer length scale for microRNA detection.Furthermore,this novel probe is expected to be widely applied in chemical biology,medical diagnostics,and biosensing fields.