Preparation Of Functional DNA Nanoprobes And Their Application In Tumor Marker Detection

microRNA(mi RNA) is an important tumor marker and its dysregulated expression is closely associated with tumorigenesis.Due to the low level of mi RNA expression in tumor cells,there is an urgent need to develop ultra-sensitive detection methods.Fluorine-modified DNA probes are widely used for mi RNA detection for their simplified design and fast response.However,conventional fluorescent DNA probes do not involve fluorescent signal enhancement and have limited ability to detect low levels of mi RNAs.Surface-enhanced Raman spectroscopy(SERS)is a non-destructive analytical method that plays an important role in the detection of biomolecules.This paper combines mesoporous silica nanomaterials(MSNs),nanometal-enhanced fluorescence technology and SERS technology to construct a functional DNA nanoprobe based on Boolean logic"AND"operation to achieve ultra-sensitive detection of mi RNA and identification of tumor cells.This paper contains the following two sections:(1)A hybridization chain reaction(HCR)-based signal amplification strategy is constructed for the sensitive detection of mi R-21 in solution using enhanced fluorescence and SERS signals.The hairpin probe DNA2 sequence is ligated with nanosilver(Ag NPs),and the hairpin probe DNA3 is labeled by ROX fluorescent molecules.In the absence of mi R-21,the fluorescence is effectively burst due to the close proximity between the ROX fluorescent molecule and the black hole quencher(BHQ).Then,in the presence of mi R-21,DNA2-Ag and hairpin DNA3-ROX-BHQ begin to hybridize,generating DNA long strands by the HCR reaction.The HCR product allows the aggregation of ROX fluorescent molecules(also Raman reporter molecules)and Ag NPs,the metal-enhanced fluorescence(MEF)effect and localized surface plasmon resonance(LSPR)effect of Ag NPs cause significant enhancement of fluorescent signals and Raman signals.This protocol provides an effective method for the sensitive detection of low expression mi R-21.(2)A Boolean logic"AND"based nanomachines(FOMNs)are designed to achieve ultra-sensitive detection of mi R-21 in cells.First,the hairpin probe DNA3-ROX-BHQ is loaded into the MSNs well and encapsulated using DNA1 in order to prevent leakage.DNA1 with telomeric extensions can extend autonomously in the presence of telomerase and d NTPs and then form a rigid hairpin structure off the MSNs surface.In the presence of both telomerase and mi R-21,the hairpin structure of DNA2-Ag is disrupted by mi R-21 and hybridized with the released DNA3-ROX-BHQ.Ultrasensitive detection of mi R-21 in solution can be achieved by recording fluorescent signals and SERS signals with a limit of detection(LOD)of 5.0×10^-13 M and5.0×10^-15 M.Intracellularly,the fluorescence signal allows the typing of different tumor cells and the localization of mi R-21;more importantly,we have made a more sensitive determination of mi R-21 expression levels in single cells by SERS technology.