Metal And Non-metal Surface-enhanced Raman Spectroscopy Biosensor Based On Enzyme-assisted Signal Amplification For MicroRNA Detection

Surface-enhanced Raman spectroscopy（SERS）,a detection method with the advantages of high sensitivity,high specificity,low sample loss and fast detection speed,has great potential in the field of biological detection and material science.Biosensors based on this technology have received extensive attention in recent years for detection of single molecules or disease markers.During the construction of these biosensors,the application of enzyme-assisted signal amplification strategy can effectively improve its sensitivity and performance.And the development of substrate materials with better enhancement effect is the fundamental to improve the detection capability of biosensors.Thus,we designed two enzyme-assisted signal amplification strategies,synthesized nanomaterial-based noble metal and semiconductor enhanced substrate,constructed different SERS biosensors to detect microRNA.The details of this paper are as follows:1.A novel recyclable surface-enhanced Raman spectroscopy platform with duplex-specific nuclease signal amplification for ultrasensitive analysis of microRNA 155Due to its complex structure,the nanomaterial used in novel SERS platform is often difficult to realize recycling.And the non-specific contact between the enhanced substrate and signal molecules often leads to the high background signal in detection.Thus,a recyclable SERS biosensor was fabricated for ultrasensitive detection of miRNA 155 by using magnetic substrate（Fe₃O₄@PDA/Pt）and duplex-specific nuclease signal amplification（DSNSA）strategy.Here,the Fe₃O₄@PDA/Pt was used as SERS platform to immobilize toluidine blue Raman reporter and capturing DNA（S1）,in which polydopamine（PDA）could avoid the aggregation of Fe₃O₄ and enhance the biocompatible of platform.When Au nanoflowers（AuNFs）modified probe DNA（S2）was partly hybridized with S1,an initial strong Raman signal（“on”status）was obtained by the approaching between AuNFs and TB.In the presence of target microRNA 155,they could completely hybridize with S2,which led to the isolation of S2 from substrate.Under the assistance of DSN enzyme,the S2 in the DNA/RNA duplex was hydrolyzed to release target miRNA 155,which could trigger another cycle.Thus the AuNFs were departed from TB with an obviously decreased signal.More important,with the application of AuNFs-modified S2,the Raman reporter was continuously assembled on the substrate throughout the detection.After the adding of S2,the S1 could re-hybridize with S2,which led the SERS biosensor return to“on”status,therefore the regeneration of biosensor was easily realized.The result shows that this biosensor could achieve a wide linear range from 1 fmol/L to 10μmol/L and the detection limit was 0.28 fmol/L,which was expected to be used in the clinical applications2.The surface-enhanced Raman scattering effect of anatase/rutile TiO2nanoparticlesThe noble metals,such as Au and Ag,with special morphology are often used as enhanced substrate in SERS.But this kind of material often has disadvantages of complex preparation process,weak stability and simple enhancement mechanism.Herein,anatase/rutile composite titanium dioxide nanoparticle was synthesized through simple hydrolysis and calcination for plasmon-free surface-enhanced Raman scattering of toluidine blue.This effect is associated with the synergistic combination of the charge transfer between the signal molecule and the substrate surface and the multi-light scattering between the substrate materials,so it has different response to laser with different excitation wavelength.Even the amorphous TiO2 has the same enhancement effect as normal Au NPs.And a better enhancement effect can be achieved by the recombination of anatase and rutile phase titanium dioxide,because of the improvement of the charge transfer efficiency between the molecule and the substrate,which can be further improved due to the recombination of different valence zone structures.To the best of our knowledge,this work is the first time,who synthesized the dual-crystal phase composite semiconductor SERS substrate.