Synthesis,Characterization And Application Of Nanoparticles For MiRNAs Detection Based On SERS

MicroRNAs?miRNA?is a class of small non-protein-coding single-stranded RNAs,which is widely found in humans and animals.Many studies have shown that abnormal miRNA expression is closely related to a variety of diseases and is an ideal tumor marker.Currently,existing detection methods for miRNA have many limitations,such as complicated operation,high cost,and easy occurrence of fluorescence bleaching and spectral overlap.Therefore,based on the surface-enhanced Raman scattering?SERS?spectral detection technology,this study intend to develop a method for detecting target miRNA with high sensitivity,high throughput,simple and fast by using SERS nano-metal labeled particles,for the early diagnosis of diseases.During the detection using SERS spectrum technology,sensitivity and reproducibility were always two key indicators,so we carried out some research work on these two topics,and the specific results were as follows:1.Developed a new method for SERS detection of miRNA based on magnetic capture.Firstly,we synthesized the magnetic particle Au@MNPs and characterized its core-shell nanostructure by means of TEM,DLS and XRD.The results show that the average particle size of Au@MNPs is about 27nm and the size distribution is narrow.Among them,the thickness of Au shell is about 7nm,the average size of MNPs is about 13nm,and its optical properties are close to pure GNPs,the surface of MNPs is almost completely covered by Au coating.In addition,under the action of magnetic field,we realized the amplification of Raman signal through GNPs/miRNA-141/Au@MNPs,which is a hybrid complex formed by magnetic aggregation hybridization reaction through target sequence miRNA-141,Au@MNPs modified by captured probe,and DSNB-GNPs modified by reported probe,in a focused beam of785 nm laser.At the same time,we also reproduced the magnetic focusing phenomenon of the hybrid complex on the microfluidic chip and confirmed the feasibility of using SERS detection.Results showed that the SERS hybridization reaction on the target sequence miRNA-141 has high specificity.Through the QCM-D monitoring of hybridization compound formation on the surface of Au,The results revealed the interaction between RNA/DNA and was positively correlated with the SERS measurement results.This method raised the detection limit of miRNA-141 to100 fM,which was only 1%of the results reported in similar literature?10pM?,up to two orders of magnitude and the LOD sensitivity was increased by 100 times.2.SERS label was improved and optimized to improve its sensitivity and stability in serum samples.Firstly,we prepared the SA@GNPs nanoparticles coated with silica and bound Raman dye molecules?DSNB?,and characterized the core and shell structure of SA@GNPs by means of TEM,DLS and uv-vis.The results showed that the silica coating encapsulated the DSNB-bound GNPs in the silicon shell,where the average particle size of SA@GNPs was 57.4±9.8nm,the average particle size of GNPs was31.2±6.2nm,and the silicon shell thickness was about 13nm.The silica shell significantly improved the stability of GNPs in various solutions.When transferred from aqueous solution to 300mm sodium chloride solution,the spectrum of SA@GNPs changed very little,indicating that it was quite stable,while the"naked"GNPs had dramatic spectral changes,even precipitating rapidly.Through SERS spectrum detection,the results also indicated that the SERS spectrum of DSNB-GNPs and SA@GNPs in aqueous solution were consistent with the characteristic spectrum of DSNB.It also showed that the silica coating process had almost no effect on the SERS activity of GNPs.However,when dispersed in 300 mM sodium chloride solution,DSNB-GNPs without silica shell showed weak spectral strength,while SERS spectral strength of SA@GNPs showed no significant change,indicating that silica shell could effectively reduce the aggregation and precipitation of DSNB-GNPs.In addition,the target miRNA-141 was detected by the hybrid complex SA@GNPs/miRNA-141/Au@MNPs,which was formed by the magnetic aggregation hybridization reaction among target sequence miRNA-141,Au@MNPs modified by captured probe,and DSNB-GNPs modified by reported probe.The experimental results once again verify the feasibility of the above method.The results showed that in the serum sample environment,the LOD of miRNA-141 was 1.8pm by using SA@GNPs tag,which was nearly 110 times higher than the detection LOD?200pM?using DSNB-GNPs tag in the previous chapter.3.A new method for SERS detection of multi-channel serum miRNA based on magnetic capture was designed.Firstly,we optimized the thickness of the silica coating and characterized its structure by means of TEM,DLS and XRD.The results show that the average shell thickness of three different silica coatings is 13nm,7nm and 3nm,respectively,and with the increase of the shell thickness,the Raman molecular signal intensity embedded on SA@GNP decreases obviously.The detection results of target miRNA-141 in serum showed that the detection sensitivity of SA@GNP tag?3nm,120fM?was 1600 times higher than that of DSNB-GNPs tag?0nm,200pm?,and 15times higher than that of SA@GNP tag?13nm,1.8pm?.In addition,we also designed a method for SERS detection of multiple serum miRNA biomarkers based on magnetic capture.Using miRNA-141,miRNA-429 and miRNA-200b as target sequences,the universal captured nano-probe Au@MNPs and three SA@GNP tags targeting different target sequences?including DSNB,MB and NB Raman molecules,respectively?were synthesized and characterized.The results showed that the 1330cm^-1 peak of DSNB,the 450 cm^-1 peak of MB and the 592cm^-1 peak of NB showed obvious distinguishing characteristics in the spectrum,and they were not disturbed by each other's adjacent peaks,so they were suitable as diagnostic markers for multiple targets.The magnetic capture experiment also proved that the probes had target specificity and could effectively and accurately identify their target sequences,Finally,multiple detection tests of miRNA-141,miRNA-429 and miRNA-200b were successfully implemented,which proved the feasibility of our technical method.In a word,combining with SERS spectrum detection technology and magnetic aggregation effect of magnetic particles,we can achieve simple,sensitive and multiple detection of biological marker miRNA.Through the design and optimization of SERS label,we can improve the reproducibility and sensitivity of detection,so as to promote its application in disease early screening and prevention.