Exosomes And MiRNAs Biomarkers Detection Methods Based On SERS

Recently,the exosomes and miRNAs have drawn extensive attention as important tumor biomarkers.Detection of the biomarkers can provide an important basis for clinical diagnosis and treatment of cancers.On account of the sensitive and multiple detection of exosomes and miRNAs,several optical nanoprobes have been fabricated based on SERS and fluorescence.A series of novel methods have been developed with these probes,and the application prospect in clinical biomarker detection have been explored.Main results include:A SERS based method for screening and simultaneous multiple detection of exosomes using magnetic substrates and SERS nanoprobes is proposed.On one hand,the capturing substrates are made of gold shell magnetic nanoballs decorated with aptamers.On the other hand,the SERS probes are achieved using gold nanoparticles modified by a specific Raman reporter and an aptamer for recognizing target exosomes.For detection of multiple kinds of exosomes,three kinds of SERS probes are first mixed with the capturing substrates.When the target exosomes are present,they form an apta-immunocomplex with the substrate and the corresponding kind of SERS probe,leaving the other non-specific SERS probes in the suspension.Therefore,a decreased SERS intensity of the corresponding kind of probe will be detected in the supernatant,indicating the presence of target exosomes.This method can be useful in the simultaneous multiple detection of exosomes and has been successfully employed for the real blood sample detection.The multiple SERS detection is a promising method for cancer screening based on exosomes.A SERS molecular beacon is fabricated for the detection of miRNA-21.The hairpin shaped DNA is modified with a thiol group for the connection of silver nanoparticle and 6-FAM as a Raman reporter.In the beginning,the hybridization of complementary ends makes the Raman reporter close to the silver nanoparticle,so that the SERS intensity is enhanced.As the target miRNA-21 concentration increases,more beacons are opened leading to the decreased SERS intensity.The detection method can sense the low concentration of miRNA in the solution with least complexity.A microfluidic chip for the detection of miRNA using the combination of SERS and fluorescence spectroscopies is presented based on the molecular beacon.First,silver nanoparticles are immobilized on a glass slide forming a substrate.Then a specifically designed molecular beacon is modified on it.The 3' end of molecular beacon is decorated with a thiol group for the attachment to the SERS substrate,while the 5' end is labled with 6-FAM as both the fluorescence and SERS reporter.Hence the addition of target miRNA-21 simultaneously induces an opposite change of fluorescence and SERS intensities.An improved calibration curve is obtained with the combination of the two intensity-concentration curves.The miRNA detection method provides a higher sensitivity and better linearity.Moreover,the microfluidic platform can reduce the reaction time and reagent dosage.The method develops new idea in the poit of care application.A sensitive RCA based detection method for multiple exosomal miRNAs is developed.The method takes the advantages of isothermal reaction and multiplex amplification abilities of RCA.First,exosomal miRNAs from two cell lines are added with specific padlocks and universal polymerase and d NTPs for RCA reaction.The production can be directly detected using molecular beacons.Normalized fluorescence intensities of molecular beacons imply the relative concentrations of corresponding miRNAs.The obtained relative expression levels of exosomal miRNAs can be used to distinguish the type of exosomes.Such exosomal miRNAs profiling and exosomes screening method is promising in cancer diagnosis.