Highly Sensitive Detection Of Heavy Metal Lead And MicroRNA Based On Enzymatic Magnetic SERS Tags

Surface enhanced Raman spectroscopy(SERS)is a powerful vibration spectroscopy technique with fingerprint characteristics,which can achieve trace detection of analytes by enhancing the Raman signal of precious metal nanostructured surface compounds.SERS technology has been widely used in bioimaging,analytical chemistry,disease diagnosis,food safety and other fields.The magnetic SERS substrate has strong Raman activity because of the advantages of the two materials,and the magnetic SERS substrate can effectively enrich from the sample and improve the SERS signal.So far,reported SERS active nanomaterials generally have the disadvantages of complex synthesis process,strong background signal,poor selectivity and so on.In response to the above problems,the combination of biosensors and SERS technology to build a new SERS sensing system has become a hot topic in research today.We used gold and silver nanoparticle assemblies prepared by nucleic acid aptamers,gold and silver nanoparticles modified by DNA molecular probes as surface-enhanced Raman substrates,and established a series of ultra-sensitive and fast new biosensors for SERS ultra-sensitive detection of heavy metal ions and tumor markers.The research content of the thesis includes the following three parts:(1)A highly sensitive SERS aptamer sensor based on DNase is used for quantitative detection of lead ions(Pb2+)in serum.First,gold nanoparticles were prepared,silver shells with different thicknesses were prepared by controlling the concentration of silver nitrate,and Au@Ag nanoparticles were prepared.Then,Fe3O4 magnetic particles were modified with polyethyleneimine(PEI),and gold/silver nanoparticles were assembled on the surface of ferroferric oxide(Fe3O4)in solution to obtain magnetic nanocomposites(Fe3O4@Au@Ag NPs).Finally,the thiolated 5’-Cy3 DNA probe was modified on the surface of Fe3O4@Au@Ag NPs,and hybridized with Pb2+ specific DNAzyme to form a SERS aptamer sensor,and used for Pb2+ quantitative detection.In the presence of Pb2+,Pb2+-specific DNAzyme bind to produce enzyme activity,which cuts the DNA probe into two segments at adenine and glyconucleotide(rA)sites,releasing Cy3 labeled ssDNA,resulting in a significant reduction in SERS signal.The SERS detection platform has a good linear relationship between 0.01 nM and 1.0 nM,and the detection limit is 5 pM.(2)Signal amplification and surface-enhanced Raman scattering based on duplex-specific nuclease(DSN)are used to detect microRNA-21(miRNA-21).Based on the double-strand specific nuclease and gold/silver nanoparticle dual signal amplification strategy,a label-free,highly sensitive and highly specific SERS biosensor was proposed.First,gold and silver nanoparticles(Au@Ag NPs)and ferroferric oxide nanoparticles(Fe3O4)were synthesized,and Au@Ag NPs were self-assembled on the surface of Fe3O4 microspheres using capture DNA chains modified at both ends to form a SERS biosensor.After adding target miRNA-21,DNA and miRNA hybridize to form heteroduplexes(DNA/RNA),which are specifically cleaved into short fragments by DSN.At the same time,miRNA-21 remains intact and can re-hybridize another DNA probe to trigger signal amplification.The method showed good linearity in the range of 0-1 nM,and the detection limit(LOD)was 0.084 fM.It can even distinguish single-base mismatch sequences or other miRNAs in the target sequence.This result provides a novel SERS method that can be successfully applied to the detection of miRNA-21 in human serum.(3)Research on SERS biosensor based on DNA modified gold nanorods and signal amplification.Based on the characteristic that DSN enzyme can specifically recognize and cleave DNA in DNA/RNA heteroduplexes and realize the amplification of circulating signals,a novel double-strand specific nuclease is proposed by taking advantage of the low background and high sensitivity of molecular beacons The auxiliary signal amplification strategy is used to detect target mRNA with high sensitivity.At the same time,the DSN enzyme has a strong single base recognition ability.In this chapter,Fe3O4@Au NRs is used as a SERS active substrate,and p-ATP is used as a Raman reporter to modify on the surface of HP@Au@Ag NPs to form the HP@APA NPs to achieve ultra-sensitive detection of the target mRNA.Based on the excellent Raman activity of the SERS biosensor,a rapid,specific,and ultra-sensitive detection method for p21 mRNA was established.Under the optimal reaction conditions,the detection linear range is 0-1 nM,and the detection limit is as low as 0.1 fM.The research results show that the SERS biosensor has high sensitivity and high specificity for the detection of small biological molecules,and is superior to the previously reported sensors.