DNA Amplification Technology-Assisted Silicon-Based SERS Aensors For Biochemical Analysis Application

Surface-Enhanced Raman scattering(SERS),as an important analytical detection technology,has several unique advantages,including high sensitivity,anti-fluorescent interference,and multi-component detection ability and so on.At present,SERS technology has been widely used in the field of chemistry,materials science,biology,and medicine.The excellent SERS substrate is the basis and prerequisite for widespread application.Silicon nanohybrids-based SERS substrates have attracted extensive attentions due to their better SERS enhancement effect and reliable signal reproducibility.In recent years,as a convenient signal amplification method,isothermal DNA amplification(e.g.,rolling circle amplification,DNA hybridization chain amplification,and Exo III enzyme-assisted DNA amplification)has been widely used in the analysis and detection of biomedicine fields.By combining DNA amplification technology with detection methods such as fluorescence,colorimetry,electrochemistry,and SERS,researchers have successfully achieved highly sensitive detection of samples such as DNA,RNA,proteins,and small molecule organics.Therefore,this paper combines the silicon nanohybrids-based SERS substrates with isothermal DNA amplification technology,with an aim to construct two kinds of SERS sensors for the analysis and detection of DNA methyltransferase(MTase)and microRNA(miRNA).The details are as follows:In the first part,by combining the polyadenine(PolyA)assembly and rolling circle amplification(RCA)technology,a SERS sensor based on a dual amplification strategy is constructed for the detection of the activity of M.SssI,a cytosine-guanine dinucleotide(CpG)methyltransferase.In the presence of M.SssI,the rolling circle amplification reaction is triggered.The long and single-stranded DNA(ssDNA)fragments are hybridized with multiple DNA signal probes(modified with dye molecules(Cy3))to form double-stranded DNA(dsDNA).As well,a kind of SERS substrate made of silver(core)-gold(satellite)nanocomposites-modified silicon wafer(Ag-Au NPs@Si)is prepared by polyadenine assembly technology.Finally,the resultant dsDNA is conjugated to the Ag-Au NPs@Si substrate,to achieve the SERS detection of DNA MTase.Of particular significance,the developed SERS sensor displays an ultrahigh sensitivity with a low limit of detection of 2.8×10-3 U/mL.The sensor also has a wide detection range,which can detect DNA MTase samples in the concentration range of 0.05 to 50 U/mL.In addition,the SERS sensor has good selectivity and signal reproducibility,and the relative standard deviation is less than 12%.Taking advantages of these merits,the developed SERS sensor is feasible for detection of M.SssI with various concentrations spiked in real samples and the signal recovery ranges from 99.6%to 107%.In the second part,by combining a silicon-based SERS substrate with Exonuclease?-assisted DNA amplification technology,a SERS sensor is constructed for miRNA detection.Firstly,silver nanoparticles modified silicon wafer(AgNPs@Si)substrate is prepared,in which AgNPs are in situ grown on silicon wafer through hydrofluoric acid-assisted galvanic displacement.And DNA signal probes are fixed to the substrate by Ag-S bond.In the presence of the target miRNA,the DNA recognition probe hybridizes with the target miRNA.The original 3'end of the DNA recognition probe is a sticky end,and exonuclease ?(Exo ?)has no catalytic activity on it.The 3'end of the hybridized DNA recognition probe becomes a blunt end,which can be recognized and hydrolyzed by Exo? to trigger Exo ?-assisted DNA amplification reaction.The amplification products are paired with the DNA signal probes fixed on the AgNPs@Si substrate,so that the dye molecules labeled on the signal probes are far away from the substrate,resulting in changes of Raman signals.The SERS sensor has high sensitivity with a low limit of detection of 1 aM.And the developed SERS sensor also has good signal reproducibility and selectivity.In addition,the sensor combined with the PDMS microfluidic chip is able to detect multiple miRNAs simultaneously.In summary,two kinds of SERS sensors are constructed by combining SERS substrate and enzyme-assisted DNA amplification technology.One is a dual signal amplyfing SERS sensor based on the combination of RCA and PolyA assembly technology,suitable for highly sensitive detection of DNA MTase.The other is a microfluidic SERS sensor constructed by combining a microfluidic silicon-based SERS substrate and Exo ?-assisted DNA amplification technology,capable of sensitive detection of miRNA.