Construction Of Surface Enhanced Raman Scattering (SERS)biosensing Platform Based On Nucleic Acid Signal Amplification And Its Application In Detection Of Cancer Biomarkers

Surface-enhanced Raman scattering?SERS?has the advantages of light stability,non-destructiveness,single-molecule level super sensitivity,and rich spectral characteristics.It has been successfully used in biomedical,clinical diagnosis,environmental monitoring,and food safety control research field.SERS-based biomedical diagnosis is essential for early biomedical monitoring and postoperative monitoring.Although various SERS active substrates can be used for biomolecular sensing and have great potential in biological detection,the plasma SERS effect can cause very large changes in the enhancement effect due to uneven distribution of hot spots,different positions and directions of signal molecules,and nanoscale changes in the shape of metal nanoparticles,and these problems often interfere with the sensitivity and accuracy of biological detection.Therefore,a potential strategy to solve these problems is to develop ultra-sensitive SERS detection based on isothermal nucleic acid amplification technology.Aiming at the above problems,this article designed multiple signal amplification strategies,combined with a variety of nucleic acid signal amplification methods,using the excellent optical properties of nanocomposite materials,noble metal core-shell nanomaterials,to construct highly sensitive and easy-to-operate for cancer biomarkers detection biosensing platform,the specific research content includes the following two aspects:?1?In this study,enzyme-free strategy for signal amplification of target circulation was introduced,which can combine the silver-dissolving reaction mediated by hydrogen peroxide mimics DNA enzyme for the construction of SERS biosensing platform to achieve CEA sensitive detection.First,under the effect of the inhibitory chain,the hairpin structure will not cause downstream chain replacement amplification.Only in the presence of the target CEA,the enzyme-free target amplification reaction can be accurately triggered,which can produce catalytic hemin/G-quadruplex HRP-mimicking DNAzymes,degrade H₂O₂ into hydroxyl radicals,and the strong oxidizing ability will corrode the carbon nanodots-silver nanoparticle?AgNPs-CDs?substrate.The SERS substrate avoids the complex manipulation of modifying Raman signaling molecules and cleverly uses the unique Raman peaks of CDs as detection signals to achieve sensitive detection of CEA.This SERS biosensing platform based on the dual signal amplification strategy simply and efficiently achieves a 1:N target signal amplification effect with a detection limit of 55 pg/mL,which provides a new idea for the construction of a platform with multiple signal amplification strategies.?2?In this study,the signal amplification strategy of concatenated hybridization chain reaction?C-HCR?was introduced into the SERS biosensing platform,and the core-shell nanomaterial Au@AgNRs synthesized by enzyme induction was used as the SERS substrate to construct the SERS platform.First,the aptamer chain probe for total prostate antigen?tPSA?was designed to have the ability to recognize the antigen at one end and can trigger the C-HCR on the other.In the C-HCR,two HCRs,one proceeds with two hairpins and the other with four biotin-modified hairpins,are coupled,making the formation of a hyperbranched DNA nanostructure with a length of more than 200 nm and a firecracker shape.This isothermal nucleic acid amplification structure can bind more than four hundred million streptavidin modified alkaline phosphatase?SA-ALP?.SA-ALP catalyzes2-phospho-l-ascorbic acid trisodium salt?AAP?to produce ascorbic acid?AA?,which will reduce silver nitrate?Ag NO₃?to form silver element deposits on gold nanorods?AuNRs?,forming a SERS substrate Au@Ag core-shell nanostructure with excellent signal amplification ability.In view of these advantages,the developed method achieves ultra-sensitive detection of PSA with a detection limit as low as 0.94 fg/mL,and has successfully achieved the detection of serum samples from clinical patients,which is consistent with clinical test results,indicating that it may have huge potential in clinical applications.In summary,in this study,we used isothermal nucleic acid amplification technology as an efficient auxiliary signal amplification strategy,with the aid of nanomaterials with excellent properties,a simple,sensitive and efficient SERS biosensing platform was constructed,which realized ultra-sensitive detection of cancer biomarkers,and established the basis for preoperative screening,intraoperative monitoring and postoperative precontrol of cancer patients.