Sers Platform Based On Raman Shift For Circulating Tumor DNA Detecting And Mechanism Study

Surface-enhanced Raman scattering(SERS)has found prosperous applications in fields such as disease diagnosis,environmental surveillance,and food safety supervision.This paper introduces our research about SERS platform based on Raman shift for circulating tumor DNA(ct DNA)detecting and mechanism study.In chapter 1,we summarize the applications for the frequency-shift assay platforms in various kinds of markers,including proteins and nucleic acids of disease,small molecules,bacteria and virus,and cells.Then,we divide indirect SERS assay platforms into two types: the traditional “sandwich” SERS assay and the frequency-shift assay.Compared to the traditional “sandwich” SERS assay,the frequency-shift assay platforms are easier to prepare.In addition,the requirement for only one single reaction step is one of the major advantages,which greatly simplifies the detection procedure.The frequency-shift SERS method shows great potential for the application in the field of biosensing.In Chapter 2,we explore an analytical method of ct DNA based on the frequency-shift assay.ct DNA is a promising noninvasive biomarker for the early diagnosis of cancers.However,it is challenging for accurate and sensitive detection of pico-to-femtomolar serum concentration of ct DNA,especially in the presence of its analogues which produce strong background noise.Herein,a DNA-r N1-DNA mediated SERS frequency-shift assay is developed.This sensing platform features in both the designed hairpin DNA-r N1-DNA probe for specific ct DNA recognition and the employed RNase HII enzyme which specifically hydrolyzes DNA-r N1-DNA/ct DNA hybrid and thus allows ct DNA recycling in the system to realize signal amplification.The detection system shows sub-femtomolar level sensitivity in phosphate buffered saline(PBS)solution and shows 0.01% specificity that represents sensitive detection of ct DNA with one single base-pair mutation(KARS G12 D mutation)from the normal ones(KARS G12 D Normal)of lung cancer.In Chapter 3,we remove the interference of amount of protein and chloridion ions from serum,and find the sensing platform above function well in both Fetal Bovine Serum(FBS)and human physiological media.In particular,the sensitive assay of ct DNA in serum samples from lung cancer patients is achieved,suggesting its high potential applications in clinical settings for early diagnosis and prognosis of lung cancer.In Chapter 4,we use nucleic acids to study the mechanism of Raman shift in the frequency-shift assay.As an emerging SERS assay platform,the frequency-shift assay shows potential for nucleic acid markers detection.However,the mechanism of Raman shift in nucleic acid detection procedure has not been reasonably explained.Based on this,we use the nucleic acid molecules to study the effect of nucleic acid length and stiffness changes on Raman shift values due to their precise molecular programmability.This study achieves a preliminary conclusion that it is mass and stiffness changes from nucleic acids hybridization alter the vibrational frequencies of Raman molecules,which thus affects the Raman frequency shift values in the nucleic acid-based Raman shift assay platform.In Chapter 5,we make a conclusion and outlook of this research.In general,we explore an analytical method of ct DNA based on the frequency-shift assay,which achieves the detection of ct DNA and the diagnosis of lung cancer.Then,we use nucleic acids to study the mechanism of Raman shift in the frequency-shift assay.Finally,we propose the prospects further that the Raman frequency-shift mechanism needs to be further explored.In addition,by combining nucleic acids which prosesses precise controllability,more diverse applications of SERS detection platform based on Raman frequency-shift have potential to be developed further.