Surface-Enhanced Raman Scattering Based Detection Methods For Tumor Biomarkers

Tumor markers are markers of tumors in specific physiological states.They are quantifiable indicators that can provide potential mechanistic information about disease occurrence.Therefore,determining their levels is crucial for early diagnosis and treatment of diseases.In recent years,SERS has gained widespread attention in the life sciences.Establishing a SERS-based method for detecting tumor markers is the focus of this thesis.This thesis focuses on prostate cancer protein biomarkers and breast cancer nucleic acid biomarkers.It constructs a SERS-based biosensing chip and method by addressing the preparation of SERS substrates,detection strategies,and pattern designs.This approach achieves ultra-sensitive,accurate,and highly selective detection of both types of tumor markers.1.Aptamer chips based on PS@Ag substrates for PSA detectionThe reproducibility of SERS spectra is essential for its detection accuracy.In this chapter,patterned PS@Ag materials were prepared by magnetron sputtering silver(Ag)film onto polystyrene colloidal microspheres(PS)using magnetic control sputtering technology.Utilizing this as a SERS substrate,an aptamer chip based on SERS was developed for the detection of prostate cancer biomarkers,including f-PSA and t-PSA.Before PSA detection,complementary DNA chains(SH-DNA)and PSA aptamers are stepwise immobilized on the PS@Ag substrate.Raman probe molecules,methylene blue(MB),are introduced through the interaction with purine bases on the aptamer.Recognition binding between PSA and the aptamer leads to aptamer release,resulting in a decrease in the quantity of MB on the substrate.The Raman peak intensity of MB is correlated with the concentration of PSA.The prepared aptamer chip exhibits high spectral reproducibility,selectivity,and sensitivity.The feasibility of this method has been validated by determining PSA levels in the serum of prostate cancer patients.2.Gene chips based on PS@Ag substrates for BRCA1 and BRCA2 detectionTo simplify the preparation process of the aforementioned PS@Ag substrates,a SERS gene chip for the detection of breast cancer biomarkers BRCA1 and BRCA2 genes was developed by directly adsorbing Ag NPs onto a PS template based on electrostatic adsorption.First,two types of detection probes with a gold(Au)core and silver(Ag)shell structure were synthesized.They were internally embedded with R6 G and MBA Raman probe molecules,externally connected to the corresponding probe DNA chains(Probe DNA)for target gene recognition.When the target gene is present,the capture DNA chains(Capture DNA)on the substrate and the detection probes bind to their corresponding target genes,forming a "sandwich" structure.Simultaneous detection of BRCA1 and BRCA2 is achieved by collecting the intensity of different Raman characteristic peaks of R6 G and MBA.The embedded Raman probe molecules are sandwiched between the Au layer and the Ag layer,ensuring the stability and reliability of the spectra.The feasibility of this method for multiple detections was validated by determining BCRA1 and BCRA2 levels in human serum.3.Magnetic immunoassay method based on signal amplification strategy for ultra-sensitive PSA detectionThe concentration and variations of disease biomarkers are crucial for disease diagnosis and monitoring.As most biomarkers have low concentrations in body fluids,introducing a signal amplification strategy significantly enhances detection sensitivity.In this chapter,a SERS-based magnetic immunoassay method is proposed by introducing a signal amplification strategy.The assay system consists of biotin-modified aptamers(B-Apt)and PSA antibodies(Ab)labeled on magnetic beads(MBs).In the presence of target PSA,it can be immobilized and captured.Sequentially adding streptavidin and signal probes,the streptavidin-biotin complex reacts to form a three-dimensional network,increasing the number of signal molecules and achieving signal amplification.This results in ultra-sensitive detection of PSA,with a detection limit as low as 0.87 pg/m L.4.Immunoassay method based on dual-mode of Raman shift and intensity for f-PSA% detectionThe ratio of free prostate-specific antigen(f-PSA)concentration to total prostatespecific antigen(t-PSA)concentration,i.e.,f-PSA%,is a more accurate prostate cancer biomarker.This chapter combines two detection modes,Raman shift and intensity,to develop a SERS immunochip for the determination of t-PSA,f-PSA,and their concentration ratio.First,4-mercaptobenzoic acid(MBA)on the immunocapture substrate is coupled with t-PSA antibodies.The binding of t-PSA induces a shift in the MBA Raman characteristic peak,allowing for quantitative determination of t-PSA based on the peak shift.Subsequently,an immunocolloidal gold with Raman probe molecules,5,5-Dithiobis(succinimidyl-2-nitrobenzoate)(DSNB),and f-PSA antibodies is introduced onto the immunocapture substrate for the immune reaction.Finally,quantitative determination of f-PSA is achieved by collecting the intensity of the DSNB Raman characteristic peak.The concentration ratio of t-PSA and f-PSA,i.e.,f-PSA%,can be calculated from the concentrations of t-PSA and f-PSA.f-PSA and t-PSA show good linear responses in concentration ranges of 0.120 ng/m L and 1200 ng/m L,respectively.This method combines Raman shift and intensity detection,simplifying the detection steps of f-PSA% while enhancing the accuracy of the results.