Study And Application Of New Biomarkers Detection Approaches Based On Surface-enhanced Raman Scattering

In recent years,the prevalence of cancer and chronic diseases has increased.Therefore,early and accurate detection of disease is very important.Biomarkers,which can be used to objectively measure and evaluate some characteristic biochemical indexes under specific physiological conditions,are very important in the prediction,diagnosis,monitoring,treatment and prognosis evaluation of diseases.Biomarkers mainly exist in tissues(cells)or body fluids.The common biomarkers can be summarized into two types:nucleic acid and protein(polypeptide).Although there are many traditional methods used in clinical diagnosis,most of them still lack of sensitivity and specificity.As a non-invasive means of detection,liquid biopsy is widely used in clinical detection.However,biomarkers in body fluids are of low abundance,and the sample matrix is complex,which means the more sensitive and specific detection methods are needed in liquid biopsy.Molecular imprinting technique(MIT)is an effective method to improve the specificity of the detection method.The molecularly imprinted polymer(MIPs)prepared by this technique is an artificial receptor with the function of target recognition and have specificity to the target molecule.MIPs have been widely used in drug delivery,sensor and disease diagnosis.In recent years,we have developed a series of boron affinity molecular imprinting techniques for the identification of cis-dio containing biomolecules.Recently,we have developed a boron-affinity anchored epitope controllable oriented surface imprinting technique,and successfully prepared MIPs for the recognition of non-glycoprotein.This method broadens the application of MIPs in the field of protein recognition and provides a specific method for biomarker recognition and detection.Surface-enhanced Raman scattering(SERS)is a detection technique of high sensitive and non-destructive,which is widely used in disease diagnosis.With the further research on the mechanism of SERS,more and more methods based on Raman detection have been proposed.In recent years,in order to achieve high sensitivity detection,we have developed two methods,i.e.plasmonic immunosandwich assay(PISA)and plasmonic affinity sandwiches assay(PASA),by utilizing plasmonic enhanced Raman scattering(PERS)generated between rough gold substrate and metallic nanoparticles.The sensitivity of these methods can reach the single molecule level.In this work,we presented two works on the specific recognition and ultrasensitive detection of biomarkers.On the one hand,for the detection of micro RN A,which is a type of bio marker in serum,we presented a core-satellite structure labeling probe based-PASA for rapid and ultrasensitive detection of circulating miRNA in human serum.Using miR-21 as the target,we verified theoretically and experimentally that due to the multiple hot spots around the core-satellite nanocomposites,the sensitivity of such labeling nano compe site-based PAS A is 3 orders of magnitude higher than that only silver Raman nanotags-based PAS A.The quantitative limit of this method is 10'14 M.By using the PAS A approach,the quantitative determination of miR-21 in human serum samples was achieved,which allowed for differentiation of a breast cancer patient from a healthy individual.This method can be easily extended for other microRNAs and circulating tumor DNA.Therefore,the core-satellite structure labeling probe-based PAS A method holds great promise for cancer diagnosis.On the other hand,we combine the boron affinity molecular imprinting technique with surface-enhanced Raman scattering technique for the detection of C-peptide,which is a biomarker of diabetes in body fluid.A dual epitope-specific MIP-based plasmonic immunosandwich assay was designed for the rapid,sensitive and specific detection of C-peptide in body fluid.In this method,the N-terminal and C-terminal epitope(12-amino acid)of C-peptide were selected as the characteristic fragments of C-peptide and saccharified by solid-phase synthesis.Preparation of N-terminal epitopedouble imprinted polymers and C-terminal epitope-double imprinted polymers by controllable oriented surface double imprinting of boronate affinity-anchored epitopes.The N-terminal epitope-double imprinted polymers with high selectivity and Raman labeling function were based on Ag/PATP@SiO2 nanoparticles(NPs)and the Cterminal epitope-double imprinted polymers with high selectivity and rapid separation capability were based on magnetic nanoparticles(MNPs).Dual MIPs can further enhanced the specificity for C-peptide,and with portable Raman spectrometer this approach can realized rapid enrichment,separation and Raman labeling of C-peptide.The method has high sensitivity,the limitation of quantitative is 1 pg/mL,and the analysis speed is fast.It takes only 20 minutes for each determination.Therefore,this method holds good promise for the diagnosis of diabetes mellitus,and also provides a new method for the detection of protein and polypeptide biomarkers.