DNA Functionalized Nanomaterials For SERS Detection Of Tumor Markers And Single Cell Analysis

Micro RNA（mi RNA）is a promising tumor marker,and the abnormal expression of mi RNA is closely related to the occurrence and development of tumors.Due to the low expression level in tumor cells,the specificity and accuracy of single mi RNA detection are not sufficient to meet the strict diagnostic criteria in clinical trials.Therefore,the simultaneous detection of multiple mi RNAs in tumor cells can not only improve the accuracy and specificity,but also avoid the waste of valuable biological samples.Meanwhile,surface-enhanced Raman technique（SERS）is widely used for mi RNA detection because of its non-destructive and ultra-sensitive nature.In this paper,we designed a dual target recognition probe（dual target recognition probe,DRP）containing a multi-hot spot gold nanocage,which combines the nonlinear hybridization chain reaction（HCR）signal amplification strategy of hairpin-free DNA with SERS technology.The DRP undergoes a nonlinear hybridization chain reaction catalyzed by the dual-target mi RNA,and finally generates a 3D multi-hot spot DNA dendrimer（3Dmh D）,realizing the simultaneous ultra-sensitive detection of two mi RNAs.The simultaneous ultra-sensitive detection of the two mi RNAs was achieved.And with the help of micro Raman imaging technology,single-cell visualization can be realized,and the spatial distribution and expression levels of two mi RNAs in tumor cells can be accurately analyzed.This thesis contains the following two main parts,and the specific work is as follows.（1）Based on the nonlinear hybridization chain reaction（HCR）,we designed a dual-target recognition probe（DRP）where the presence of the target mi RNA successfully catalyzed the multi-branched DNA hybridization chain reaction,enabling the simultaneous detection of two mi RNAs with a single probe.DRP is a novel multifunctional Raman probe with a gold nanocage core modified by two nucleic acid probes and labeled with Raman signaling molecules ROX and Cy3.The mi RNAs detection system includes super double-stranded branching DNA（F1 and G1）with Raman reporter molecules,a short toe and two single-stranded helper DNAs（f4/g4 and f7/g7）.When the trigger target mi RNAs（mi R-21 and mi R-155）are present,the multi-branched DNA reaction process is triggered.The corresponding Raman reporter molecules on the initially formed Y-type DNA are also multiplied,and in the absence of spatial effects,the multi-branched structure will grow in an exponentially growing stranded branching fashion,generating a multi-branched polymer with 2ⁿ trigger target micro RNA exposure sites after n cycles.Due to the metal enhancement effect of gold nanocages（Au NCs）,ultra-sensitive detection of mi R-21 and mi R-155 can be achieved with the help of SERS.（2）A DRP dual target recognition probe based on following an"AND"cascade logic strategy was designed to achieve simultaneous in situ detection of mi R-21 and mi R-155 in tumor cells.The gold nanocage-based core DRP probe is not degraded by enzymes and stably performs multi-branched DNA hybrid strand amplification reactions to generate 3Dmh D.The3Dmh D can significantly enhance the Raman signal for ultra-sensitive detection of the target by SERS technique.mi R-21 has a linear range of 10^-16-10^-11 M and a LOD value of 3.5×10^-17M.3.5×10^-17 M;similarly,mi R-155 has a linear range of 10^-16-10^-11 M and a LOD of 4.75×10^-17 M.In addition,laser confocal micro Raman spectroscopy showed that the DRP probe can be used for in situ visualization and analysis of tumor cells,enabling the differentiation of different tumor cells.