Development And Applications Of The Biosensors Integrating Surface Enhanced Raman Scattering And DNA Cycled Amplification Technologies

Advances in human genomics and proteomics have revealed critical roles of specific genes and proteins in disease occurrence and progression.Detection of those target molecules as potential biomarkers is of great value in diagnostics or prognostics.However,the biomarkers appear to be in extrenely low concentrations,especially at the early stage of diseases.It remains challenging to develop highly sensitive and specific detection methods for early diagnosis and personalized medicine.This thesis describes my research efforts to combine surface enhanced Raman scattering(SERS)technique and enzymatic cycling amplification technology for bioanalysis.First,I focus on the "proof-of-concept," demonstration of a sensitive platform by integrating SERS technique and Exo Ⅲ-assisted DNA amplification.I design a group of DNA sequences to construct the assay.Capture DNAs are immobilized on the SERS substrate(Au NPs@Si)via the Au-S bonds.A single copy of the target DNA can initiate many cycles of cleavage reaction of the probe DNA with assistance of Exo III,leading to generation of multiple copies of the residual DNA.Subsequent hybridization of the residual DNA with the capture DNA on the Au NPs@Si substrate allows for generating intensive SERS signals.I have demonstrated that the target DNAof MnSOD gene in the concentration as low as 1 aM can reproducibly be detected.It offers a detection limit several orders of magnitude better than the previous reports in the literature.The new biosensor exhibits an excellent specificity in differentiating DNA sequences with a single-base mismatch.As a robust,flexible,and ultrasensitive approach,it promises important applications in clinical diagnostics and DNA identification where only a very limited amount of the biological sample is available.My second project moves the above concept forward by the development of a powerful method to detect target cell membrane proteins.In this new design,the incubation of aptamer sgc8 with CCRF-CEM cells enables high-affinity binding to the target PTK7 protein on the cell membranes.The binding events can trigger conformational alteration of the sgc8 aptamer;thus exposing a short sequence as the target DNA for initiation of Exo Ⅲ-assisted amplification.Therefore,detection of the low-abundance PTK7 protein can be transformed into the accumulated DNA signals by Exo Ⅲ-assisted amplification and SERS technique.In my preliminary tests,CCRF-CEM cells as few as 1000 cells mL-1 can effectively be detected by this new approach.I have demonstrated robust performance of the integrated platform using tumor cell-spiked blood samples.This method may find important applications in detection of rare tumor cells or the target membrane proteins for clinical research.In conclusion,I have developed a new integrated platform based on SERS technology and Exo III enzymatic cycling amplification.It promises highly sensitive detection of nucleic acids or cell membrane proteins.My research progress provides a new analytical tool with a potential promoting disease diagnosis and clinical research.