Isothermal Cyclic Amplification Detection Of Active Substances In Cells

With the development of medical treatment,the average lifetime of human beings has be extended.However,the threat of cancer to human beings has become increasingly prominent and the cancer has gradually become the first cause of death for Chinese residents.The survival of cancers patients is strongly associated with the stage of the tumor diagnosis.Therefore,early diagnosis of cancers is essential for human health.In this thesis,we combined the advantages of the nano-particles and the common amplification method to detect the active substances in cancers cells in order to realise the early diagnosis of cancers.The telomerase and miRNA may function as universal biomarkers for cancer diagnosis because of the different concentration of miRANs and telomerase between the cancer cells and normal cells.In this thesis,we have developed different biosensors for detecting telomerase and miRNA in cells.This thesis mainly includes three aspects as following:(1)We have developed a new approach for detecting miRNA based on strand displancement amplification and hybridization chain reaction.With the assistant of polymerase and nicking endonuclease(NEase),target miRNA combined with the single stranded template DNA to generate a great amount of trigger DNA which can induce HCR.Consequently,the proposed DNA amplification strategy combining with the inherent high sensitivity of the SERS technique enabled low detection limit of miRNA.With high sensitivity,universality,rapid analysis,and high selectivity,this method has a great potential in bioanalysis and clinical biomedicine for detecting biomolecules with trace amounts.(2)We have designed an efficient strategy based on signal amplification to detect telomerase activity.The strand displacement amplification and the catalytic hairpin assembly can generate more MB-DNA and thus connect more Raman probes to amplify the signal.At the same time,the nanoparticles can improve intensity of the Raman signal and the magnetic beads can remove the redundant Raman signal.This method is simple,sensitive,and no need of PCR,which may be promising to provide a clinical protocol for cancers diagnosis and contribute to the ultimate goal of improvinghuman health.(3)We designed a new biosensor for detecting telomerase based on the specific structure of the triple helix and the fluorescence resonance energy transfer between nanoparticles with Cy5.In this strategy,target identification,enzyme amplification and signal transduction can be accomplished at the same time,which make the whole strategy much easier.This method mainly consists of two parts: the first part is the construction of the triple helix.The fluorescence intensity of Cy5 was reduced when near the gold nanoparticle due to the special structure of the triple helix,however,the fluorescence signal was restored in the presence of telomerase.The second part is the catalytic hairpin assembly reaction,which can increase the intensity of the fluorescence signal.Compared with the traditional method,this method is easier to operate and it might open a new perspective for developing a highly selective,stable and sensitive cancers diagnosis and treatment system.