Enzymatic Signal Amplification Based Electrochemical Sensing Method For Detection Of Tumour Markers

Early screening and treatment of cancer have important influence on the improvement of survival rate.Accurate quantitative testing of tumour markers can help with early screening and post-operative monitoring.Tumour markers are certain substances produced and released by tumour cells,often in the form of nucleic acids,antigens and enzymes in body fluids,and are a class of substances that can reflect the presence of tumour cells.Traditional assays can only detect morphological species in the mid to late stages of cancer,or when the abundance of a marker in a sample such as blood reaches a certain level.In recent years,with the improvement of detection technology and the introduction of various molecular probes,sensing platforms and amplification tools,rapid,sensitive and immediate quantitative detection of tumour markers in blood has become a reality.Electrochemical sensing technology plays an important role in real-time detection based on the advantages of fast signal response and low cost.The specific research content of this work is as follows:(1)An exonucase Ⅲ driven electrochemical sensor was designed for the efficient detection of micro RNA-155 combined with multiple signal amplification strategies.In the presence of miRNA-155,DNA hairpin probes with exposed bases at both ends are recognised,binding to them to form flat ends that can be hydrolysed by nucleic acid exonuclease III.The resulting single-stranded DNA proceeds to the next cycle where further hydrolysis produces more single-stranded DNA.The end product can initiate a hybridised chain reaction on the electrode surface,using methylene blue as an electrical signal indicator for electrochemical detection.Under optimal reaction conditions,the logarithm of the target concentration has a good linear relationship with the current signal,with a linear range of 0.1 f M ～ 0.1 n M and a detection limit as low as 0.035 f M.(2)Electrochemical biosensors based on Exo III for sensitive detection of circulating tumor DNA(ct DNA).Ct DNA is released from various parts of tumor cells through apoptosis and necrosis,is present in blood and body fluids and is an important tumor marker.In the presence of ct DNA,it can be hybridized with the template complex strand.After exonuclease III hydrolyzes the complex along the 3 ’-5’ line,the shed target can be combined with other complex strands for further hydrolysis.The remaining portion of the composite chain after cutting can also form a hairpin structure and continue to be hydrolyzed by exonuclease III,which produces more single-stranded DNA and is hydrolyzed by hybridization with stem loop DNA fixed on the electrode surface.The open stem loop introduces single-stranded DNA labeled with ferrocene(ss DNA-Fc),which generates an electrical signal.Under optimal conditions,the logarithm of ct DNA concentration has a good linear relationship with the electrical signal,the concentration range is 50 f M ～ 10 pM,and the detection limit is as low as 16 fM.