Novel Nucleic Acid Signal Amplification Strategies For Electrochemiluminescent Aptasensor Fabrication To Detect Mucin 1

Nucleic acid signal amplification strategy as a kind of novel amplification method,which transfers the input target molecular into masses of output nucleic acid molecular for target molecular detection with the help of nucleic acid amplification,enzyme,ribozyme and strand replacement amplification.Nucleic acid signal amplification strategy is widely used in biosensing,and its rational ultization contribute to realize the highly sensitive and selective detection of target molecular.The electrochemiluminescence?ECL?aptasensor is a kind of novel biosensor with the combination of the aptamer as recognition and the ECL technology,possessing the advantages of low cost,easy operation,quick response,high sensitivity and good selectivity.In this work,three kinds of novel nucleic acid signal amplification strategies were designed to fabricate ECL aptasensors for highly sensitive and selective detection of cancer biomarker mucin 1?MUC1?.The researches mainly includes the following aspects:1.Electrochemiluminescence aptasensor based on cascading amplification of nicking endonuclease-assisted target recycling and rolling circle amplifications for mucin 1 detectionIn this work,a novel and sensitive ECL aptasensor for MUC1 was designed based on cascading amplification of nicking endonuclease-assisted target recycling and rolling circle amplifications?RCA?.In the presence of target MUC1,Nt.BbvC I-assisted target recycling process was triggered to generate massive single-stranded primers which were available for subsequent RCA reactions on the as-prepared electrode interface.With rationally sequence designed,massive long DNA molecules with multiple G-quadruplex units were formed to generate the G-quarduplex/hemin complexes in the presence of hemin,which could enhance the ECL intensity of luminol-H₂O₂ system due to its strongly catalytic performance toward the coreactant H₂O₂.The proposed aptasenor for MUC1detection showed a limit of detection down to 7.1 fg mL^-11 and a wide linear range from 10 fg mL^-11 to10 ng mL^-11 with acceptable stability,sensitivity,and possessed promising potential applications in clinical diagnostics.2.In situ generation of electrochemiluminescent DNA nanoflowers as signal tag for mucin 1detection based on a strategy of target and mimic target synchronous cycling amplificationThe mimic target generated in most of the previously reported target conversion strategies was not able to further participate in the synchronous target cycling process,which limited the conversion efficiency to a certain extent.Herein,a sensitive ECL aptasensor for MUC1 was developed,which consisted of novel ECL signal tag and highly efficient target conversion strategy.On one hand,the novel ECL signal tag of DNA nanoflowers?DNA NFs?was in situ generated on the electrode interface by RCA,which not only simplified the operation procedures,but also increased the stability for luminophore loading.On the other hand,a highly-efficient target conversion strategy was implemented by target and mimic target synchronous cycling amplification process to improve conversion efficiencey effectively.The experimental data indicated the as-proposed biosensor had a wide linear range from 1 fg mL^-11 to 10 ng mL^-11 with a detection limit of 0.23 fg mL^-1.Significantly,the fabricated ECL aptasensor had huge potential to be applied in clinical diagnosis.3.Enzyme-freetargetrecyclinganddouble-outputamplificationsystemfor electrochemiluminescent assay of mucin 1 with MoS₂ nanoflowers as co-reaction acceleratorMost of the previously reported enzyme-free target recycling amplication strategies indicated that one mimic target was obtained in one circle of target recycling,which affected the amplication efficiency to a certain extent.In this work,a sensitive electrochemiluminescent assay of MUC1 was developed with the advantages of target recycling amplification strategy based on the acquirement of two mimic targets in one circle of target recycling and effective MoS₂ nanoflowers?MoS₂NFs?-based signal probe.Briefly,the target MUC1 triggered enzyme-free recycling and double-output amplification process was executed to acquire masses of single-stranded DNA?ssDNA?as mimic target,which further participated the catalytic hairpin assembly?CHA?process for signal amplification.Meanwhile,MoS₂ NFs were prepared as an effective co-reaction accelerator,which not only possessed excellent catalytic performance for H₂O₂ decomposion to largely enhance the luminous intensity of N-?aminobutyl?-N-ethylisoluminol?ABEI?-H₂O₂ ECL system,but also offered a desirable platform for ABEI functionalized Ag nanoparticles?ABEI-Ag complexes?loading via Ag-S binding.The experimental results showed the proposed aptasensor had a good linear relationship in the range of 1 fg mL^-11 to 10 ng mL^-11 for MUC1 detection,and the limit of detection was 0.58 fg mL^-1.In addition,the aptasensor exhibited good stability and selectivity and it had the potential to be applied in clinical research.