| Sensitive detection of tumor biomarkers is of great importance for clinical diagnosis and prognosis evaluation of multiple diseases.However,tumor biomarkers usually present in clinical samples at extremely low abundance,it is essential to develop simple,rapid,specific methods for sensitive detection of tumor biomarkers.Isothermal amplification technology has the advantages of high amplification efficiency and mild reaction conditions,and has been widely applied to detect various tumor biomarkers.In this thesis,we develop isothermal signal amplification-based biosensors for sensitive detection of tumor biomarkers including flap endonuclease 1（FEN1）and legumain（LGMN）.The detail is described as follows:（1）We demonstrate label-free and homogeneous detection of FEN1 based on ligation-promoted hyperbranched rolling circle amplification（HRCA）.The structure-specific endonuclease FEN1 participates in various genome maintenance pathways in eukaryotes including base excision repair,DNA replication,and maintenance of telomeres.The aberrant expression of FEN1 has been linked to a variety of human diseases.FEN1 has become a potential biomarker for diseases diagnosis and therapeutic.In this assay,a flapped padlock probe hybridizes with an assistant probe to form a circular DNA substrate with a 5′-flap for the recognition of FEN1.When FEN1 is present,5′-flap of circular DNA substrate is cleaved by FEN1,and the nicked padlock probe with 5′-phosphate is linkable by Taq DNA ligase to form a circular padlock probe,which can serve as a template to initiate HRCA reaction in the presence of Vent（exo-）DNA polymerase,primers 1 and 2.The generated ds DNA fragments with varying length can stain with SYBR Green I to generate an enhanced fluorescence signal.Due to the high specificity of FEN1-induced cleavage and the high amplification efficiency of HRCA reaction,the proposed method exhibits good specificity and high sensitivity with a detection limit of 1.51×10-6 U/μL.It can be performed isothermally and homogeneously without the involvement of costly specific antibodies,fluorophore-labeled probes,complicated nanomaterial synthesis,high-precision temperature cycling and additional separation steps.In addition,this method can also be used to screen FEN1inhibitors and detect FEN1 activity quantitatively in human cancer cells,holding great potential applications in the clinic diagnosis and drug discovery.（2）We develop a fluorescent biosensor based on magnetic separation and cyclic enzymatic repairng amplification for ultrasensitive detection of LGMN.LGMN is involved in multiple important life processes such as protein catabolism and antigen presentation.Aberrant expression of LGMN is associated with a variety of human diseases.Therefore,there is an urgent need to develop efficient methods for sensitive detection of LGMN suitable for early diagnosis of related neoplastic diseases.In this assay,when LGMN is present,the substrate（DNA-peptide conjugate）can be specifically cleaved,generating trigger.After magnetic separation,the resultant triggers can hybridize with the template to initiate the UDG/Endo IV-assisted cyclic enzymatic repairng amplification,producing more and more triggers.The resultant triggers can hybridize with the signal probes,initiating Endo IV-mediated cyclic cleavage of signal probes to release large amounts of Cy3 fluorescent molecules.LGMN activity can be quantified by simply measuring the released Cy3 fluorescent molecules.The improved sensitivity of this assay can be ascribed by the good specificity of LGMN-induced cleavage,high efficiency of cyclic enzymatic repairng amplification,and Endo IV-mediated cyclic cleavage of signal probes.This assay exhibits high sensitivity,and can be performed without complex thermal cycling process.Moreover,this method can sensitively detect LGMN acticity in colon cancer cells at the single-cell level,and it can be further applied to screen LGMN inhibitors,holding great potential in clinical diagnosis and biomedical research. |
PDF Full Text Request