Construction Of Signal Amplification-Based Enzymatic Biosensors For Biomedical Applications

The maintenance of genome stability and integrity is essential for all organisms.DNA damage may lead to genomic instability,premature aging,developmental disorders and the high risk of cancer.DNA repair enzymes are involved in DNA damage repair.Their main functions are to specifically recognize DNA damage caused by various internal or external factors and to repair them through different repair ways.The abnormal expression of DNA repair enzymes is closely related to aging,cardiovascular disease,human immune deficiency,neurodegenerative diseases and even cancers.They are regarded as the important biomarkers for a variety of diseases including cancers.The conventional methods for the detection of DNA repair enzymes include enzyme-linked immunosorbent assay?ELISA?,gel electrophoresis in combination with radioactivity labeling,high performance liquid chromatography?HPLC?,mass spectrometry?MS?and polymerase chain reaction?PCR?-based amplification.These methods may inevitably suffer from poor sensitivity,radioactive pollution,complex and expensive instruments,long analysis time,and tedious operation steps,greatly limiting their further clinical applications.Therefore,it is highly desirable to develop a simple,rapid and ultra-sensitive methods for the detection of various DNA repair enzymes,facilitating basic biomedical research and early clinical diagnosis.In this thesis,we use telomerase,uracil DNA glycosylase?UDG?,human alkyladenine DNA glycosylase?hAAG?and human 8-oxoguanine DNA glycosylase?hOGG1?as the models to develop a series of simple,sensitive and selective methods for simultaneous detection of a variety of DNA repair enzymes in combination with isothermal amplification technology,fluorescence spectrum analysis,single-molecule detection technology,quantum dots?QDs?and magnetic beads.The detailed contents are as follows:?1?We develop a triple-amplification strategy for the sensitive detection of telomerase activity from cancer cells using telomere-based primer generation-triggered rolling circle amplification?RCA?in combination with enzyme-assisted cyclic signal amplification.This protocol includes following four steps:?1?a telomere extension reaction,?2?apurinic/apyrimidinic endonuclease?APE1?-assisted cyclic cleavage of assistant probes for the generation of abundant primers,?3?a RCA reaction,and?4?APE1-catalyzed cyclic cleavage of signal probes for the generation of an enhanced fluorescence signal.In the first step,the telomerase substrate?TS?primer is recognized and elongated by the telomerase extracted from HeLa cells,and a number of telomeric repeat units?TTAGGG?are incessantly added to the 3'end of the primer to form a long single-stranded DNA?ssDNA?.In the second step,the NH₂-modified assistant probe with an AP site can completely hybridize with the resultant extended product to form a stable double-stranded DNA?dsDNA?.APE1 prefers to cut the AP site in dsDNA,leading to the breakage of assistant probe and the generation of a new DNA primer with a free 3'-OH end,but APE1 exhibits no activity towards the AP site in ssDNA.Notably,the resultant extended product may subsequently hybridize with new assistant probes to initiate the cyclic cleavage processes and generate abundant new DNA primers.In the third step,the new DNA primer with a free 3'-OH end may hybridize with the circular template to initiate a RCA reaction in the presence of Bst DNA polymerase,generating a large number of long repetitive ssDNAs that are complementary to the signal probes.In the fourth step,the signal probe hybridizes with the RCA product to form a DNA duplex with an AP site,initiating APE1-induced cyclic cleavage of signal probes to release numerous fluorophores.In this research,each resultant extended telomeric product can induce the generation of abundant new DNA primers which can trigger numerous RCA reactions to produce extremely long ssDNA molecules.Each RCA amplification product has many repetitive sequence units and each repetitive unit can induce cyclic cleavage of abundant signal probes to release numerous fluorophores,eventually resulting in an amplified fluorescence signal,which can be used for quantitative detection of telomerase even in single cell.?2?We construct a single quantum dot-based nanosensor with multi-layer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase?hAAG?using APE1-assisted cyclic cleavage-mediated signal amplification in combination with the DNA polymerase-assisted multiple Cy5-mediated fluorescence resonance energy transfer?FRET?.The hAAG recognizes the I/T base pairs and cleaves the N-glycosidic bond between the sugar and the hypoxanthine base,releasing the hypoxanthine base to form an AP site.Then APE1 cleaves the AP site,leading to the break of hairpin probe into two portions,generating a trigger.The resultant triggers can hybridize with the AP probes to form the AP probe/trigger dsDNAs.Subsequently,APE1 enzyme induces cyclic cleavage of dsDNAs,releasing the triggers and a large number of primers with 3'-OH.The released primers can initiate the polymerization with the biotinylated capture probe as the template in the presence of Klenow Fragment,Cy5-dATP,dCTP,dGTP and dTTP,generating stable dsDNAs with the incorporation of multiple Cy5molecules.These biotin-/multiple Cy5-labeled dsDNAs can self-assemble onto the QD surface via specific biotin-streptavidin binding to form the QD-dsDNA-Cy5 nanostructure.Under the excitation of 405 nm,efficient FRET occurs with the 605QD as the donor and Cy5 as the acceptor,and the Cy5 signals can be simply measured by total internal reflection fluorescence?TIRF?microscope for the quantification of hAAG activity.In contrast to the typical QD-based FRET approaches,the assembly of multi-layer of multiple Cy5 molecules onto a single QD significantly amplifies the FRET signal.We further verify the FRET model with one donor and multi-layer acceptors theoretically and experimentally.This single QD-based nanosensor can sensitively detect hAAG with a detection limit of as low as 4.42×10^-12 U/?L.Moreover,it can detect hAAG even in single cancer cell,and distinguish cancer cells from normal cells.Importantly,this single QD-based nanosensor can be used for kinetic study and inhibition assay,and it may become a universal platform for the detection of other DNA repair enzymes by designing appropriate DNA substrates.?3?We develop a single-molecule detection method for simultaneous measurement of hOGG1and UDG based on excision repair-initiated endonuclease IV?Endo IV?-assisted signal amplification.We design two assistant probes modified with two 8-oxoG bases and five uracil bases,which can hybridize with two trigger probes to form the DNA duplex substrates for hOGG1 and UDG,respectively.In addition,we design two magnetic bead-conjugated signal probes,with one being modified with an AP site and labeled with Alexa Fluor488?AF488?for hOGG1 assay and the other being modified with an AP site and labeled with cyanine 5?Cy5?for UDG assay.The presence of hOGG1 and UDG may induce the base excision repair reaction to release the corresponding trigger probes from the detection probes.The released trigger probes may hybridize with the corresponding signal probes,respectively,initiating the Endo IV-assistant cyclic cleavage of signal probes.After magnetic separation,the released fluorophores in the supernatant can be accurately quantified by single-molecule detection,with AF488 indicating the presence of hOGG1 and Cy5 indicating the presence of UDG.This method is very sensitive with a detection limit of as low as 1.892×10^-5 U/mL for hOGG1 and 1.736×10^-5 U/mL for UDG,and it can even detect multiple DNA glycosylases at the single-cell level.Moreover,this method can be applied for simultaneous measurement of enzyme kinetic parameters and the screening of both hOGG1 and UDG inhibitors,holding great potentials in biomedical research and clinical diagnosis.?4?We demonstrate a simple and sensitive method for simultaneous detection of multiple DNA repair enzymes based on the integration of single-molecule detection with RCA-driven encoding of different fluorescent molecules.The hAAG and UDG are used as the target analytes.We design a bifunctional dsDNA substrate with a hypoxanthine base?I?in one strand for hAAG recognition and an uracil?U?base in the other strand for UDG recognition,whose cleavage by APE1 generates two corresponding primers.The resultant two primers can hybridize with their respective circular templates to initiate RCA,resulting in the incorporation of multiple Cy3-dCTP and Cy5-dGTP nucleotides into the amplified products.After magnetic separation and exonuclease cleavage,the Cy3 and Cy5 fluorescent molecules in the amplified products are released into the solution and subsequently quantified by TIRF-based single-molecule detection,with Cy3 indicating the presence of hAAG and Cy5 indicating the presence of UDG.This strategy greatly increases the number of fluorescent molecules per concatemer through the introduction of RCA-driven encoding of different fluorescent molecules,without the requirement of any specially labelled detection probes for simultaneous detection.Due to the high amplification efficiency of RCA and the high signal-to-ratio of single-molecule detection,this method can achieve a detection limit of 6.10×10^-9 U/mL for hAAG and 1.54×10^-9 U/mL for UDG.It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and the screening of DNA glycosylase inhibitors,holding great potential in early clinical diagnosis and drug discovery.