Development Of Quantum Dot-based Fluorescence Resonance Energy Transfer Nanosensor For Biochemical Analysis

In comparison with organic dyes and fluorescent proteins,semiconductor quantum dots(QDs)have significant advantages including high brightness,high quantum yield,good stability against photobleaching,narrow emission bands and size-tunable emission spectra.In addition,the dimensions of QDs are comparable to those of biomolecules.The combination of QDs with fluorescence resonance energy transfer(FRET)has promising applications in biochemical research.In this thesis,we developed a series of new biosenors for sensitive detection of various biomolecules including transferase,circulating tumor DNA,mi RNAs,nucleases and methyltransferases based on QD-based FRET in combination with the single-molecule detection and isothermal amplification technology,microparticles and three-dimensional(3D)DNA nanostructure.This thesis contains following contents:1.Single quantum dot-based FRET nanosensor for sensitive detection of O-Glc NAc transferase(OGT)activityWe develop a single QD-based nanosensor for sensitive OGT assay.OGT is an intracellular enzyme responsible for O-linked N-acetylglucosamine(O-Glc NAc)glycosylation,and the deregulation of OGT activity occurs in cancer,diabetes,and neurodegenerative disease.We design a Cy5/biotin-modified peptide with a serine hydroxyl group for sensing OGT and a protease site adjacent to the glycosylation site for proteinase cleavage,with a universal nonradioactive UDP-Glc NAc as the sugar donor and a Cy5/biotin-modified peptide as the substrate.In the presence of OGT,it catalyzes the glycosylation reaction to generate a glycosylated peptide that is a protease-protection peptide.The resultant glycosylated Cy5/biotin-modified peptides may assemble on the surface of the streptavidin-coated QD to obtain a QD-peptide-Cy5 nanostructure in which the FRET from the QD to Cy5 can occur,leading to the emission of Cy5 which can be quantified by single-molecule detection.This method exhibits high sensitivity with a limit of detection of 3.47 × 10-13 M,and it is very simple and straightforward without the involvement of any enzyme purification,radioisotope-labeled sugar donors,specific antibodies,and the synthesis of fluorescent UDP-Glc NAc analogues.Moreover,this method can be used for enzyme kinetic analysis,quantitative detection of cellular OGT activity,and the screening of OGT inhibitors,holding great potential for further application in drug discovery and clinical diagnosis.2.Quantum dot-based fluorescence resonance energy transfer with the integration of microparticles for sensitive detection of circulating tumor DNAWe integrated the microparticles(MP)with the QD-based FRET for the sensitive detection of circulating tumor DNA(ct DNA).The ct DNA fragments carrying tumor-specific sequence alterations usually represents a variable small fraction of the total circulating DNA.We designed a double biotin-labeled link probe for the binding of streptavidin-conjugated MP and 605 nm emission QDs(605QDs).In the presence of ct DNA,the target ct DNA can hybridize with the capture and reporter probes to form the sandwich hybrids.The resultant sandwich hybrids can assemble on the surface of MP-Link-605 QD to form the MP-link-605QD-ds DNA-Cy5 complexes in which the FRET occurs from the 605 QD to Cy5,leading to the improved acceptor sensitized emission efficiency for Cy5 and the enhanced Cy5 fluorescence intensity.The Cy5 counts can be simply quantified by single-molecule detection.This assay has significant advantages of simplicity,and high sensitivity,low sample consumption,holding great potential for early cancer diagnosis.3.Integration of isothermal amplification with quantum dot-based fluorescence resonance energy transfer for simultaneous detection of multiple micro RNAsMicro RNAs(mi RNAs)are small non-coding RNAs that regulate important physiological processes,and their dysregulation is associated with various human diseases.Simultaneous sensitive detection of multiple mi RNAs may facilitate early clinical diagnosis.In this research,we demonstrate for the first time the integration of hyperbranched rolling circle amplification(HRCA)with QD-based FRET for the simultaneous detection of multiple micro RNAs with a single-color QD as the donor and two fluorescent dyes as the acceptors.We used mi R-21 and mi R-221 as target mi RNAs.We designed two circular templates which may specifically hybridize with mi R-21 and mi R-221,respectively,for the initiation of the HRCA reaction.The products of the HRCA reaction may hybridize with both capture probes and reporter probes to form the biotinylated acceptor-labeled sandwich hybrids.The resultant sandwich hybrids can assemble on the surface of the QD,enabling efficient FRET between the QD and the acceptors,with the Cy3 signal indicating the presence of mi R-21 and the Texas Red signal indicating the presence of mi R-221.This assay has significant advantages of simplicity and low cost.The HRCA reaction can be performed under isothermal conditions with the same reverse primer for different target mi RNAs,and the products of the HRCA reaction for both mi R-21 and mi R-221 can specifically hybridize with the same capture probes.This assay exhibits excellent specificity and high sensitivity with a detection limit of 7.2 × 10-16 M for mi R-21 and 1.6 × 10-17 M for mi R-221,and it can be used for simultaneous detection of multiple mi RNAs in human cancer cells,holding great potential in biomedical research and clinical diagnosis.4.Construction of tetrahedral DNA-quantum dot nanostructure with the integration of multiple fluorescence resonance energy transfer for multiplex enzymes assayWe develop a construction of tetrahedral DNA-QD nanostructure with the integration of multiple FRET for multiplex enzymes assay.We design a 3D DNA nanostructure by integrating a streptavidin-conjugated 525 nm emission QD(525QD)with a tetrahedron-structured DNA labeled with biotin,Cy3,Texas Red and Cy5,respectively.The tetrahedron-structured DNA is constructed by four oligonucleotide strands via a simple annealing process.The resultant biotin-Cy3-Texas Red-Cy5 tetrahedron DNA may subsequently conjugate to a streptavidin-coated 525 QD through specific biotin-streptavidin interaction to obtain a 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA.This 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure enables the generation of multiple FRET between the 525 QD and three dyes(i.e.,Cy3,Texas Red,and Cy5).Especially,we realize the precise and programmable arrangement of fluorophores using many tetrahedron DNA model systems,with the functionality of manipulating the energy-transfer path and FRET efficiency.The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure has well-defined dye-to-dye spacing and high controllability for energy transfer between intermediary acceptors and terminal acceptor,enabling simultaneous emission of four fluorophores for multiplexed assay.As a proof-of concept,we demonstrate the use of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure for simultaneous detection of multiple endonucleases and methyltransferases even in complex biological samples as well as the screening of multiple enzyme inhibitors.