Construction Of Fluorescent Biosensors And The Applications In The Detection Of NAD~+ And DNA

Biomolecules include nucleic acids,proteins,enzymes,and small biological molecules.Abnormal concentration of biomolecules is closely related to a variety of human diseases and cancers.Hence,it is important to determine biomolecules in the fields of biochemistry and clinical diagnosis.The conventional methods for biomolecular assays usually have disadvantages such as long analysis time,low sensitivity,and complicated operation steps.In addition,low-abundant analytes are difficult to be accurately quantified by the conventional detection methods.There is an urgent need to develop simple and sensitive analytical tools for quantitative detection of biomolecules.Biosensors have the advantages of high sensitivity,good selectivity,and fast analysis speed,with wide applications in the fields of biochemistry and clinical diagnosis.In this thesis,we develop new fluorescent methods for sensitive detection of small biomolecule nicotinamide adenine dinucleotide(NAD~+)and Human T-lymphotropic virus types I and II DNA(HTLV-?/? DNA).The main contents are outlined below:(1)A new fluorescent method is developed to simultaneously detect HTLV-I DNA and HTLV-II DNA on the basis of RNase H-mediated cyclic cleavage of signal probes.We designed Cy3-labeled signal probe 1 and Cy5-labeled signal probe 2,which can be assembled on the magnetic beads(MBs)to form signal probe-MBs.The signal probes can separately hybridize with HTLV-?/? DNA to form DNA-RNA duplex.Then Ribonuclease H(RNase H)can specifically cleave the RNA of DNA-RNA duplex to initiate cyclic cleavage of signal probes,releasing large amounts of Cy3 and Cy5 fluorescent molecules.The released Cy3 and Cy5fluorophores can be quantitatively measured by single-molecule detection,with Cy3 indicating HTLV-I DNA and Cy5 indicating HTLV-II DNA.This method exhibits high sensitivity with a detection limit of 66.1 a M for HTLV-I DNA and 82.8 a M for HTLV-II DNA.Moreover,this method can be applied to simultaneously detect HTLV-I DNA and HTLV-II DNA in Hu T-78cells,providing a new approach for biomedical research and early clinical diagnosis.(2)A trifunctional split dumbbell probe coupled with ligation-triggered isothermal rolling circle amplification is designed for label-free and sensitive detection of nicotinamide adenine dinucleotide.In this assay,a single trifunctional split dumbbell detection probe simultaneously acts as a probe for NAD~+recognition,a template for RCA reaction,and a substrate for SYBR Green I binding.Upon the addition of NAD~+,the E.coli DNA ligase is activated to catalyze the ligation of 5?-phosphoryl terminus(5?P)and 3?-hydroxyl terminus(3?OH)of the split dumbbell probe,forming a closed dumbbell probe.The resultant circular dumbbell probe can serve as a template to trigger RCA reaction through phi29 DNA polymerase-catalyzed elongation of single stranded primer,generating numerous copies of dumbbell probes which can be simply monitored by using SYBR Green I as the fluorescent indicator in a label-free manner through the binding of SYBR Green I with the double-stranded stem region of dumbbell probe amplicons.This method shows high sensitivity and good selectivity.The limit of detection is calculated to be 85.6 f M.This method can be applied for accurate and sensitive detection of NAD~+in complex biological samples and cancer cells.