Study Of New Methods For Detection Of Enzymes Activity Based On Extension Of DNA

DNA extension is usually used as an effective mean of signal amplification for biosensor.And strategies based on extension of DNA have the advantages of sensitive,simple operation and easy signal readout,making it widely used in many analytical science.Reported methods of T4PNK is always complex,time-consuming and costly.In this thesis,we reported a TdT-triggered extension of surface DNA for the assay of T4PNK,which is simple,sensitive and cheap.Besides we extended the application of DNA extension.DNA strand extension is used as a molecular switch to control the DNA strand displacement reaction,at the same time,realizing sensitive and simple detection of telomerase.The major contents of the thesis were as follows:1.Study of New Methods for Detection of T4 polynucleotide kinase Activity.In this work,we report a TdT-triggered extension of surface DNA for the assay of DNA 3'-phosphatases(take T4PNK as an example).Specially,substrate DNA probes with 5' thiol terminals and 3' phosphoryl terminals are immobilized on gold electrode surface via gold-thiol chemistry.In the presence of T4PNK,the 3'-phosphoryl end of the substrate DNA is hydrolyzed to a hydroxyl group,followed by addition of a long DNA sequence on its 3' terminal hydroxyl in the mixing solution containing both TdT and deoxythymidine triphosphates(dTTPs).Thus,the long ssDNA nanotail product can adsorb amounts of hexaammineruthenium chloride(RuHex)to the electrode surface via electrostatic interaction,producing amplified electrochemistry signal.Consequently,a wide linear range from 1mU/mL to1U/mL can be achieved with a low limit of detection(LOD)of 1 mU/mL.Also of note,the strategy has the advantages of simple operation,low price,high sensitivity,making it possible to be used in some resource-limited area in the future.2.Study of New Methods for Detection of Telomerase Activity.In this work,we report a DNA extended-based strategy for the detection of telomerase.The primer sequence of the telomerase with 5'quenching group and the complementary nucleotide chain with 3' fluorescent group have the domains complementary to each other,and the complementary nucleotide chain contains additional unpaired domains(CCCTAA),which can used as toehold.After the formation of duplex,the fluorescence was quenched.Invading strand can initiate DNA displacement reaction through toehold binding,recovering the fluorescence.In the presence of telomerase,the telomerase reaction products could paired with toehold,which can hidden toehold and suppress DNA displacement reaction.By measurement of the intensity of fluorescence spectra,the telomerase activity was detected with a wide linear range from 8 × 10-10 to 8 × 10-5 IU and the low limit of detection(LOD)is 8 ×10-10 IU.It is not only a strategy for telomerase detection but also a new method to control DNA displacement.