Sensitive Detection Of T4 Phosphatase Activity Based On The Portable Blood Glucose Meter And Graphene Oxide Biosensor

T4 polynucleotides phosphatase(T4 PNP)can hydrolyze the 3' terminal phosphate group of nucleic acid molecules into hydroxyl groups and play a very important role in the repair of the terminal damage of nucleic acid molecules.If the abnormal expression of T4 PNP is involved in normal cell growth,it will cause a series of diseases.Therefore,the detection of T4 PNP enzyme activity is of great significance for biological research and clinical diagnosis.In this paper,two protocals for detect detecting T4 PNP enzyme activity are developed by using a portable glucose meter and graphene oxide-based fluorescence sensing platform.The specific contents are as follows:First,the strategy based on portable glucose meter is developed for the detection of the activity of T4 PNP enzyme.In the experiment,the magnetic microspheres modified by Streptavidin(STV)were used as the carrier of the signal enrichment.Using the specific binding of STV and biotin(biotin),the template DNA chain labelled by the biotin in 5' terminal and the phosphate group in 3' terminal is immobilized to the surface of magnetic microspheres.Due to the catalyzing of T4 PNP enzyme,the template DNA chain with the 3 'terminal phosphoric acid group occurs dephosphorylation in the present of T4 PNP enzyme and extends a rich T base sequence at the 3' end after adding terminal deoxynucleotidyl transferase(TdT)and dTTP.Then the extended products are used as primers to capture a number of the rich-A DNA probes modified with invertase,and then the enrichment of invertases on the suface of magnetic microspheres is realized.After adding sucrose,sucrose is converted to glucose by the catalyzing of the invertase and the activity of T4 PNP enzyme is detected indirectly by measuring glucose concentration in the system.The signals of blood glucose meter are linearly proportional to the concentration of T4 PNP enzyme in the range of 10-6 U/m L~1 U/m L and 1 U/mL~10U/m L and the detection limit was about 5.5×10-7 U/mL.The method is simple and sensitive,and has been applied to the screening of T4 PNP inhibitors.Second,the strategy based on graphene oxide biosensor is developed for the detection of the activity of T4 PNP enzyme.In the experiment,a template DNA chain with phosphate groups at both ends is designed.In the presence of T4 PNP enzyme,3'-terminal phosphate group of the template DNA is specifically identified and the phosphoric acid group is hydrolyzed into hydroxyl group.Then the chain extension is performed at the 3' end of the template DNA under the combined action of TdT and dTTP.The extension product captures probe P2,a rich-A DNA fragment,modified with the fluorescent dye molecule to form a double-stranded structure,and simultaneously probe P1 is added for hybridization with the template DNA chain.After the hybridization reaction is completed,a certain concentration of graphene oxide is added and graphene oxide adsorbs single-stranded DNA more quickly than double-stranded DNA.Therefore,a strong fluorescence signal is generated in the system.Conversely,when there is no T4 PNP enzyme in the system,the dephosphorylation reaction can not occur and the template DNA can not be extended.When the graphene oxide is added,a lot of the single-strand DNA contained in the solution is adsorbed on the surface of the graphene oxide,fluorescence resonance energy transfer between the graphene oxide and the fluorescent molecules modified by the probe P2 is performed,and the fluorescence is quenched.The fluorescence intensity are linearly proportional to the concentration of T4 PNP enzyme in the range of 0.001 U/mL~0.5 U/m L and 0.5 U/mL~20 U/mL and the detection limit was about 0.0008 U/m L.This method is initially used for the screening of T4 PNP enzyme inhibitors and satisfactory results were obtained.