New Methods For Detection Of Tumor Markers:8-OHdG And PARP-1

Tumor markers play an important role in the early screening,diagnosis and prognosis of tumors.With the development of biotechnology,new tumor markers are constantly found.The levels of 8-OHdG and PARP-1 in lung cancer,breast cancer and ovarian cancer patients are significantly higher than those in healthy people.Therefore,8-OHdG can be used as a tumor marker to reflect the extent of DNA oxidative damage caused by carcinogenesis.PARP-1 can be used as a biomarker for the diagnosis and prognosis of cancer.In this paper,the detection of tumor markers by constructing electrochemical bioanalysis based on electrodes and solid-state nanochannels has been studied.The main contents of this paper are as follows.(1)A simple electrochemical biosensor for ultrasensitive detectionof 8-OHdG was proposed based on it triggered polyaniline(PANI)deposition on tetrahedral DNAnanostructure(TDN).TDN was immobilized onto a gold electrode surface based on self-assembly betweenthree thiolated nucleotide sequences.8-OHdG-aptamer on the top of TDN formed a hemin/G-quadruplexstructure in the presence of 8-OHdG and hemin,which have high catalytic activity totrigger PANI deposition.Numerous negative charges on the duplex DNA contained in hemin/G-quadruplexand TDN supplied exquisite environment for PANI deposition.The response signals correlated linearly with the concentration of 8-OHdGranging from 10 pM to 2nM,with a detection limit of 1 pM(S/N=3).The sensitivity was improved toalmost 300-fold when compared with most of previously reported electrochemical methods.Themethod was also simple and reliable,avoiding complex,expensive label procedures and nanomaterial.(2)A facile and label-free method to detect PARP-1 activity was first constructed by using PAR based PANI deposition.The strategy was shown in Scheme 1.The gold electrode was modified with thiolated specific double-stranded DNA(dsDNA)through gold-sulfur bond.When PARP-1 bound to specific dsDNA structures,it possessed an NAD~+-dependent catalytic activity to help the synthesis of a negatively charged polymer(PAR).PAR were linear or branched polymer that possessed double charge density than that of DNA.Positively charged polyaniline(PANI)were prone to adsorbed on the negatively charged PAR due to electrostatic interaction.The reduction of PANI produced strong electrochemical signal when DPV detection method was applied.The DPV signal was linearly increased with the concentrations of PARP-1.When an inhibitor was in incubation with PARP-1,its activity was confined,and prevented PARylation process.As a result,the deposition of PANI became less and the electrochemical signal decreased.The method was sensitive and reliable for detection of PARP-1 activity and evaluation of its inhibitors.(3)The large size of hyperbranched PAR increased the steric hindrance in the AAO nanopore and resulted in a reduced flux of probe ions inside the nanochannels.Taking advantage of the large size of these hyperbranched PAR,the novel strategy for PARP-1detection has been proposed based on its great impacts on the diffusion flux of probe ions(ferricyanide)in the artificial nanochannels.It's the first time to be reported that hyperbranched polymer had great steric hindrance effects on the flux of probe ions in the nanochannels.It is also proved that the electrostatic repulsion effect is very important.The method is label-free,simple and sensitive.Quantitative detection of PARP-1 activity was achieved with the detection limit of 0.006 U,which is lower or comparable to the most reported methods.On the other hand,the method has good accuracy,selectivity and reproducibility.The strategy has been used to detect PARP-1 activity in real breast cancer cells and to evaluate PARP-1 inhibitors with satisfactory results,indicating that it is a potential powerful method for clinical diagnosis and drug development in the future.