The Application Of Hemin-Graphene Nanocomposites In The Detection Of Cancer-related Enzymes

There are many types of biomarkers associated with cancer,including microRNA,DNA,enzymes,metabolites and so on.These markers are produced by tumor cells.Therefore,the early diagnosis of cancer is carried out by the detection of these markers.Due to the low concentration of the biomarker in the early stage of cancer,it is of great concern to develop accurate,reliable,sensitive and effective detection methods for tracing cancer markers.In recent years,graphene has been explored deeply due to their excellent physical and chemical properties.Functionalized graphene were also introduced into the detection of cancer markers.In this paper,we focus on the fabrication of colorimetric biosensors for sensitive detection of telomerase and PARP-1 by using Hemin-graphene composites.The details are summarized as follows:(1)A simple colorimetric strategy for label-free quantification of human telomerase activity by using excellent properties of Hemin-graphene nanomaterial(H-GNs)was developed.In this strategy,H-GNs were adjusted to coagulate to appropriate degree by carefully selecting the contained NaCl amount in the presence of original TS primer.Under the action of telomerase,TS primer was elongated with repeating sequences of(TTAGGG)_x.These negatively charged DNA enhanced individual H-GNs electrostatic repulsion and resisted salt-induced H-GNs coagulation.As a result,the supernate of the corresponding solution contained more dispersed H-GNs and showed dark blue color after chromogenic reaction.Thus,telomerase activity could be quasi-quantified by naked eye and precise quantified by UV spectrometer.The proposed method had the linear range from 100 to 2300HeLa cells/mL and the detection limit was 60 cells/mL.Compared with the method of detecting telomerase activity constructed by gold nanoparticles,this method was not only simple but also had higher sensitivity.And it has been successfully applied to detect telomerase activity in real urine samples,which was of great significance for the detection and prevention of cancer.(2)We developed a simple,label-free colorimetric method to detect PARP-1 activity based on the interactions of Hemin-graphene composites(H-GNs)and PAR.Firstly,the designed ds/ssDNA was still adsorbed on H-GNs by the?-?stacking interactions between H-GNs and unmatched 18 bases.And it was found that selecting the proper ds/ssDNA and NaCl concentration could adjust H-GNs coagulate to appropriate degree.However,in the presence of PARP-1,the auto-PARylation functions of PARP-1 were activated by the coexisting specific dsDNA and subsequently formed a homodimer.A negatively charged PAR polymer was formed by using NAD~+as substrate.PAR had ample negative charges and its charge density was even as two-folds of as single strand DNA,which had greatly impacts on the dispersibility of H-GNs due to the strong electrostatic repulsion.As a result,H-GNs existed stably in the same concentration of salt solution.The method does not require expensive marking process.The proposed method had the linear range from 0.05 to 1 U with a detection limit of 0.034 U.In addition,this new method has been successfully applied to detect PARP-1 activity in human serum and different cancer cells.