The Application Of Nanomaterials In The Detection Of Tumor-related Enzymes

Tumor-related enzymes are widely present in tumor cells,and their presence and quantitative changes can reveal the nature of tumors.The quantitative detection of tumor-related enzymes has important research significance and practical value in many aspects such as early diagnosis,treatment and prognosis of cancer.Therefore,it is extremely important to establish a low-cost,high-sensitivity,specific,and easy-to-operate tumor-related enzyme detection method.In recent years,DNA damage repair enzyme,poly（ADP-ribose）polymerase-1（PARP-1）,has been confirmed to be closely related to the occurrence and development of tumors.And it has been overexpressed in patients with lung cancer,breast cancer,oral cancer,ovarian cancer and prostate cancer compared to normal people.At the same time,it’s indicated that the overexpressed flap endonuclease 1（FEN1）,another DNA damage repair enzyme,could promote the proliferation and differentiation of tumor cells.And the function loss of FEN1 may lead to genomic instability and cancer susceptibility.In addition,compared to normal cells,the content of FEN1was higher in tumor cells,and the overexpressed FEN1 was related to tumor size and degree of differentiation.Therefore,PARP-1 and FEN1 can be regarded as original markers to predict the occurrence and development of tumors by detecting their content in the body.This paper mainly uses nanomaterials to construct two different biosensors for the quantitative activity detection of PARP-1 and FEN1.The main research contents of this paper are as follows:（1）Based on the negatively charged poly（ADP-ribose）polymer（PAR）and cationic polymer（PFP）,we have constructed a label-free,high-sensitivity photoelectrochemical（PEC）biosensor for the quantitative detection of PARP-1activity.To the best of our knowledge,it is the first time that PEC has been used to monitor PARP-1 activity.In this method,PARP-1 was firstly activated by specific double-stranded DNA and catalyzed the broken of glycosylic bond that linked nicotinamide with ribose.The obtained ADP-ribose unites polymerized to linear or hyper branched PAR containing 200 ADP-ribose moieties with abundant phosphate groups(PO₄^3-),resulting in much more negative charges.Therefore,the positively charged PFP could be adsorbed on the surface of PAR by electrostatic interaction.Under light conditions,PFP with good optoelectronic properties produced a photocurrent that can be detected.The larger the amount of PARP-1,the larger the photocurrent generated.Although double-stranded DNA also contained PO₄^3-,it was small in quantity and could only adsorb less PFP,producing a weaker photocurrent signal.This method has a wider detection range（0.01 U-2.0 U）,and the detection limit is 0.007 U.In addition,this method can also detect PARP-1 activity in ovarian cancer cells（A2780）and breast cancer cells（MCF-7）.At the same time,the method is also compared with the Human PARP ELISA Kit,and both have obtained satisfactory results.（2）Based on the high affinity of gold nanoparticles（Au NPs）to poly A and the selectivity of Oli Green to thymine（T）,we constructed a simply prepared multifunctional nanoprobe for ultrasensitive detection of FEN1.First of all,poly A at3’-end of ss DNA was absorbed on the surface of Au NPs effectively,with the 5’-end of ss DNA overhanging because of the weak affinity of Au NPs to thymine,resulted in a poly A-mediated nanoprobe（Au NPs-poly A-poly T）.This nanoprobe not only worked as effective fluorescence quencher but also as the original nano-substrate of FEN1.In the absence of FEN1,Au NPs could quench the green fluorescence emitted by Oli Green-poly T complex.In the presence of FEN1,it recognized and cleaved the overhanging poly T segment efficiently,turning on the fluorescence of Oli Green-poly T complex.This indicated that the assembled nanoprobe was an effective artificial substrate to FEN1,although it was completely different from previously reported substrates that are all composed of ds DNA with a flap strand.The linear range of this method was from 0.05 U to 2.0 U,with the LOD of 0.007 U.At the same time,cancer cells and normal cells can be distinguished by confocal imaging,indicating that this method has broad application prospects in clinical diagnosis.