Research On New Detection Methods Of Tumor-associated Enzymes Activity

Early detection,early diagnosis,and early treatment are the main means to reduce cancer mortality and saves lives,improve the quality of life of patients.Therefore,scientists are working to develop new technologies to improve the level of early detection of tumors.There are many types of cancer biomarkers,such as nucleic acids,proteins,sugars,small metabolites,and cytogenetic and cytokinetic parameters,as well as entire tumour cells found in the body fluid.They can be used for risk assessment,diagnosis,prognosis,and for the prediction of treatment efficacy and toxicity and recurrence.Combined nucleic acids,nanomaterials with various detection instruments,a series of novel sensors have been developed for the detection of cancer-related enzyme markers telomerase,poly（ADP-ribose）polymerase-1（PARP-1）and flap endonuclease（FEN1）.The main points of this article are summarized as follows:（1）A novel,visual and label-free method was designed for telomerase detection.First,repeating（TTAGGG）n sequences were extented on telomerase substrate（TS）primer in the presence of telomerase extracts from Hela cells,K⁺and d NTPs to form the G-quadruplex,when hemin was added,it formed G-quadruplex/Hemin.Then,the obtained horseradish peroxidase mimicking G-quadruplex/Hemin catalyzed the H₂O₂-mediated etching of GNRs to generate a series of distinct color changes due to their plasmon-related optical response.As a result,telomerase activity can be quantitatively detected ranging from 200 to 15000 He La cells/m L.The limit of detection（LOD）was 90 He La cells/m L（S/N=3）.The application of this method in bladder cancer samples was in agreement with the clinical results.（2）A novel circulating-flow quartz crystal microbalance（QCM）biosensing method has been established to detect telomerase based on the principle of hybridization of oligonucleotide-functionalized gold nanoparticles（GNPs-c DNA）with target sequences to achieve frequency signal amplification.The detection limit was as low as 37 cells/m L.Moreover,the assay platform exhibited excellent selectivity.（3）An efficient method for the determination of PARP-1 by using mass-sensitive QCM was constructed.Once activated by the specific DNA,PARP-1 cleaves nicotinamideadenine dinucleotide（NAD⁺）into nicotinamide and ADP-ribose to synthesize a hyperbranched poly（ADP-ribose）polymer.Positively charged cetyltrimethylammonium bromide（CTAB）-coated gold nanorods（GNRs）were introduced to increase the frequency change significantly because of the strong electrostatic interaction between them with negatively charged PAR.PARP-1 ranging from 0.06 to 3 n M can be facilely detected with a low detection limit of 0.04 n M.The strategy has been used to detect PARP-1 activity in real cancer cells lysate with satisfactory results.（4）TOTO-1 is well-known for differentiating ss-DNA from ds-DNA because it is virtually non-fluorescent without DNA and exhibits very low fluorescence with ss-DNA,while it emits strong fluorescence with ds-DNA.In this paper,we found that TOTO-1 has different fluorescent intensities for different deoxyribonucleoside triphosphates（d ATP,d GTP,d CTP,d TTP）and adenosine-5’-diphosphate（ADP）.Moreover,different types of polydeoxynucleotides（Poly（d G）,Poly（d A）,Poly（d C）,Poly（d T））also have different fluorescence intensity.Interestingly,in the presence of telomerase,the G-rich DNA sequence formed at the 3’end of its primer,and when PARP-1 is present,it will be activated by specific double-stranded DNA,then PAR is formed on the PAPP-1 itself.Therefore,TOTO-1 was explored in detecting both of them by combining with the G-rich sequence and PAR to generate fluorescence.More importantly,the use of TOTO-1 to detect telomerase and PARP-1 at the same time will significantly increase the specificity of cancer screening and reduce the proportion of false positives,which makes this method a promising application in clinical diagnosis and drug screening.（5）The main method for evaluating FEN1 enzyme expression in biology is western blot analysis,but it is usually more complicated and has limited sensitivity.Here,we developed a method for sensitive detection of FEN1 on the basis of Nt.Bst NBI nuclease actuated Bst DNA polymerase mediated strand displacement amplification reaction.Once the 5’flap of double-flap DNA substrate was cleaved by target FEN1,the cleaved single strand DNA（5’flap）initiated strand-displacement amplification to produce plenty of G-rich DNA sequences,These G-rich sequences that self-assembled into G-quadruplexes revealed horseradish-peroxidase（HRP）like catalytic activities in the presence of hemin as well as fluorescence enhancement of thioflavin T（Th T）.This innovative method opened the way for sensitive,accurate,and reliable diagnoses of clinical diseases.