A Novel Biosensor Based On Entropy-driven Enzyme-free Amplification System For Quick Detection Of HPV-18 E6/E7 MRNA

Objective:An entropy driven enzyme-free amplification system based on nucleic acid strand replacement reaction was used to detece HPV-18 E6/E7 mRNA.A large amount of nucleic acid substrates was converted into products through the loop reaction.Lateral flow immunochromatography was used to construct a biosensor to capture amplified products and amplify the signal in the same time.The method can be applied for rapid screening of clinical human papillomavirus infection.Methods:1.Construction and evaluation of entropy-driven enzyme-free amplification system:HPV-18 genome was analyzed and specific sequences of conservative segments are selected as target sequences.An entropy-driven enzyme-free amplification system was designed for target sequences.Theoretically analyze the feasibility of the method using mathematical models.Non-denaturing polyacrylamide gel electrophoresis was applied to verify the programmatic hybridization scheme between nucleic acid strands.A fluorescence detection system based on the amplification method was constructed by modifying the nucleic acid chains in the system,and the programmatic response of the system was verified.The system was optimized to obtain the optimal reaction conditions.The system was evaluated using single-stranded DNA analogs to analyze the sensitivity and specificity.2.Construction and preliminary evaluation of entropy-driven enzyme-free amplification system fluorescence sensor:A lateral flow immunochromatography was used to further amplify the amplified signal and convert it into a fluorescent signal using fluorescence microsphere.An entropy-driven enzyme-free amplification system-based fluorescence biosensor was constructed.Trizol was used to lyse the cells and stabilize the nucleic acid.Hela,A549,MCF-7,and HEK293 cells were used to analyze the sensitivity and specificity of the sensor.Different types of HPV-infected clinical cervical cancer patient samples were used as a negative group to evaluate the clinical detection effect of the sensor.Results:1.Construction and evaluation of entropy-driven amplification system:The entire gene sequence of HPV18 was queried through the NCBI database,and specific sequences were selected from its E6/E7 as detection targets.A nucleic acid calculation software NUPACK was used to assist in designing an Enzyme-free isothermal amplification cycle system based on strand displacement reaction.Theoretical calculation results show that the product conversion efficiency can reach99%.Stepwise analysis of its reaction process by non-denaturing gel electrophoresis proved that it can react programmly.The system is transformed into a fluorescence sensing system by fluorescent groups and quenching groups.Step-by-step analysis of the reaction process of fluorescence proves that the reaction is consistent with the theory,and consistent with the results of non-denaturing gel electrophoresis.The investigation of the reaction conditions determined the optimal reaction conditions.The concentration of the complex SBO and chain F were 100 n M,and the reaction was performed at 37℃in the dark for 60 minutes.The relationship between the fluorescence intensity and the target concentration was analyzed.When the target concentration was between 100 p M to10 n M,the fluorescence intensity in the amplification system has a linear relationship with the logarithm of the target concentration.The linear correlation equation is Y=395042log C-276257,R~2=0.9753.The minimum detection limit was about 5 p M.The system was highly specific,and a single base mismatch on the target sequence can prevent the reaction.The system also had excellent stability,and the test result remains unchanged within 30 days of freeze-drying the probe.2.Construction and preliminary evaluation of a fluorescence sensor based on entropy-driven enzyme-free amplification system:A fluorescent test strip was used to detect a nucleic acid double-stranded labeled with biotin and digoxin.It was proved that it can specifically detect the labelled nucleic acid complex.The fluorescence sensing system introduced above was used to prove that Trizol has the function of lysing cells and stabilizing nucleic acids at the same time and the amplify system act well in cell lysates.Experiments show that when the number of cells was between 10~3 and10~6,the logarithm of the number of cells is linearly related to the sensor count.The regression equation is Y=6597log N-19351 R~2=0.997,and the correlation coefficient R~2=0.997,where N is the number of cells.The limit of detect is about 300cells.The sensor has extremely high specificity,P<0.01.Diagnostic tests on clinical samples show that entropy-driven enzyme-free amplification cycle fluorescent biosensors have a very high diagnostic effect on clinical samples and can distinguish HPV-18 E6/E7 mRNA-expressing patients significantly(χ~2=49.1,P<0.01).The diagnostic sensitivity was 95.2%,the specificity was 100%,the positive predictive value was 100%,and the negative predictive value was 88.9%.Conclusions:Based on the strand displacement reaction between nucleic acid strands,we have constructed a specific and sensitive isothermal enzyme-free nucleic acid amplification method.We further amplify the amplified signal and convert it into fluorescence signal through immunological methods.A fluorescence sensor based on entropy-driven enzyme-free amplification system was constructed.The sensor has the advantages of high sensitivity,good specificity,simple method,low cost,and rapid detection.It can quickly detect HPV-18 E6/E7 mRNA and has broad application prospects.