Cs-can Redox Polymerization-Assisted Signal Amplification In The Application Of Lesion P53 Gene Diagnosis

Molecular diagnosis plays an important role in the early diagnosis and treatment of diseases. The existing DNA detection methods have advantages of high sensitivity and specificity, however,These techniques also have limit such as large instrument, complex operation and high cost. And polymerization-assisted amplification has recently been attracted attention as a promising signal amplification technique for use in molecular diagnostic. Because polymerization reaction allows direct correlation of biological binding events to polymer growth, this approach is shown to yield macroscopically observable polymer, obtaining high sensitivity and eliminating the need for any detection equipment.Herein we established a CS-CAN redox polymerization-assisted amplification system, and its application in the DNA detection of disease p53 gene. To achieve the purpose of signal amplification of target DNA,we use the natural substances chitosan which been connected to the target as reductant, and ceric ammonium nitrate(CAN) as oxidizer, generating redox reaction to to initiate polymerization in extremely mild conditions at room temperature, atmospheric pressure and aqueous solution. The optimum detection conditions are:the reaction pH was 5.0, SA-biotin binding time for at least 1 h, concentration of CS-biotin was 1.0 mg/mL, CS-biotin binding to SA for at least 1.5 h, concentration of CAN was 5-10 μmol/mL, redox polymerization time was 2 h. Streptavidin (SA) as model detected biomolecule was investigated here, as a result, detection limit of SA was demonstrated that 0.05 ng/mL SA was differentiable from the background using ellipsometry.The results showed that the limit of detection reached to 0.05 nM DNA with difference from the background via ellipsometry measurement, and the results can be distinguish by naked eye when DNA concentrations greater than 1.0 nM.Then we used CS-CAN RP system to detecte the lesion p53 gene DNA sequences that cause of non-small cell lung cancer, The lesion type of p53 gene was the first base mutation in codon 248 and the third in codon 249, a polymer film was visibly formed on the surface with the perfectly matched sequences between target lesion p53 gene and sigal probe, and its thickness was 300 nm when 1 μM target lesion p53 gene was given, and the thickness was 15 nm when 1 μM target p53 gene was given; The detection result of lesion p53 gene which the third base mutation in codon 249 was 300 nm polymer film when 1 μM target lesion p53 gene was given, and 40 nm when 1 μM target p53 gene was given. The results showed that the limit of detection reached to 0.5 nM lesion p53 DNA sequences with difference from the background via ellipsometry measurement, and the results can be distinguish by naked eye when lesion p53 DNA sequences concentrations greater than 1.0 nM. Detection result is based on CS-CAN RP system can distinguish single-base mismatches DNA from 12 bases. And the anti-interference ability is excellent of CS-CAN redox polymerization-assisted amplification system, target DNA can be detected when 50 times impurities DNA in the solution.