| Point mutation in genomic DNA has a direct link with human disease, Genetic mutation analysis plays an important role in early clinical diagnosis of cancer. It is a hot topic for experts to develop novel and high sensitive methods in detection of single nucleotide polymorphisms. Aiming at the problems including gene mutation identification and fabrication of sensing interfaces that using click chemistry, several novel methods have been developed for electrochemical, piezoelectric DNA biosensors. The detailed contents are described as follows.(1) In chapter 2,a novel electrochemical method for SNP detection is proposed based on allele-specific extension and enzymatic-induced silver deposition. Briefly, allele-specific capture probe was firstly immobilized on the gold electrode; such a probe can perfectly match with wild gene and can be extended, whereas mutant genes which mismatch with the probe at 3' terminal bases cannot be extended. After the formed duplexes unfolded, the target sequences dissociate from the electrode surface. Because the extended sequence perfectly matches with biotin-modified detection probe, hybridization takes place, and then streptavidin-conjugated alkaline phosphatase can be captured to the electrode surface due to the specific interaction of biotin and streptavidin. The enzyme-induced silver deposition is used to amplify the response. The electrochemical signal is obtained by using linear sweep voltammetry. The present approach has been demonstrated with the identification of single-base mutation in -28 site (A to G) forβ-thalassemia gene and the wild type target can be determined in the range from with 3.0×10-16-3.0×10-8 M with a low detection limit of 1.0×10-16 M.(2) In chapter 3, a novel biosensing technique for highly specific identification of gene with single base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe, which tethered onto the QCM utilizing gold nanoparticles modification and then with a biotinylated allele-specific detection DNA. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 4-chloro-1-naphthol by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single base mutation in -28 site of theβ-thalassemia gene. The target gene can be determined in the range from 0.1 nM to 100 nM with a detection limit of 0.1 nM.(3) In chapter 4, we have developed a novel sensing interface for the immobilization of nucleic acid molecules based on the copper-catalyzed click chemistry. Meanwhile, a electrochemical method for indirectly detecting metal ions was proposed via taking copper ion as a model analyte, choosing methylene blue as electrochemical indicator. Firstly, the azido capture probe was immobilized on the gold electrode surface, then the methylene blue labeled alkynyl hairpin probe was added to initiate click reaction. This method utilizes the catalytic activity of Cu(I) for electrochemical quantitative detection. In addition, the hairpin molecular beacon immobilized on the gold electrode surface can also act as a capture probe to hybridize with single-strand DNA, and it can be used for the specific gene analysis. |
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