Novel Methods Of Preparation Of DNA Probes For Gene Detection And SNP Identification And Biosensors Based On Aptamers

With the implement of the Human Genome Project and the development of the functional proteomics research, it's an important domain that high sensitive assay methods were developed for the detection of the proteins and DNA. Because point mutations in genomic DNA have direct link with human diseases,there has been considerable interest in developing rapid and sensitive methods to detect point mutations.DNA aptamers are short nucleic acid ligands artificially selected for their high specificity and affinity for various targets including proteins, small molecules and even cells. Because of their numerous advantages over antibodies such as adaptability to various targets, ease in synthesis and storage, and versatility in labeling, immobilization, signaling and regeneration, the widespread application of aptamers to biosensors is expected to hold potential in the detection of various proteins and small molecules.Electrochemical and piezoelectric biosensors attract significant attention and become the research hotspot for their low-cost, fast response, being simple, sensitive and compatible with microfabrication technologies. Aiming at the key technique including probe immobilization method and detection method used in electrochemical biosensor, several novel methods been developed for electrochemical, piezoelectric DNA biosensors and electrochemical aptasensor. The detailed content is described as follows.1) In chapter 2,CeO2/Chitosan composite matrix was firstly developed for the single-stranded DNA (ssDNA) probe immobilization and the fabrication of DNA biosensor related to the colorectal cancer gene. The preparation method is quite simple and inexpensive. Such matrix combined the advantages of CeO2 and chitosan, with good biocompatibility, nontoxicity and excellent electronic conductivity. The matrix was used to immobilize completely complementary ssDNA probe with the target sequence and showed the enhanced loading of ssDNA probe on the surface of electrode. The established biosensor has high detection sensitivity, a relatively wide linear range from 1.6×10-11-1.2×10-7 M and the ability to discriminate completely complementary target sequence and four-base-mismatched sequence. The limit of the detection is 1.0×10-11 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. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. 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 3, 3-diaminobenzidine (DAB) 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 0.7 nM to 100 nM with a detection limit of 0.1 nM.3) In chapter 4,a novel electrochemical method for SNP detection is proposed based on allele-specific extension and enzymatic-induced silver deposition. Briefly, allele-specific capture probe firstly immobilized on the gold electrode, which perfectly matches with wild gene and can be extended, whereas mismatching with the mutant gene at 3' terminal bases cannot be extended. After denaturation with 1M NaOH, the formed duplexes unfold and target sequences dissociate from the electrode surface. Because the sequence extended perfectly match with biotin-modified detection probe, hybridization take 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 of silver 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.4) In chapter 5 , a label-free electrochemical sensor using aptamer based on target-induced displacement is reported with adenosine as the model analyte. The sensing substrate is prepared using a gold electrode modified with a self-assembled monolayer of 1, 6-hexanedithiol that mediates the assembly of a gold nanoparticle film, which can increase the surface loading of capture probe and enhance the signal. An aptamer for adenosine is applied to hybridizing with the capture probe, yielding a double-stranded complex of the aptamer and the capture probe on the surface. The interaction of adenosine with the aptamer displaces the aptamer sequence and causes it to dissociate from the interface. This results in a decrease of absorption state of methylene blue. Then, the redox current of the indicator can reflect the concentration of the analyte. The fabricated sensor is shown to exhibit high sensitivity, desirable selectivity. The linear range for target detection is 5-1000 nM with a detection limit of 1 nM. The regeneration of the developed biosensor is simple and fast. 5) In chapter 6, an electrochemical immunosensor is reported by usingaptamer-based enzymatic amplification with immunoglobin E (IgE) as the model analyte. The IgE-antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target captured, the biotinlynated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinlynated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then, silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range from 0.1-100 nM with a low detection limit of 0.02 nM, which possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.6) In chapter 7, based on the principle of antibody-aptamer sandwich in chapter 6, we reported a label-free electrochemical biosensor for thrombin detection. A novel nanogold-chitosan composite film using the electrochemical deposition method was prepared to immobilize thrombin antibody. Combined the special properties of gold nanoparticles and chitosan, the composite film showed enhanced conductivity and loading ability. The electrochemical deposition conditions of the composite film was investigated and its surface morphology was characterized by SEM. Methylene blue (MB) as the electrochemical active marker intercalating in the probing aptamer was applied to offer the response signal by differential pulse voltammetry (DPV). With appropriate extended design of the aptamer sequence, the amount of MB intercalating in the aptamer was increased and the signal response was also enhanced. The fabricated sensor was simple and low-cost and without the need of labeling. The linear response range for thrombin detection covered 1-60 nM with a detection limit of 0.5nM.