Studies On Biosensor Based On Enzyme And Aptamer

Compared to the complex operation, high cost, low sensitivity and precision of the early diagnosis of tumour, biosensors based on aptamers with a high sensitivity, short analytical time were in great need. Several amplifying techniques, including screen- printed electrodes (SPE), nanoparticles and bio-bar-code, have been employed to develop biosensors. Probes fabricated based on electrode surfaces, modified by nano-technologies and aptamers, were used for antigen identification. Sensing strategies for highly sensitive detection of small molecule compounds and proteins were developed by use of switching structures as well as high specificity and affinity of aptamers. This paper contained five chapters.In the first chapter, the principle and classification of DNA biosensor was introduced. Research status of DNA biosensor and research progress of aptamer sensor were reviewed.In the second chapter, the thionine (Th) doped screen-printed electrodes were developed by screen-printed technique and thionine (Th) doped carbon ink at the chosen temperature and time. The ssDNA on SPE was hybridized with target DNA and signal DNA modified with horseradish peroxidase (HRP). An electrochemical method to directly detect DNA hybridization was developed by detecting the peak current of 2-aminophenoxazin-3-one (AP) which was produced by HRP catalyzed oxidative reaction of o-aminophenol (OAP) by H2O2. Results showed that the change in peak current was linear with target DNA concentration in the range from 1.0×10?12 mol·L-1 to 1.2×10?11 mol·L-1. The detection limit was 7.8×10-13 mo1·L-1 (3σ).In the third chapter, an photochemical sensing strategy for highly sensitive detection of thrombin was developed based on switching structures of aptamers from DNA/DNA duplex to DNA/target complex and aptazyme which was used as novel chemiluminescent enhancer. The magnetic beads were modified by thrombin aptamer sequences, and then hybridized with hemin aptamer sequences. In the presence of thrombin, the thrombin aptamer part bound with thrombin and folded to the complex structure. As a result, hemin aptamer sequences were released into solution. In the presence of hemin, the aptazyme which was used as novel chemiluminescent enhancer in the Luminol-H2O2 chemiluminescene system was formed. The thrombin was detected with a linear range from 5.0×10-12 mo1·L-1 to 5.0×10-11 mo1·L-1 and a detection limit of 3.1×10-12 mo1·L-1 (3σ).In the fourth chapter, a dual-aptamer electrochemical biosensor based on bio-bar- code technique and anodic stripping voltammetry (ASV) for the simultaneous detection of adenosine and thrombin was developed. An Au electrode as the sensing surface was modified with a thiolated capture probe containing both adenosine aptamer and thrombin aptamer. Then the adenosine aptamer sequence hybridized with its complementary DNA, which was loaded on the Au Nanoparticles (AuNPs) associated with signal DNA labeled with CdS nanoparticles. In the presence of adenosine and thrombin, the stronger binding affinity of the adenosine aptamer with adenosine than that with complementary DNA destroyed the double-helix structure assembled on the electrode, with the dissociation of the CdS NPs into the solution. Then a secondary thrombin aptamer, which was loaded on the AuNPs together with PbS NP labeling signal DNA, was introduced. The metal sulfide nanoparticles were measured by ASV, and the concentrations of adenosine and thrombin were proportional to the signals of either metal ion. With the dual-aptamer based sensor, adenosine could be quantified over the range from 1.0×10-11 mo1·L-1 to 2.0×10-9 mo1·L-1 with a detection limit of 8.8×10-12 mo1·L-1 (3σ); thrombin could be quantified over the range from 1.0×10-12 mo1·L-1 to 3.0×10-10 mo1·L-1,with a detection limit of 7.6×10-13 mo1·L-1 (3σ). The sensor could be used in biological samples with excellent selectivity.In the last chapter, it was summarized for the whole thesis.