Experimental Study Of Detection System Of Quartz Oscillation Gene Sensor Array With Strand Extension Reaction And PNA

Objectives:To construct detection system of quartz oscillation gene sensor (QOGS) array and to improve its sensitivity with bis-PNA probe and strand extension technique. And the possibility of directly detecting clinical samples was also explored. Methods:1. Technology of fine processing was firstly used to make the solo QOGS and then the gold electrode was etched around two sides of sensor. Different thickness and diameter of electrodes were compared in order to determine its influence to the detection of gene hybridization. Based on it, glued 2 5 style QOGS array and clamped 2 5 style QOGS array were constructed successively. Also we developed the self-oscillating circuit and the PESA version 2.0 software applying to record and analyze frequency. So the detection system of QOGS array was constructed by combing all these components.2. HPV probe was immobilized on the gold electrode surface of QOGS array by thiol method and biotin-avidin method, respectively. Advantages and disadvantages of two methods were compared specifically.3. Two kinds of mecA and femA probes of staphylococcus aureus were immobilized on the gold electrode surface of QOGS array. Klenow large fragment was added to extend the strand in room temperature after the probes were hybridized with corresponding target DNA.4. Bis-PNA probe for detecting HBV was immobilized on the goldelectrode surface of QOGS array. The optimal pH value, ion concentration andprobe concentration were determined through a series of experiments. And the curve was statistically drawn up about the relationship between quantity of target and decrease of frequency. Also the possibility of using bis-PNA probe to directly detect the extracted genomic DNA from 28 positive clinical samples caught hepatitis and 12 negative sera samples was explored on the late stage of experiment. Comparison was processed with quantitative PCR to summarize the advantages and disadvantages of bis-PNA method and finally determined the detection limit of this method to clinical sera samples. Results:1.1 Quartz crystal with AT cutting style was selected to be elementary plate of sensor because of its benefit for stable oscillation in liquid phase of sensor. The thickness of gold electrode among 50nm to 100nm would be most suitable for detection of gene hybridization, while the diameter 4.0mm would be less insert loss and afford more quality burden. Many advantages such as convenience, less energy, economy and so on would be possessed with glued style QOGS array which was constructed based on solo QOGS, but it would strongly effect the oscillating stability in liquid phase of sensor array. Clamping style QOGS array, however, successfully overcame the disadvantages such as randomized distribution of the response force that was caused by glued style, and hence realized the stable oscillation in liquid phase of sensor array.1.2 Self-oscillating circuit that was developed by ourselves had strong vibromotive ability in liquid phase and its stability of frequency could also meet the request of experiment.1.3 Self-developed PESA version 2.0 software applying to record and analyze frequency had many functions such as setting up primary parameters, recording change of frequency in real-time, plotting figures of frequency change automatically, and so on. Data would be transferred to computer automatically after the collection and analysis.2. The sensitivity of two methods was comparative. The biotin-avidin method took less time to react with target sequence and could immobilize more probes and hence combine more quantities of target sequence, but the procedure was tedious. While thiol method was quite simple but it took much more time for reaction.3. Before the strand extension reaction, the sensitivity of the QOGS array was 0.5nM, while it improved to 0.05nM after strand extension reaction.4.1 Frequency change increased firstly and then decreased when pH values of hybridization buffer varied from 5.8 to 8.0, and pH6.8 was the watershed. As for equilib...