葡萄球菌肠毒素基因的DNA传感器研究

葡萄球菌肠毒素B（SEB）DNA探针自组装膜研究；压电DNA传感器重复使用的条件优化；基于引物链延伸反应并将流动注射、人工神经网络与石英谐振式测量方法相结合，构建具有DNA咏焕┰龉δ芎投嗷蛑悄芑锒系难沟鏒NA传感系统；建立SEB基因的测定方法。此研究对称肺兰旒际跛降奶岣哂兄匾饔茫⑼贫渌Э浦蠨NA检测技术的发展。

In this study, we established a new technique to detect SEB DNA hybridization by the piezoelectric DNA sensor empolying Staphylococcal enterotoxin B (SEB) gene as a molecular recognizing component and piezoeletric quartz crystal as a mass sensitive component.The research work includes five parts and main results are as follows: 1 Piezoelectric DNA sensor and effects of non-chemical factor on the sensor Based on the piezoelectric DNA sensor and its component, working principle, we have researched effects of non-chemical factor on sensor, such as temperature, humidity, fluid viscosity,.fluid density, electromagentic on the sensor and the measures to eliminate those effects. Those experiments will be a solid foundation for the further study of piezoelectric DNA sensor. 2 Preparation of SEB DNA probe and its application.The objective of this study is to establish a method for the preparation of SEB DNA probe and the detection of SEB gene. Based on the experimental objective, two strand oligonucleotides were.synthesized,and then DNA fragment of 169 bp and 700 bp were obtained by polymerase chain reaction (PCR). After they were purified，the fragments were ligated into T vector ，transformed and then selected the positive clone. The sequence of the gene fragments amplified by PCR was.proved correctly by sequencing. It provides a scientific basis for the research on piezoelectric DNA.sensor, to establish a sensitive and specific dot hybridization method with biotinylated SEB gene probe by PCR..3 Studies on APTES immoboilized ssDNA method for piezoelectric DNA sensor.development A new method has been developed to monitor DNA hybridization by using piezoeletric DNA sensor system.AT-cut crystals, with a based resonant frequency of 3.58MHz and 10MHz, were modified by a series of surface pretreatment as follows: gold plated, anoidical oxidation and 3-aminopropyltriethoxysilane treatment. Single stranded DNA probe was electro-blotted onto the surface of crystal electrodes. The DNA probe was further fixed on the crystal surface by UV.crosslinking. The crystal sensor is dipped into the hybridization solution and the increased surface mass induced by hybridization is measured by the decrease in the resonant freqency of crstal..Immobililzed efficiency was observed by active methods, passive methods, and dot hybridization method. The piezoelectric DNA sensor could be repeatedly used, the complementary ssDNA of the SEB DNA probe could be removed from the crystal by washing with 0.5 mol/L NaOH denaturant..After the hybridized crystal was immersed into 1.2mol/L NaOH and then 1.2mol/L HCl, all substances could be removed from the crystal surface, and the quartz crystal could be reused for many times. The APTES-coated ssDNA crystal was stable for three weeks when stored at 4℃ in.buffer. 4 Studies on self-assembled monolayers technology for piezoelectric DNA sensor Self-assembled monolayer technology has been applied in biosensor or chemical sensor. In this 国家自然科学基金资助项目结题报告 6 chapter, two ways which immobilized DNA probe on gold electrode were studied, one way was that.the thiolated oligonuleotide was assembled on gold electrode surface by self-assembled characteristic.The other was that 3 3`-dithiopropionic acid was applied to immobilize DNA,.1-ethyl-3-[3-(dimethy -lamino) propyl]carbodiimide (EDC) and N-hydroxysuccinimide (NHS) can coat avidin on gold electrode and then avidin can combine to different segment (25-mer biotinylated oligonuleotide and 169bp biotinylated DNA probe by PCR) biotinylated nucleotide on gold electrode.surface. The consistency and specificity of the piezoelectric DNA sensor adopted above two immobilization methods were well. The sensor can reuse three times or more than three times by electrode regeneration. 5 Study on dual parameter piezoelectric DNA sensor for liquid detection Piezoelectric DNA sensor for liquid detection was developed by 25-mer biotinylted oligonucleotides immobilized on gold electrode surface of quartz crystal by avidin–biotin syste