摘要
目的探讨并建立量子点核酸传感器检测微生物样本时缓冲液中最佳阳离子价态及最优离子强度。方法采用巯基法固定金黄色葡萄球菌16S rDNA探针于石英晶体微阵列上,并用石英晶体微天平(QCM)检测样品频率漂移量以确定最佳离子强度,并用荧光显微镜观察不同反应体系中杂交完成后的荧光强度和荧光效率,以此判断单价和二价阳离子对荧光效率和杂交效率的影响,并运用SPSS软件对数据进行统计分析。结果该量子点传感器检测平台具有良好的性噪比;对于普通的短链核酸杂交反应,随着离子强度增加,杂交效率呈现先增后减的趋势,当离子强度为20mmol/L时,杂交效率达到峰值,荧光强度约为0.95AU;对于MgCl2缓冲液,当离子强度为15mmol/L时,杂交效率达到最大,约为0.82AU;对于CuCl2缓冲液,离子强度为15mmol/L时,杂交效率达到峰值,约为0.72AU。结论单价阳离子NaCl缓冲液是基于荧光检测的微生物传感器的首选,其最佳离子强度为20mmol/L,缓冲液体系中应尽量避免添加二价阳离子。
OBJECTIVE To discuss and establish the optimal ionic strength and its valence state in the hybridization buffer during the detection of microorganism biosensor. METHODS Hydrosulfide group was adopted to immobilize the 16S rDNA probes of Staphylococcus aureus on the quartz crystal biosensor array. We detected the frequency shifts induced by nucleic acid hybridization. Then we detected the total fluorescence intensity with fluorescence microscopy. The first step was used to make sure the optimal ionic strength and the second one was for the influence of two-valence cation on fluorescence quenching. All of the data were analyzed by the SPSS software. RESULTS The quantum dot based microorganism biosensor had good signal-noise ratio. The hybridization efficiency increased gradually and then falled down with the increase in ionic strength. The hybridization efficiency got the peak when the ionic strength reached 20mmol/L, this peak fluorescence intensity was 0.95 AU. Similarly, the peak fluorescence intensities produced were 0. 82 AU and 0. 72 AU at 15 rnmol/L ionic strength for MgCl2 and CuCl2 buffers, respectively. CONCLUSIONS One valence cation buffer is the first choice of quantum dot based microorganism biosensor, and the optimal ionic strength is 20 mmol/L. Addition of two valence cation buffer must be avoided to the great extent.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2010年第11期1527-1530,共4页
Chinese Journal of Nosocomiology
基金
国家"863"项目(2007AA02Z416)
国家自然科学基金(30900348)
全军科技成果推广扩试项目(08JKS01)
军队科技攻关(08G089)
重庆市科技攻关项目(CSTC
2008AC0001)
重庆市自然科学基金(CSTC
2007BB5067)
第三军医大学军事医学专项(2008XG24)
福建省医疗器械和医药技术重点实验室开放基金(09001)
关键词
离子强度
微生物传感器
核酸杂交
量子点
Ionic strengths Microorganism biosensors Nucleic acid hybridizations Quantum dots
