利用光波导模式光谱生物传感器研究二级结构对固-液界面DNA杂交动力学的影响  

The effects of secondary structure on solid-liquid interface kinetics of DNA hybridization analyzed by OWLS

作　　者：钟连声[1] 齐华文[1] 潘忠诚[1] 马汝海[2] 何群[1] 姜雪[3] 赵雨杰[1] 

机构地区：[1]中国医科大学生物芯片中心,沈阳110001 [2]中国医科大学化学教研室,沈阳110001 [3]中国医科大学附属盛京医院康复中心,沈阳110022

出　　处：《中国医药生物技术》2011年第5期341-346,共6页Chinese Medicinal Biotechnology

摘　　要：目的探讨探针和靶分子二级结构对基因芯片杂交过程的影响,并完善基因芯片设计方法,提高基因芯片的重复性和特异性。方法应用二级结构分析软件Mfold设计3对25-mer完全互补的具有不同二级结构的探针和靶分子(P0T0、P3T3、P4T4)及1对尾端有2个碱基错配的探针和靶分子(P4T3)。制备醛基化传感器芯片,将终浓度为1、2、5、10、20μmol/L的探针分别固定在芯片上,与浓度为5μmol/L的N,N′-对羧苄基吲哚三菁(Cy5)标记靶分子杂交,荧光图像扫描仪检测确定最佳杂交探针浓度;以最佳探针浓度构建芯片,分别与终浓度为0.1、0.2、0.5、1.0、2.0、5.0μmol/L的Cy5标记靶分子杂交,荧光图像扫描仪检测确定最佳杂交靶分子浓度;同时用光波导模式光谱生物传感器(OWLS)实时监测其杂交过程。以0.4%SDS和0.1%NaOH分别处理杂交后芯片10、20、30、60min,荧光图像扫描仪检测芯片再生稳定性。控制靶分子进样速度和杂交时间分别为15μl/h、7h和180μl/h、0.5h,OWLS测定杂交复合物质量、杂交效率和反应速率。结果荧光图像扫描结果显示,最佳杂交探针和靶分子浓度分别为10、1μmol/L,传感器芯片构建成功且具有再生稳定性。15μl/h、7h和180μl/h、0.5h条件下,P0T0、P3T3、P4T4、P4T3杂交复合物的质量(ng/cm2)分别为45和30、43和20、40和13、42和18;杂交效率(%)分别为40.9和27.3、39.1和18.2、36.4和11.8、38.2和16.4;杂交7h时的反应速率常数[K,105L/(mol·s)]值分别为0.465、0.247、0.081、0.247。结论在一定时间内,探针和靶分子二级结构增多可导致杂交时间延长,杂交速率和效率降低。应用OWLS成功建立了一种研究固-液界面DNA杂交过程的新平台,为研究基因芯片的杂交过程提供了一个新方法。Objective To study the effects of secondary structure of probes and target molecules on gene chip hybridization and to improve the design methods as well as the reproducibility and specificity of gene chips. Methods Several 25-mer probes and target molecules which had varying degree of secondary structures （with P0, P3, P4 perfectly matching with T0, T3, T4 respectively, but P4 and T3 having two terminal mismatches） were designed by Mfold, a secondary structure analysis software online. 1, 2, 5, 10 or 20 μmol/L probes were fixed to the surface of chips and hybridized with 5 μmol/L corresponding target molecules labeled with Cy5. The fluorescence signal was detected with fluorescence image scanner to determine the optimal probe concentration. The optimal concentration of probe was fixed to the surface of chips and hybridized with 0.1, 0.2, 0.5, 1.0, 2.0 or 5.0μmol/L target molecules labeled with Cy5 respectively. The fluorescence signal was detected with fluorescence image scanner to determine the optimal target molecule concentration. At the mean time, the hybridization signal was monitored by OWLS. 0.4% SDS and 0.1% NaOH was used to regenerate the hybridized chips for 10, 20, 30, 60 min and the regeneration stability of the chips was detected with fluorescence image scanner. The inject speed of target molecules and the hybridization time were controlled at 15 μl/h for 7 h and 180 μl/h for 0.5 h, respectively. The quality of hybrid complexes, the hybridization efficiency and the reaction rate were measured by OWLS. Results The signal obtained by the fluorescence image scanner showed that the optimal concentration of probe and target molecule were 10 and 1 lamol/L respectively. The censor chip was successfully established with regeneration stability. At the condition of 15 μl/h for 7 h and 180μl/h for 0.5 h, the hybrid quality of P0T0, P3T3, P4T4, P4T3 complexes （ng/cm2） was 45 and 30, 43 and 20, 40 and 13, 42 and 18, respectively, and the hybrid efficiency （%） was 40.9 and 27.3, 39.1 and 18.2,

关 键 词：生物传感技术 寡核苷酸序列分析 核酸杂交 

分 类 号：R440[医药卫生—诊断学]