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荧光共振能量转移(fluorescence resonance energy transfer,FRET)是指供体荧光分子发射光谱与受体分子的吸收光谱有显著的重叠且分子间距小于10 nm时发生的一种非放射性的能量转移[1],导致供体荧光淬灭而受体荧光增强或不变。近些年,FRET技术以其精准高效的特点被广泛应用在分析检测领域,为检测生物分子提供了重要的分析方法。基于FRET技术,实现了活细胞内ATP分子的检测[2],金属离子如汞离子的检测[3-4],许多疾病相关基因[5-6]以及酶活性的检测等[7-8]。
银纳米簇(AgNCs),作为一种新型的低毒性“绿色”荧光标记材料,具有量子产率高、毒性低、生物相容性好等特点[9],使得其被广泛应用在多个研究领域。在银纳米簇的合成过程中,相较于其他的合成模板,DNA更具优势,如DNA具有分子识别的功能(包括对于互补链和小分子的识别),不同序列的DNA模板可调谐不同的发射波长等[10]。
研究发现,G碱基可以增强DNA/银纳米簇(DNA/AgNCs)的荧光强度[11-12],基于此,我们设计了系列非银簇模板部分的互补链,考察G碱基个数对于银簇荧光强度的影响,实验结果显示,暴露的G碱基个数与银簇的荧光强度呈正相关关系。该实验不仅验证了G碱基对于银簇荧光的增强作用,还提示我们在设计含有G-四联体适配体的荧光探针时,可通过改变非银簇部分的互补链长短来调控荧光的淬灭及恢复程度,以获得最佳的检测效果。
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VS-100C恒温混匀仪(无锡沃信仪器制造有限公司);FL-6500荧光分光光度计(PerkinElmer);ZEN3600粒径电位测定仪(英国马尔文公司);精密电子天平(北京赛多利斯仪器系统有限公司);Vortex-Genie2多功能旋涡混合器(美国Scientific Industries公司);TGL-16C离心机(上海安亭科学仪器厂);实验室pH计 FE20[梅特勒-托利多仪器(上海)有限公司];Tecnai G2 F30高分辨率电子显微镜(荷兰FEI公司)
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硝酸银、硼氢化钠、盐酸(国药集团化学试剂有限公司);三羟甲基氨基甲烷(Tris,大连美仑生物技术有限公司);氯化镁、氯化钠、氯化钾(上海泰坦科技股份有限公司);试剂均为分析纯。实验用水为屈臣氏蒸馏水。
相关DNA序列由生工生物工程(上海)股份有限公司合成。合成DNA-AgNCs的模板序列及P1A5C5的互补序列如表1、表2所示。
表 1 DNA/AgNCs的模板序列
名称 序列(5′—3′) P1C5 GGAGGTGGTGGGGCCCCCTAATTCCCCC P1AC5 GGAGGTGGTGGGGACCCCCTAATTCCCCC P1A5C5 GGAGGTGGTGGGGAAAAACCCCCTAATTCCCCC P1N GGAGGTGGTGGGGCCCTAACTCCCC P1Y GGAGGTGGTGGGGCCCTTAATCCCC 表 2 P1A5C5的互补序列
名称 序列(5′—3′) 0G CCTCCACCACCCCTTTTT 1G CTCCACCACCCCTTTTT 2G TCCACCACCCCTTTTT 4G TCCACCCCTTTTT 5G CACCCCTTTTT 6G ACCCCTTTTT 7G CCCTTTTT
The effect of different guanine base number on fluorescence intensity of DNA/ silver nanoclusters
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摘要:
目的 通过C-G碱基互补配对的方式,考察不同鸟嘌呤碱基(G)数目对DNA/银纳米簇荧光信号强度的影响,以此来探究新的荧光探针开关构建的方法。 方法 利用核酸碱基互补配对原则,设计了一系列银纳米簇的适配体部分的互补序列,考察了银纳米簇的适配体序列中暴露的G碱基个数对荧光信号的影响。 结果 碱基G可增强银纳米簇的荧光信号强度,且荧光信号强度与G碱基个数呈现正相关关系,拟合线性方程为Y=1726.1X+8972.5,r=0.9789。 结论 该实验研究对于调节银纳米簇的荧光强度以及设计适配体为G四联体的荧光探针开关具有借鉴意义。 Abstract:Objective To investigate the effect of different guanine base numbers on the fluorescence intensity of DNA/ silver nanoclusters through C-G base complementary pairing, in order to explore a new method for the construction of fluorescent probe switches. Methods Designed complementary sequences of aptamer parts of a series of silver nanoclusters by using the nucleic acid base complementary pairing principle, and investigated the effect of the number of G bases exposed in the aptamer sequence on the fluorescence signal. Results Base G could enhance the fluorescence signal intensity of silver nanoclusters, and the fluorescence signal strength was positively correlated with the number of G bases. The fitting linear equation was Y=1726.1X+8972.5, r=0.9789. Conclusion This study is a great reference for the regulation of fluorescence intensity of silver nanoclusters and the design of G quadruplet aptamer fluorescent probe switch. -
Key words:
- DNA/AgNCs /
- complementary pairing /
- fluorescence probe /
- aptamer
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表 1 DNA/AgNCs的模板序列
名称 序列(5′—3′) P1C5 GGAGGTGGTGGGGCCCCCTAATTCCCCC P1AC5 GGAGGTGGTGGGGACCCCCTAATTCCCCC P1A5C5 GGAGGTGGTGGGGAAAAACCCCCTAATTCCCCC P1N GGAGGTGGTGGGGCCCTAACTCCCC P1Y GGAGGTGGTGGGGCCCTTAATCCCC 表 2 P1A5C5的互补序列
名称 序列(5′—3′) 0G CCTCCACCACCCCTTTTT 1G CTCCACCACCCCTTTTT 2G TCCACCACCCCTTTTT 4G TCCACCCCTTTTT 5G CACCCCTTTTT 6G ACCCCTTTTT 7G CCCTTTTT -
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