脱氧核糖核酸-银纳米簇荧光探针用于沙眼衣原体的定量检测  

Deoxyribonucleic Acid-Silver Nanoclusters Fluorescent Probe for Quantitative Detection of Chlamydia Trachomatis

作　　者：李丹靓 周朴帆 刘璐瑶 张雪冰 梁好 陈丽丽 LI Danliang;ZHOU Pufan;LIU Luyao;ZHANG Xuebing;LIANG Hao;CHEN Lili(Department of Public Health Laboratory Sciences,School of Public Health,Hengyang Medical School,University of South China,Hengyang421001,China;Zhuzhou Hetang District Center for Disease Control and Prevention,Zhuzhou 412000,China)

机构地区：[1]南华大学衡阳医学院公共卫生学院卫生检验与检疫系,衡阳421001 [2]株洲市荷塘区疾病预防控制中心,株洲412000

出　　处：《理化检验（化学分册）》2023年第5期506-511,共6页Physical Testing and Chemical Analysis(Part B:Chemical Analysis)

基　　金：衡阳市科技计划项目(衡财教指[2020]67号)。

摘　　要：采用化学法合成脱氧核糖核酸-银纳米簇(DNA-AgNCs)荧光探针,并用于沙眼衣原体色氨酸合成酶β链基因(目标序列)的选择性荧光检测。在pH 6.6乙酸盐缓冲溶液中加入两条DNA探针和硝酸银,在还原剂硼氢化钠作用下合成了荧光强度大的DNA-AgNCs。在pH 5.5乙酸铵缓冲溶液中加入目标序列和DNA-AgNCs溶液,使混合溶液体积达到100μL,其中DNA-AgNCs的浓度达到1.5μmol·L^(-1)(以DNA浓度计),于15℃反应10 min,目标序列与其中一条DNA探针杂交,DNA-AgNCs结构被破坏,检测体系荧光猝灭。结果显示:DNA-AgNCs颗粒呈类球形,粒径约为2 nm,分散性良好且未发生团聚,荧光激发、发射波长分别在558,610 nm处。猝灭程度ΔF(目标序列添加后、前的检测体系荧光强度的差值)与目标序列浓度在0.23~1.10μmol·L^(-1)内呈线性关系,检出限(3s/k)为57 nmol·L^(-1),测定值的相对标准偏差(n=11)为1.5%~4.9%。添加目标序列以及10倍目标序列浓度的同源序列、症状相似序列、衣原体序列和随机序列,仅目标序列加入时,检测体系荧光猝灭,说明合成的荧光探针具有优异的选择性;以海拉细胞裂解液和胎牛血清作模拟基质进行加标回收试验,回收率为98.0%~100%。Deoxyribonucleic acid-silver nanoclusters(DNA-AgNCs)fluorescent probes were synthesized by chemical method and used for selective fluorescence detection of tryptophan synthetaseβchain gene of Chlamydia trachomatis(the target sequence).Two DNA probes and argenti nitras were added into the pH 6.6acetate buffer solution to synthesize DNA-AgNCs with high fluorescence intensity in the presence of reducing agent of sodium borohydride.The target sequence solution and DNA-AgNCs solution were added into the pH 5.5ammounium acetate buffer solution,and the mixed solution was made its volume up to 100μL[the concentration(caculated by DNA)of DNA-AgNCs reached to 1.5μmol·L^(-1)],and reacted for 10min at 15℃.The target sequence was hybridized with one of the 2 DNA probes,and the structure of DNA-AgNCs was destroyed,resulting in fluorescence quenching.It was shown that DNA-AgNCs particles were spherical in shape with particle size of about 2nm,good dispersion and no aggregation.The excitation and emission wavelengths of fluorescence were at 558,610nm,respectively.Linear relationship between values of the concentration of the target sequence and theΔF(the difference between fluorescence intensities of the detection system after and before adding of the target sequence)was kept in the range of 0.23-1.10μmol·L^(-1),with detection limit(3s/k)of 57nmol·L^(-1),and RSDs(n=11)of the determined values ranged from 1.5%to 4.9%.Adding the target sequence and 10times of the target sequence concentration of homologous sequence,symptom similarity sequence,Chlamydia sequence and random sequence,the detection system showed fluorescence quenching effect when the target sequence was added,indicating that the synthesized fluorescent probe had excellent selectivity.Test for recovery was made by standard addition method using HeLa cell lysate and fetal bovine serum as simulated matrices,giving recoveries in the range of 98.0%-100%.

关 键 词：脱氧核糖核酸-银纳米簇 沙眼衣原体 荧光检测 

分 类 号：O657.32[理学—分析化学]