应用恒电位在金基底表面电化学沉积纳米金，通过Au—S键将巯基修饰DNA探针固定在纳米金表面，并与互补靶序列杂交，构建计时库仑电化学DNA传感器，并检测急性早幼粒细胞白血病（APL）PML/RARα融合基因. 采用电子扫描显微镜（SEM）与电化学交流阻抗技术（EIS）观察纳米金和表征DNA传感器的构筑过程. 以氯化六氨合钌（[Ru(NH3)6]Cl3, RuHex）作电化学杂交指示剂，由计时库仑法检测人工合成APL的PML/RARα融合基因. 结果表明，纳米金能放大RuHex检测信号，杂交前后电量差值（?Q）与靶标链DNA浓度的对数（lgC）在1.0×10-13 mol?L-1 ~ 1.0×10-9 mol?L-1 范围内呈线性关系，检出下限3.7×10-14 mol?L-1 (S/N = 3). 该法操作简便、特异性好，有望用于实际样品的检测.
王丽满
,
林丽清
,
翁少煌
,
林新华
,
陈元仲
. 纳米金放大计时库仑法的PML/RARα融合基因检测[J]. 电化学, 2012
, 18(3)
: 286
-290
.
DOI: 10.61558/2993-074X.2917
potential. The sulfydryl modified probe was immobilized onto surface of gold nanoparticles via Au-S covalent bond and hybridized with complementary target DNA sequences, then the DNA sensor was fabricated for the detection of PML/RARα fusion gene by chronocoulometry in acute promyelocytic leukemia (APL). Scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) were used to characterize the surface morphology of gold nanoparticles and the constructing process of sensor. With hexaammineruthenium(Ⅲ) chloride (RuHex) as a novel electrochemical indicator, the chronocoulometric DNA biosensor was employed to monitor artificial APL PML/RARα fusion gene fragment．Experimental results showed that the gold nanoparticles amplified the detection signal of RuHex. The linear relationship between the charge difference in the RuHex after and before hybridization and logarithmic concentrations (log C) of target sequence was observed in the concentration range of 1.0×10-13 ~ 1.0×10-9 mol?L-1, and the detection limit had been estimated to be 3.7×10-14 mol?L-1 (S/N=3). This approach is simple, has good specificity, and is promising to apply for the detection of APL in the real sample for medical diagnostics.
[1] Wang J, Zhang S J, Zhang Y Z. Fabrication of chronocoulometric DNA sensor based on gold nanoparticles/poly(L-lysine) modi?ed glassy carbon electrode [J]. Analytical Biochemistry, 2010, 396(2): 304-309.
[2] Ren R, Leng C C, Zhang S S. A chronocoulometric sensor based on screen-printed electrode doped with ionic liquid and polyaniline nanotubes [J].Biosensors and Bioelectronics, 2010, 25(9): 2089-2094.
[3] Jiang W (姜炜), Huang L (黄蕾), Zhang Y Z (张玉忠). Fabrication of dioxyribonucleic acid chronocoulometric biosensor based on gold nanoparticles/poly(ferulic acid)/multiwalled carbon nanotubes modified glassy carbon electrode [J].Chinese Journal of Anaylytical Chemistry (分析化学), 2011, 39(7): 1038-1042.
[4] Wilson H, Miller J, Kristi L, et al. Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RARα fusion mRNA [J]. Blood, 1993, 82(6): 1689-1698.
[5] Zhang J Y (张建瑜), Tang J M (唐加明). Clinic study about 21 cases of acute promyelocytic leukemia [J]. International Medicine ＆ Health Guidance News (国际医药卫生导报), 2007, 13(12): 28-31.
[6] Seishi O, Kinuko M, Yuko S, et al. Detection of the PML/RARα fusion gene in acute promyelocytic leukemia with a complex translocation involving chromosomes 15, 17, and 18 [J]. Cancer Genetics and Cytogenetics, 1993, 69(2): 113-117.
[7] Mokany E, Todd A V, Fuery C J, et al. Diagnosis and monitoring of PML-RARalpha-positive acute promyelocytic leukemia by quantitative RT-PCR [J]. Methods in Molecular Medicine, 2006, 125: 127-147.
[8] Jin Y H, Kyong E K, Kyeong H K, et al. Identification of PML-RARα rearrangement by RT-PCR and sequencing in an acute promyelocytic leukemia without t(15;17) on G-banding and FISH [J]. Leukemia Research, 2007, 31(2): 239-243.
[9] Daniela D, Roberta R, Franco M, et al. The PML/RARa fusion gene in the diagnosis and monitoring of acute promyelcocytic leukemia [J]. Molecular basis of disease, 1995, 80: 155-160.
[10] Steel A B, Herne T M, Tarlov M J. Electrochemical quantitation of DNA immobilized on gold [J]. Analytical Chemistry, 1998, 70(22): 4670-4677.
[11] Trasatti S, Petrii O A. Real surface area measurements in electrochemistry [J]. Electroanalytical Chemistry, 1992, 327(1/2): 353-376.
[12] Gao W, Xia X H, Xu J J, et al. Three-dimensionally ordered macroporous gold structure as an efficient matrix for solid-state electrochemiluminescenec of Ru(bpy)32+/TPA system with high sensitivity [J]. Journal of Physical Chemistry C, 2007, 111(33): 12213-12219.
[13] Wang H M (汪红梅), Weng S H (翁少煌), Lin L Q (林丽清), et al. Study on the detection of PML/RARα fusion gene in acutepromyelocytic leukemia based on chronocoulometric [J]. Journal of Instrumental Analysis (分析测试学报), 2011, 30(8): 872-876.
