基于聚合酶辅助信号放大的电化学发光DNA传感器研究
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摘要
构建一种新型基于聚合酶辅助电致化学发光DNA传感器,利用循环链置换聚合反应、辅助目标mRNA循环以及量子点的信号放大,实现超灵敏检测目标mRNA。将巯基修饰的发卡型捕获探针(Capture DNA,CP)通过Au-S键组装到Fe_3O_4@Au表面,并通过磁性组装到磁控玻碳电极上。目标mRNA存在时,目标mRNA打开发卡CP,与之杂交形成dsDNA;然后加入聚合酶、引物链(DNA1)及碱基,引物链开始扩增,将目标mRNA取代,释放的目标mRNA重新结合CP,引发下一轮扩增循环,使信号循环放大,最后加入TGACd Te量子点标记的DNA2,与打开后的CP末端序列通过碱基互补配对结合,进行电化学发光检测。在1×10~(-15)~1×10~(-11)mol/L范围内,目标mRNA浓度的对数与ECL信号呈良好的线性关系,检出限为3.4×10~(-16)mol/L。人体血清样加标回收率为97.2%~102.3%。本方法通过加入聚合酶使目标mRNA循环检测以及结合量子点标记的信号放大协同提高了检测的灵敏度。结果表明,此传感器具有良好的选择性、稳定性和重现性。
A novel polymerase-based electrochemiluminescence DNA sensor was constructed for messenger RNA( mRNA) detection by cyclic chain displacement polymerization,assisted by target mRNA cycle,and quantum dots signal amplification. Firstly,the mercapto-modified capture-type capture DNA( CP) was immobilized on the surface of a magneto-controlled glassy carbon electrode via Au-S bond. After adding the target mRNA,CP was opened and hybridized with mRNA to form ds DNA. After adding polymerase,primer chain( DNA1) and the base,the primer chain was extended to replace the target mRNA. After one cycle,the mRNA chain could open another hairpin in order to carry out next cycle of amplification. Finally,electrochemical luminescence detection was carried out by adding DNA2 labeled TGA-Cd Te quantum dots. The amplification of the target mRNA by the addition of polymerase and the signal combined with the quantum dot mark improved the sensitivity of the sensor greatly. The result showed that the logarithm of target mRNA concentration had a good linear relationship with the corresponding ECL signal in the range of 1 × 10~(-15)-1 ×10~(-11)mol/L,with the detection limit of 3. 4 × 10~(-16)mol/L( S/N = 3). Under the optimal conditions,the recoveries of mRNA spiked in human serum sample were 97. 2%-102. 3%. This sensor exhibited good selectivity,stability and reproducibility.
A novel polymerase-based electrochemiluminescence DNA sensor was constructed for messenger RNA( mRNA) detection by cyclic chain displacement polymerization,assisted by target mRNA cycle,and quantum dots signal amplification. Firstly,the mercapto-modified capture-type capture DNA( CP) was immobilized on the surface of a magneto-controlled glassy carbon electrode via Au-S bond. After adding the target mRNA,CP was opened and hybridized with mRNA to form ds DNA. After adding polymerase,primer chain( DNA1) and the base,the primer chain was extended to replace the target mRNA. After one cycle,the mRNA chain could open another hairpin in order to carry out next cycle of amplification. Finally,electrochemical luminescence detection was carried out by adding DNA2 labeled TGA-Cd Te quantum dots. The amplification of the target mRNA by the addition of polymerase and the signal combined with the quantum dot mark improved the sensitivity of the sensor greatly. The result showed that the logarithm of target mRNA concentration had a good linear relationship with the corresponding ECL signal in the range of 1 × 10~(-15)-1 ×10~(-11)mol/L,with the detection limit of 3. 4 × 10~(-16)mol/L( S/N = 3). Under the optimal conditions,the recoveries of mRNA spiked in human serum sample were 97. 2%-102. 3%. This sensor exhibited good selectivity,stability and reproducibility.
引文
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33 Zhao F L,Xie Q Y,Xu M F,Wang S Y,Zhou J Y,Liu F.Biosens.Bioelectron.,2015,66:238-243
2 Pang H X,Wang C W,Wang J,Sun Z W,Xiao R,Wang S Q.Biosens.Bioelectron.,2016,79:574-580
3 Wang H S,Yang M S,Xu J,Zou B K,Zhou Q,Bian J S,Wang X W.Tumor.Biol.,2016,37(1):723-727
4 Martens-Uzunova E S,B9ttcher R,Croce C M,Jenster G,Visakorpi T,Calin G A.Eur.Urol.,2014,65(6):1140-1151
5 Tran D A,Bai A Y,Singh P,Wu X W,Szabo P E.Nucleic Acids Res.,2014,42(3):1772-1783
6 Buxbaum A R,Wu B,Singer R H.Science,2014,343(6169):419-422
7 Yang X F,Yang J H,Wang J L,Wen Q,Wang H,He J C,Hu S F,He W T,Du X L,Liu S D,Ma L.Sci.Rep.,2016,6:38963
8 Zhang N,Bing J,Liu X J,Qi C,Shen L Y,Wang L L,Shangguan D H.Chem.Sci.,2015,6(7):3831-3838
9 Yang D,Gao L,Wang T F,Qiao Z D,Liang Y J,Zhang P.Febs.Lett.,2014,588(23):4448-4456
10 Liu Y Q,Zhang M,Yin B C,Ye B C.Anal.Chem.,2012,84(12):5165-5169
11 Adinolfi B,Pellegrino M,Giannetti A,Tombelli S,Trono C,Sotgiu G,Varchi G,Ballestti M,Posati T,Carpi S,Nieri P,Baldini F.Biosens.Bioelectron.,2017,88:15-24
12 Wang S,Zhang L Q,Wan S,Cansiz S,Cui C,Liu Y,Cai R,Hong C Y,Teng I T,Shi M L,Wu Y,Dong Y Y,Tan W H.ACS.Nano,2017,11(4):3943-3949
13 Li J P,Sun M,Wei X P,Zhang L P,Zhang Y.Biosens.Bioelectron.,2015,74:423-426
14 Bellón S,Vicent S V,Falcón R,Martín T P,Bursa J.J.Clin.Virol.,2016,82:S63-S64
15 Clancy E,Coughlan H,Higgins O,Boo T W,Cormican M,Barret L,Smith T J,Reddington K,Barry T.J.Microbiol.Meth.,2016,127:197-202
16 Baby N,Faust A C,Smith T,Sheperd L A,Knoll L,Goodman E L.Antimicrob.Agents.Ch.,2017,61(4):e02432
17 Hu R,Liu T,Zhang X B,Huan S Y,Wu C C,Fu T,Tan W H.Anal.Chem.,2014,86(10):5009-5016
18 Hu Y H,Xu X Q,Liu Q H,Wang L,Lin Z Y,Chen G N.Anal.Chem.,2014,86(17):8785-8790
19 Chen J H,Zhang J,Guo Y,Li J,Fu F F,Yang H H,Chen G N.Chem.Commun.,2011,47(28):8004-8006
20 Rand A C,Jain M,Eizenga J M,Brown A M,Olsen H E,Akeson M,Paten B.Nat.Methods,2017,14(4):411-413
21 Liu Y,Yu J.Microchim.Acta,2016,183(1):1-19
22 Samanta A,Banerjee S,Liu Y.Nanoscale,2015,7(6):2210-2220
23 Coopersmith K,Han H,Maye M M.Langmuir,2015,31(27):7463-7471
24 Lin C S,Wu Y F,Luo F,Chen D G,Chen X.Biosens.Bioelectron.,2014,59:365-369
25 Peng H P,Hu Y,Liu P,Deng Y N,Wang P,Chen W,Liu A L,Chen Y Z,Lin X H.Sens.Actuators B,2015,207:269-276
26 Ren W R,Gao Z Y,Li N B,Luo H Q.Biosens.Bioelectron.,2015,63:153-158
27 Yang L L,Tao Y Z,Yue G Y,Li R B,Qiu B,Guo L H,Lin Z Y,Yang H H.Anal.Chem.,2016,88(10):5097-5103
28 Cai H D,Li K G,Shen M W,Wen S H,Luo Y,Peng C,Zhang G X,Shi X Y.J.Mater.Chem.,2012,22(30):15110-15120
29 Fang Q L,Zhang X Y,Xu H J,Qian H S,Xuan S H.Chem.Lett.,2016,45(5):535-537
30 Aliberti A,Cusano A M,Battista E,Causa F,Netti P A.Analyst,2016,141(4):1250-1256
31 Xu H,Xue C,Zhang R B,Chen Y R,Li F,Shen Z F,Jia L,Wu Z S.Sens.Actuators B,2017,243:1240-1247
32 Li D D,Cheng W,Yan Y R,Zhang Y,Yin Y B,Ju H X,Ding S J.Talanta,2016,146:470-476
33 Zhao F L,Xie Q Y,Xu M F,Wang S Y,Zhou J Y,Liu F.Biosens.Bioelectron.,2015,66:238-243