基于滚环合成的聚分子信标用于凝血酶的靶向检测

doi:10.7503/cjcu20190450

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (12): 2465.doi: 10.7503/cjcu20190450

基于滚环合成的聚分子信标用于凝血酶的靶向检测

张志庆(),汪珊珊,张子辰,马杰,王秀凤(),周亭,王芳,张国栋

中国石油大学(华东)理学院, 青岛 266580

收稿日期:2019-08-12 出版日期:2019-12-04 发布日期:2019-12-04
通讯作者: 张志庆,王秀凤 E-mail:zhangzq@upc.edu.cn;xfwang@upc.edu.cn
基金资助:
山东省自然科学基金(ZR2019MB063);山东省自然科学基金(ZR2017MB045);山东省自然科学基金(ZR2016BL14);国家自然科学基金(21603276);国家自然科学基金(21703286);青岛市应用基础研究项目(17-1-1-74-jch);中央高校基本业务项目(19CX02060A);中央高校基本业务项目(19CX02049A);中央高校基本业务项目(19CX02057A)

Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin ^†

Zhiqing ZHANG(),Shanshan WANG,Zichen ZHANG,Jie MA,Xiufeng WANG(),Ting ZHOU,Fang WANG,Guodong ZHANG

College of Science, China University of Petroleum(East China), Qingdao 266580, China

Received:2019-08-12 Online:2019-12-04 Published:2019-12-04
Contact: Zhiqing ZHANG,Xiufeng WANG E-mail:zhangzq@upc.edu.cn;xfwang@upc.edu.cn
Supported by:
? Supported by the Natural Science Foundation of Shandong Province, China(ZR2019MB063);the Natural Science Foundation of Shandong Province, China(ZR2017MB045);the Natural Science Foundation of Shandong Province, China(ZR2016BL14);the National Natural Science Foundation of China(21603276);the National Natural Science Foundation of China(21703286);the Applied Basic Research Project of Qingdao, China(17-1-1-74-jch);the Fundamental Research Funds for the Central Universities, China(19CX02060A);the Fundamental Research Funds for the Central Universities, China(19CX02049A);the Fundamental Research Funds for the Central Universities, China(19CX02057A)

摘要/Abstract

摘要：

采用滚环扩增(RCA)合成得到的DNA长链打开带适配体的分子信标, 由于RCA长链上带有多个与分子信标(MB)互补的重复序列, 其打开分子信标的能力比单一互补短链提高了上百倍. 所形成的聚多价分子信标组装体, 在分子信标浓度相同的情况下, 打开后的荧光强度也大幅上升; 并且由于组装体上多价适配体的存在, 聚分子信标对凝血酶的靶向能力显著增强. 实验结果表明, 聚分子信标结合凝血酶后, 其荧光信号与凝血酶浓度呈线性关系, 检测灵敏度达到0.2 nmol/L, 该体系的构建有利于实现对凝血酶的高灵敏、特异性检测.

关键词: 滚环扩增合成, 聚分子信标, 适配体, 凝血酶

Abstract:

A long chain structure of DNA synthesized by rolling circle amplification(RCA) was used to turn on aptamer-based molecular beacons(MBs). Owing to the multiple complementary repeat units, the RCA scaffold can hybridize tens or hundreds of MBs to form polyvalent aptamer probes. The unfold ability and the fluorescence intensity of MBs were both improved by RCA compared to short single chains. Experimental results demonstrated that the system is significantly more effective than monovalent molecular beacon in targeting thrombin and signal sensitivity because of multivalent effects. Once the poly valent molecular beacon binding to the thrombin, there was a linear relationship between fluorescence signal and thrombin concentration, and the detection limit of thrombin concentration was 0.2 nmol/L. The establishment of this system is conducive to the realization of highly sensitive and specific detection of thrombin.

Key words: Rolling circle amplification, Polyvalent molecular beacon, Aptamer, Thrombin

中图分类号:

O657.3
O648

TrendMD:

张志庆,汪珊珊,张子辰,马杰,王秀凤,周亭,王芳,张国栋. 基于滚环合成的聚分子信标用于凝血酶的靶向检测. 高等学校化学学报, 2019, 40(12): 2465.

Zhiqing ZHANG,Shanshan WANG,Zichen ZHANG,Jie MA,Xiufeng WANG,Ting ZHOU,Fang WANG,Guodong ZHANG. Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin ^†. Chem. J. Chinese Universities, 2019, 40(12): 2465.

图/表 7

Table 1 DNA Sequences

Name	DNA sequence(5'-3')	Source
Circle DNA1	TGTCTTCGCCTTCTTGTTTCCTTTCCTTGAAACTTCTTCCTTTCT-	TaKaRa Biotechnology(Dalian) Co., Ltd.
	TTCTTTCGACTAAGCACC
Primer DNA2	GGCGAAGACAGGTGCTTAGTC	TaKaRa Biotechnology(Dalian) Co., Ltd.
Repeated sequence of	GGTGCTTAGTCGAAAGAAAGAAAGGAAGAAGTTTCAAGGAA-	TaKaRa Biotechnology(Dalian) Co., Ltd.
RCA product	AGGAAACAAGAAGGCGAAGACA
MB	(FAM)GGAAGAAGTGGTTGGTGTGGTTGGCCCCTGTCTTCGC-	Sangon Biotechnology(Shanghai) Co., Ltd.
	CTTCTTGTTTCCTTTCCTTGAAACTTCTTCC(DABCYL)
FC short chain	TTTCTTTCTTTCGACTAAGCACC	Sangon Biotechnology(Shanghai) Co., Ltd.
63-cDNA	GGTGCTTAGTCGAAAGAAAGAAAGGAAGAAGTTTCAAGGAA-	Sangon Biotechnology(Shanghai) Co., Ltd.
	AGGAAACAAGAAGGCGAAGACA

Fig.1 Electrophoresis gel(A) and UV-Vis spectrum(B) of RCA products

Fig.2 Illustration of the signal-amplifying of molecular beacon(A) and poly(MB) detection of thrombin(B)

Fig.3 Fluorescence spectra(A) of different concentrations of FC and the signal-to-background ratio(S/B)(B) cMB=30 nmol/L. a. control; cFC:cRCA:cMB : b. 0:1:3; c. 1:1:3; d.10:1:3; e. 30:1:3; f. 100:1:3; g. 300:1:3; h.1000:1:3.

Fig.4 Fluorescence spectra(A, B) and relative fluorescence intensity ratios(C, D) under different conditions (A) a. control; cRCA:cMB : b. 1:100; c.1:30; d.1:10; e.1:5; f. 1:3; g. 1:1. cMB=30 nmol/L. (B) a. Control; c63-cDNA:cMB: b. 1:100; c.1:30; d.1:10; e.1:3; f. 1:1; g. 3:1; h. 10:1; i. 30:1; j. 100:1. cMB=30 nmol/L. (C) RCA-MB assembly and 63-cDNA-MB assembly with 30 nmol/L MB; (D) RCA-MB assembly with different concentrations of MB.

Fig.5 Detection sensitivity of MB-RCA for thrombin (A) Fluorescence spectra. cMB =100 nmol/L. cThrombin/(μmol·L-1): a. 0; b. 0.0001; c. 0.0003; d. 0.001; e. 0.003;f. 0.01; g. 0.03; h. 0.1; i. 0.3; j. 1. (B) Relative fluorescence intensity ratio vs. the concentration of thrombin.

Table 2 Comparison of different methods for detection of thrombin

Method	Linear range/(nmol·L^-1)	Detection limit/(nmol·L^-1)	Ref.
Electrochemical sensor based on aptamers	40—100	10	[26]
Aptamer biosensor based on guanine-quenching		0.81	[27]
Fluorescence detection based on molecular beacon	0—30	0.83	[23]
Fluorescence detection based on dual quenching molecular beacon	0.4—40	0.18	[28]
Polyvalent molecular beacon probe base on RCA	0.1—30	0.20	This work

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基于滚环合成的聚分子信标用于凝血酶的靶向检测

Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin ^†

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基于滚环合成的聚分子信标用于凝血酶的靶向检测

Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin †

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Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin ^†