基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒

引用本文: 田梓佚, 徐牧岑, 胡顺铭, 张楚妍, 陈琛, 李永新. 基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒[J]. 分析化学, 2023, 51(1): 93-101. doi: 10.19756/j.issn.0253-3820.221154 shu

Citation: TIAN Zi-Yi, XU Mu-Cen, HU Shun-Ming, ZHANG Chu-Yan, CHEN Chen, LI Yong-Xin. A Biosensor Based on Catalytic Hairpin Assembly and Fluorescence Resonance Energy Transfer for Detection of Enterovirus 71[J]. Chinese Journal of Analytical Chemistry, 2023, 51(1): 93-101. doi: 10.19756/j.issn.0253-3820.221154 shu

基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒

四川大学华西公共卫生学院/四川大学华西第四医院, 成都 610041

通讯作者: 李永新,E-mail:liyongxin_2006@163.com

基金项目:
成都市科技局项目(No.2022-YF05-00296-SN)和四川大学大学生创新创业训练计划项目(No.20221291L)资助。

摘要: 以肠道病毒71型(EV71)为检测对象,建立了基于催化发夹组装(CHA)和荧光共振能量转移(FRET)的生物传感器。设计了检测EV71 VP1基因的CHA信号放大策略,该体系由一对发夹探针(H1、H2)构成,H1和H2分别标记了荧光基团Cy3和Cy5。当体系中有VP1基因时,可引发H1和H2催化自组装反应,从而使得Cy3和Cy5相互靠近并发生FRET,导致Cy3荧光信号降低,Cy5信号增强;当体系中没有VP1基因时,H1和H2则稳定存在于反应体系中,不发生FRET,只检测到Cy3的荧光信号。优化了缓冲液浓度、H1与H2的比例、反应温度和反应时间。在最优条件下,所构建的传感器的Cy5与Cy3的荧光强度比值与EV71 VP1基因浓度在0.5~20nmol/L范围内呈良好的线性关系,检出限为73pmol/L(3σ)。咽拭子样品的加标回收率为99.6%~103.1%,相对标准偏差在0.3%~1.7%之间。本方法具有较好的特异性及抗干扰能力,在EV71监测和手足口病早期诊断方面具有良好的应用前景。

关键词:

English

A Biosensor Based on Catalytic Hairpin Assembly and Fluorescence Resonance Energy Transfer for Detection of Enterovirus 71

West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China

Corresponding author: LI Yong-Xin, liyongxin_2006@163.com

Received Date: 30 March 2022
Revised Date: 24 September 2022
Fund Project:
Supported by the Chengdu Science and Technology Bureau Project (No.2022-YF05-00296-SN) and the Innovation and Entrepreneurship Training Program for College students of Sichuan University (No.20221291L).

Abstract: A biosensor based on catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET) was established for detection of enterovirus 71 (EV71). A CHA signal amplification strategy was designed to detect the VP1 gene of EV71, which was consisted of a pair of hairpin probes (H1 and H2). The fluorophores Cy3 and Cy5 were labeled on H1 and H2, respectively. When the VP1 gene was present, the catalytic assembly reaction of H1 and H2 was triggered, Cy3 and Cy5 got closer and FRET occurred, leading to a reduced Cy3 signal and an enhanced Cy5 signal. In the absence of VP1 gene, H1 and H2 existed stably in the reaction system and could not generate FRET. Experimental conditions including reaction buffer concentration, the ratio of H1 to H2, reaction temperature and reaction time were optimized. Under the optimized conditions, the fluorescence intensity ratio of Cy5 to Cy3 showed a good linear relationship with EV71 VP1 gene concentration in the range of 0.5-20 nmol/L, and the detection limit was 73 pmol/L (3σ). The spiked recoveries of throat swab samples ranged from 99.6% to 103.1% with the relative standard deviations of 0.3% to 1.7%, showing good specificity and anti-interference ability. The proposed sensor had a good application prospect in EV71 virus monitoring and early diagnosis of hand, foot and mouth disease.

Key words:

Enterovirus 71
/ Catalytic hairpin assembly
/ Fluorescence resonance energy transfer
/ Biosensor
/ Hand, foot and mouth disease

1. [1]
  HU Xiao-Xing, PENG Jie, FENG Yue, LIU Li, ZHANG A-Mei, XIA Xue-Shan. Life Sci. Res., 2016, 20(3):189-195. 胡晓星, 彭杰, 冯悦, 刘丽, 张阿梅, 夏雪山. 生命科学研究, 2016, 20(3):189-195.
2. [2]
  LI J, YIN X Z, LIN A W, NIE X Z, LIU L Y, LIU S H, LI N, WANG P, SONG S S, WANG S N, XU D Y. Hum. Vaccines Immunother., 2021, 17(7):2097-2100.LI J, YIN X Z, LIN A W, NIE X Z, LIU L Y, LIU S H, LI N, WANG P, SONG S S, WANG S N, XU D Y. Hum. Vaccines Immunother., 2021, 17(7):2097-2100.
3. [3]
  FAN Rong-Jun, LI Xiao-Peng, LIANG Shuang, LIANG Ying, HUAI Qing-Jie, REN Li-Na. China Med. Pharm., 2012, 2(11):14-15, 29. 范荣军, 李晓鹏, 梁爽, 梁英, 怀清杰, 任莉娜. 中国医药科学, 2012, 2(11):14-15, 29.
4. [4]
  MA Hong-Bin, HOU Jun, LI Lu-Ping, YANG Jing, GUO Jing-Xia, LIU Ai-Xia, XU Jun, NIE Wei-Min, LI Bo-An, MAO Yuan-Li. Med. J. Chin. People's Liberation Army, 2010, 35(3):239-240. 马洪滨, 侯俊, 李鲁平, 杨静, 郭静霞, 刘爱霞, 徐军, 聂为民, 李伯安, 毛远丽. 解放军医学杂志, 2010, 35(3):239-240.
5. [5]
  WU Hai-Bo, GUO Chao-Tan. Int. J. Epidemiol. Infect. Dis., 2008, 35(3):188-190. 吴海波, 郭潮潭. 国际流行病学传染病学杂志, 2008, 35(3):188-190.
6. [6]
  TAN E L, YONG L L G, QUAK S H, YEO W C A, CHOW V T K, POH C L. J. Clin. Virol., 2008, 42(2):203-206.TAN E L, YONG L L G, QUAK S H, YEO W C A, CHOW V T K, POH C L. J. Clin. Virol., 2008, 42(2):203-206.
7. [7]
  YU He-Juan, XU Bian-Li, HUANG Xue-Yong, WEI Hai-Yan, LI Xing-Le, DU Yan-Hua. Mod. Prev. Med., 2010, 37(8):1517-1519, 1523. 于贺娟, 许汴利, 黄学勇, 卫海燕, 李幸乐, 杜燕华. 现代预防医学, 2010, 37(8):1517-1519, 1523.
8. [8]
  WANG Y, LI K J, XU G L, CHEN C, SONG G Q, DONG Z Z, LIN L, WANG Y, XU Z Y, YU M X, YU X G, YING B W, FAN Y B, CHANG L Q, GENG J. Research, 2021, 2021:2813643.WANG Y, LI K J, XU G L, CHEN C, SONG G Q, DONG Z Z, LIN L, WANG Y, XU Z Y, YU M X, YU X G, YING B W, FAN Y B, CHANG L Q, GENG J. Research, 2021, 2021:2813643.
9. [9]
  ALI M M, LI F, ZHANG Z Q, ZHANG K X, KANG D K, ANKRUM J A, LE X C, ZHAO W A. Chem. Soc. Rev., 2014, 43(10):3324-3341.ALI M M, LI F, ZHANG Z Q, ZHANG K X, KANG D K, ANKRUM J A, LE X C, ZHAO W A. Chem. Soc. Rev., 2014, 43(10):3324-3341.
10. [10]
  WANG Z Y, WANG Y, LIN L, WU T, ZHAO Z Z, YING B W, CHANG L Q. Biosens. Bioelectron., 2022, 195:113663.WANG Z Y, WANG Y, LIN L, WU T, ZHAO Z Z, YING B W, CHANG L Q. Biosens. Bioelectron., 2022, 195:113663.
11. [11]
  DIRKS R M, PIERCE N A. Proc. Natl. Acad. Sci. U. S. A., 2004, 101(43):15275-15278.DIRKS R M, PIERCE N A. Proc. Natl. Acad. Sci. U. S. A., 2004, 101(43):15275-15278.
12. [12]
  WU J T, LV J R, ZHENG X Q, WU Z S. Talanta, 2021, 234:122637.WU J T, LV J R, ZHENG X Q, WU Z S. Talanta, 2021, 234:122637.
13. [13]
  YIN P, CHOI H M, CALVERT C R, PIERCE N A. Nature, 2008, 451(7176):318-322.YIN P, CHOI H M, CALVERT C R, PIERCE N A. Nature, 2008, 451(7176):318-322.
14. [14]
  LIU J M, ZHANG Y, XIE H B, ZHAO L, ZHENG L, YE H M. Small, 2019, 15(42):1902989.LIU J M, ZHANG Y, XIE H B, ZHAO L, ZHENG L, YE H M. Small, 2019, 15(42):1902989.
15. [15]
  WU Y, FU C C, SHI W B, CHEN J Y. Talanta, 2021, 235:122735.WU Y, FU C C, SHI W B, CHEN J Y. Talanta, 2021, 235:122735.
16. [16]
  ZHANG D Y, TURBERFIELD A J, YURKE B, WINFREE E. Science, 2007, 318(5853):1121-1125.ZHANG D Y, TURBERFIELD A J, YURKE B, WINFREE E. Science, 2007, 318(5853):1121-1125.
17. [17]
  LI Y X, LUO Z W, ZHANG C Y, SUN R, ZHOU C, SUN C J. TrAC, Trends Anal. Chem., 2021, 134:116142.LI Y X, LUO Z W, ZHANG C Y, SUN R, ZHOU C, SUN C J. TrAC, Trends Anal. Chem., 2021, 134:116142.
18. [18]
  ZHANG X J, HU Y, YANG X T, TANG Y Y, HAN S Y, KANG A, DENG H S, CHI Y M, ZHU D, LU Y. Biosens. Bioelectron., 2019, 138:111314.ZHANG X J, HU Y, YANG X T, TANG Y Y, HAN S Y, KANG A, DENG H S, CHI Y M, ZHU D, LU Y. Biosens. Bioelectron., 2019, 138:111314.
19. [19]
  SASMAL D K, PULIDO L E, KASAL S, HUANG J. Nanoscale, 2016, 8(48):19928-19944.SASMAL D K, PULIDO L E, KASAL S, HUANG J. Nanoscale, 2016, 8(48):19928-19944.
20. [20]
  CHEN L, ZHANG X W, ZHANG C L, ZHOU G H, ZHANG W P, XIANG D S, HE Z K, WANG H Z. Anal. Chem., 2011, 83(19):7316-7322.CHEN L, ZHANG X W, ZHANG C L, ZHOU G H, ZHANG W P, XIANG D S, HE Z K, WANG H Z. Anal. Chem., 2011, 83(19):7316-7322.
21. [21]
  CHEN L, ZHANG X W, ZHOU G H, XIANG X, JI X H, ZHENG Z H, HE Z K, WANG H Z. Anal. Chem., 2012, 84(7):3200-3207.CHEN L, ZHANG X W, ZHOU G H, XIANG X, JI X H, ZHENG Z H, HE Z K, WANG H Z. Anal. Chem., 2012, 84(7):3200-3207.
22. [22]
  XIONG L H, HE X W, ZHAO Z, KWOK R T K, XIONG Y, GAO P F, YANG F, HUANG Y L, SUNG H H, WILLIAMS I D, LAM J W Y, CHENG J Q, ZHANG R L, TANG B Z. ACS Nano, 2018, 12(9):9549-9557.XIONG L H, HE X W, ZHAO Z, KWOK R T K, XIONG Y, GAO P F, YANG F, HUANG Y L, SUNG H H, WILLIAMS I D, LAM J W Y, CHENG J Q, ZHANG R L, TANG B Z. ACS Nano, 2018, 12(9):9549-9557.
23. [23]
  CHI Xu, SHENG Chuan-Lun, WANG Jin-Hong. Chin. J. Lab. Diagn., 2017, 21(4):702-704. 迟旭, 盛传伦, 王锦鸿. 中国实验诊断学, 2017, 21(4):702-704.
24. [24]
  GUKASSYAN V, HSU Y Y, KUNG S H, KAO F J. J. Biomed. Opt., 2007, 12(2):024016.GUKASSYAN V, HSU Y Y, KUNG S H, KAO F J. J. Biomed. Opt., 2007, 12(2):024016.
25. [25]
  LU W W, KUNG F Y, DENG P A, LIN Y C, LIN C W, KUNG S H. Arch. Virol., 2017, 162(3):713-720.LU W W, KUNG F Y, DENG P A, LIN Y C, LIN C W, KUNG S H. Arch. Virol., 2017, 162(3):713-720.
26. [26]
  REN D X, SUN C J, HUANG Z J, LUO Z W, ZHOU C, LI Y X. Sens. Actuators, B, 2019, 296:126577.REN D X, SUN C J, HUANG Z J, LUO Z W, ZHOU C, LI Y X. Sens. Actuators, B, 2019, 296:126577.
27. [27]
  ZHOU C, ZOU H M, SUN C J, REN D X, XIONG W, LI Y X. Anal. Bioanal. Chem., 2018, 410(12):2981-2989.ZHOU C, ZOU H M, SUN C J, REN D X, XIONG W, LI Y X. Anal. Bioanal. Chem., 2018, 410(12):2981-2989.
28. [28]
  ZHOU C, SUN C J, LUO Z W, LIU K P, YANG X J, ZOU H M, LI Y X, DUAN Y X. Sens. Actuators, B, 2018, 254:956-965.ZHOU C, SUN C J, LUO Z W, LIU K P, YANG X J, ZOU H M, LI Y X, DUAN Y X. Sens. Actuators, B, 2018, 254:956-965.
29. [29]
  HUNAG Ke-Qiang, ZHANG Yong, XU Wen-Bo. Chin. J. Virol., 2017, 33(3):469-476. 黄克强, 张勇, 许文波. 病毒学报, 2017, 33(3):469-476.
30. [30]
  OU Xin-Hua, SUN Bian-Cheng, WANG Pei-Rong, CHEN Jing-Fang, LE Jia-Chang. Chin. J. Health Lab. Technol, 2015, 12:1932-1934. 欧新华, 孙边成, 王佩荣, 陈静芳, 乐加昌. 中国卫生检验杂志, 2015, 12:1932-1934.
31. [31]
  LI Sha, ZHANG Ru-Sheng, CHEN Jing-Fang. Int. J. Lab. Med., 2017, 38(4):470-472. 李莎, 张如胜, 陈静芳. 国际检验医学杂志, 2017, 38(4):470-472.
32. [32]
  LEE K Y. Korean J. Pediatr., 2016, 59(10):395-401.LEE K Y. Korean J. Pediatr., 2016, 59(10):395-401.
33. [33]
  MA Dong-Li, LIU Bi-Chen, ZHONG Shan. Int. J. Lab. Med., 2017, 38(22):3159-3161. 马东礼, 刘必忱, 钟山. 国际检验医学杂志, 2017, 38(22):3159-3161.

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沈阳化工大学材料科学与工程学院沈阳 110142

基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒

通讯作者: 李永新,E-mail:liyongxin_2006@163.com

English

A Biosensor Based on Catalytic Hairpin Assembly and Fluorescence Resonance Energy Transfer for Detection of Enterovirus 71

Corresponding author: LI Yong-Xin, liyongxin_2006@163.com

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒

通讯作者: 李永新,E-mail:liyongxin_2006@163.com

English

A Biosensor Based on Catalytic Hairpin Assembly and Fluorescence Resonance Energy Transfer for Detection of Enterovirus 71

Corresponding author: LI Yong-Xin, liyongxin_2006@163.com

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs