Citation: FU Xin, ZOU Ting, ZHANG He, ZHANG Pei-Rou, JIANG Xu-Chun. Ultrasensitive Colorimetric Sensor for Detection of Thrombin Based on Loop Mediated Isothermal Signal Amplification Triggered by Aptamer Sandwich-Mediated Enzymatic Digestion[J]. Chinese Journal of Analytical Chemistry, ;2022, 50(7): 1022-1031. doi: 10.19756/j.issn.0253-3820.210793 shu

Ultrasensitive Colorimetric Sensor for Detection of Thrombin Based on Loop Mediated Isothermal Signal Amplification Triggered by Aptamer Sandwich-Mediated Enzymatic Digestion

Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling, College of Materials and Chemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China

Corresponding author: ZHANG He, mzhang_he@126.com
Received Date: 14 October 2021
Revised Date: 20 December 2021

Fund Project: Supported by the National Natural Science Foundation of China (No.21005067) and the Natural Science Foundation of Hunan Province, China (No.2019JJ40055).

An ultrasensitive colorimetric sensor for detection of thrombin was designed based on loop mediated isothermal signal amplification (LAMP) triggered by aptamer sandwich-mediated enzymatic digestion. In the presence of thrombin, aptamer capable of recognizing thrombin heparin site and fibrinogen site was used to recognize thrombin and construct a sandwich structure. The Nb.BsrDI nicking endonuclease could recognize and cleave this complementary sequence to start-up loop mediated isothermal amplification. G-quadruplex-hemin DNAzyme could catalyze the oxidation of ABTS to ABTS^·+ in the presence of H₂O₂, and the color changed to green. Under the optimal experimental conditions, the linear detection range of this method for thrombin was 0.01-1.0 ag/mL, the detection limit (3σ) was 0.008 ag/mL, and the regression equation was ΔA_{420 nm}=0.187C_thrombin+0.171 (R²=0.991). When there were a large number of other interfering proteins in the serum sample, the sensor still had a high selectivity to thrombin. When this method was applied to the detection of thrombin content in serum samples, the recoveries were 96.1%-103.2%. This method innovatively integrated aptamer-based sandwich assay, loop-mediated isothermal amplification technology, and G-quadruplex-hemin DNAzyme enzymatic signal amplification technology that caused the ultrasensitive detection of thrombin. The method showed many advantages such as lower cost, simple operation and good stability, and could be used for highly sensitive clinical detection of thrombin in human serum.
- Thrombin,
- Loop mediated isothermal amplification,
- G-quadruplex-hemin DNAzyme,
- Colorimetric analysis
1. [1]
  PEACOCK R B, MCGRANN T, TONELLI M, KOMIVES E A. Sci. Rep., 2021, 11(1):9354.
2. [2]
3. [3]
  SAPSFORD K E, BERTI L, MEDINTZ I L. Angew. Chem., Int. Ed., 2006, 45(28):4562-4589.
4. [4]
  HERNANDEZ-RODRIGUEZ N A, CAMBREY A D, CHAMBERS R C, GRAY A J, MCANULTY R J, LAURENT G J, HARRISON N K, SOUTHCOTT A M, DUBOIS R M, BLACK C M. Lancet, 1995, 346(8982):1071-1073.
5. [5]
  KITAMOTO Y, IMAMURA T, FUKUI H, TOMITA K. Kidney Int., 1998, 54:1767-1768.
6. [6]
  NIU S, QU L, ZHANG Q, LIN J. Anal. Biochem., 2012, 421(2):362-367.
7. [7]
  MANN K G. J. Thromb. Haemost., 2006, 4(1):58-59.
8. [8]
  RAND M D, LOCK J B, VAN'T VEER C, GAFFNEY D P, MANN K G. Blood, 1996, 88(9):3432-3445.
9. [9]
  OBUBUAFO A, BALAMURUGAN S, SHADPOUR H, SPIVAK D, MCCARLEY R L, SOPER S A. Electrophoresis, 2008, 29(16):3436-3445.
10. [10]
  CHOI J H, CHEN K H, STRANO M S. J. Am. Chem. Soc., 2006, 128(49):15584-15585.
11. [11]
12. [12]
  XU H, CUI H, YIN Z, WEI G, LIAO F, SHU Q, MA G, CHENG L, HONG N, XIONG J, FAN H. Bioelectrochemistry, 2020, 134:107522.
13. [13]
  CHENG L, XU C, CUI H, LIAO F, HONG N, MA G, XIONG J, FAN H. Anal. Chim. Acta, 2020, 1111:1-7.
14. [14]
  GUO W J, YANG X Y, WU Z, ZHANG Z L. J. Mater. Chem. B, 2020, 8(16):3574-3581.
15. [15]
  ZHAO Q, LU X, YUAN C G, LI X F, LE X C. Anal. Chem., 2009, 81(17):7484-7489.
16. [16]
17. [17]
  SUN Y, ZHU X, LIU H, DAI Y, HAN R, GAO D, LUO C, WANG X, WEI Q. ACS Appl. Mater. Interfaces, 2020, 12(5):5569-5577.
18. [18]
  YU N, WU J. Biosens. Bioelectron., 2019, 146:111726.
19. [19]
  LI X, WU Y, NIU J, JIANG D, XIAO D, ZHOU C. J. Mater. Chem. A, 2019, 7(34):5161-5169.
20. [20]
  LIU Y Z, JIANG X K, CAO W F, SUN J Y, GAO F. Sensors-Basel, 2018, 18(12):4289.
21. [21]
  ZHAO Q, GAO J. Biosens. Bioelectron., 2015, 63:21-25.
22. [22]
  BEZUNEH T T, FEREJA T H, ADDISU K S, LI H J, JIN Y D. Microchem. J., 2021, 160:105649.
23. [23]
  LIN X, CHEN Q, LIU W, LI H, LIN J M. Biosens. Bioelectron., 2014, 56:71-76.
24. [24]
  YIGIT M V, MAZUMDAR D, LU Y. Bioconjugate Chem., 2008, 19(2):412-417.
25. [25]
  KIM K S, LEE H S, YANG J A, JO M H, HAHN S K. Nanotechnology, 2009, 20(23):235501.
26. [26]
  WALKER G T, FRAISER M S, SCHRAM J L, LITTLE M C, NADEAU J G, MALINOWSKI D P. Nucleic Acids Res., 1992, 20(7):1691-1696.
27. [27]
  LIZARDI P M, HUANG X H, ZHU Z R, BRAY-WARD P, THOMAS D C, WARD D C. Nat. Genet., 1998, 19(3):225-232.
28. [28]
  NIMITPHAK T, KIATPATHOMCHAI W, FLEGEL T W. Nucleic Acids Res., 2000, 28(12):e63.
29. [29]
  VINCENT M, XU Y, KONG H. EMBO Rep., 2004, 5(8):795-800.
30. [30]
  LIN Q, XU P, LI J, YIN C, FENG J. Microb. Pathog., 2017, 109:183-188.
31. [31]
  XUE T, MA Z, LIU F, DU W, AN C. BMC Pulm. Med., 2020, 20(1):70.
32. [32]
  ULEP T H, DAY A S, SOSNOWSKI K, SHUMAKER A, YOON J Y. Sci. Rep., 2019, 9:9629.
33. [33]
34. [34]
  ZHOU W, GONG X, XIANG Y, YUAN R, CHAI Y. Biosens. Bioelectron., 2014, 55:220-224.
35. [35]
36. [36]
  LI Z, ZHAO J, WANG Z, DAI Z. Anal. Chim. Acta, 2018, 1008:90-95.
37. [37]
  XU S Y, ZHU Z, ZHANG P, CHAN S H, SAMUELSON J C, XIAO J, INGALLS D, WILSON G G. Nucleic Acids Res., 2007, 35(14):4608-4618.
38. [38]
  ZHANG H, HU X J, FU X. Biosens. Bioelectron., 2014, 57:22-29.
39. [39]
40. [40]
  ZHANG H, LEI Z X, FU X, DENG X C, WANG Q, GU D Y. Sens. Actuators, B, 2017, 246:896-903.
1. [1]
  Liwei Wang , Guangran Ma , Li Wang , Fugang Xu . A Comprehensive Analytical Chemistry Experiment: Colorimetric Detection of Vitamin C Using Nanozyme and Smartphone. University Chemistry, 2024, 39(8): 255-262. doi: 10.3866/PKU.DXHX202312094
2. [2]
  Conghao Shi , Ranran Wang , Juli Jiang , Leyong Wang . The Illustration on Stereoisomers of Macrocycles Containing Multiple Chiral Centers via Tröger Base-based Macrocycles. University Chemistry, 2024, 39(7): 394-397. doi: 10.3866/PKU.DXHX202311034
3. [3]
  Quanliang Chen , Zhaohui Zhou . Research on the Active Site of Nitrogenase over Fifty Years. University Chemistry, 2024, 39(7): 287-293. doi: 10.3866/PKU.DXHX202310133
4. [4]
  Zhexue Lu , Ping Wu , Huihui Li , Libai Wen . 四“味”一体的无机及分析化学课程思政. University Chemistry, 2025, 40(6): 333-340. doi: 10.12461/PKU.DXHX202405196
5. [5]
  Heng Zhang . Determination of All Rate Constants in the Enzyme Catalyzed Reactions Based on Michaelis-Menten Mechanism. University Chemistry, 2024, 39(4): 395-400. doi: 10.3866/PKU.DXHX202310047
6. [6]
  Yerong Chen , Bingbin Yang , Xinglei He , Yuqi Lin , Keyin Ye . Enzyme-Directed Evolution Enables Bioconversion of Organosilicon Compounds. University Chemistry, 2025, 40(10): 121-129. doi: 10.12461/PKU.DXHX202411054
7. [7]
  Tengyue ZHANG , Jingjing FENG , Zili LIANG , Jia′nan DAI , Jing MA . Optimization of C-doped BiVO₄ performance for tetracycline degradation using response surface methodology-assisted orthogonal experiments. Chinese Journal of Inorganic Chemistry, 2025, 41(12): 2561-2574. doi: 10.11862/CJIC.20250104
8. [8]
  Chunmei GUO , Weihan YIN , Jingyi SHI , Jianhang ZHAO , Ying CHEN , Quli FAN . Facile construction and peroxidase-like activity of single-atom platinum nanozyme. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1633-1639. doi: 10.11862/CJIC.20240162
9. [9]
  Yu Dai , Xueting Sun , Haoyu Wu , Naizhu Li , Guoe Cheng , Xiaojin Zhang , Fan Xia . Determination of the Michaelis Constant for Gold Nanozyme-Catalyzed Decomposition of Hydrogen Peroxide. University Chemistry, 2025, 40(5): 351-356. doi: 10.12461/PKU.DXHX202407052
10. [10]
  Pengli GUAN , Renhu BAI , Xiuling SUN , Bin LIU . Trianiline-derived aggregation-induced emission luminogen probe for lipase detection and cell imaging. Chinese Journal of Inorganic Chemistry, 2025, 41(9): 1817-1826. doi: 10.11862/CJIC.20250058
11. [11]
  Jianjun LI , Mingjie REN , Lili ZHANG , Lingling ZENG , Huiling WANG , Xiangwu MENG . UV-assisted degradation of tetracycline hydrochloride by MnFe₂O₄@activated carbon activated persulfate. Chinese Journal of Inorganic Chemistry, 2024, 40(10): 1869-1880. doi: 10.11862/CJIC.20240187
12. [12]
  Junjie Zhang , Yue Wang , Qiuhan Wu , Ruquan Shen , Han Liu , Xinhua Duan . Preparation and Selective Separation of Lightweight Magnetic Molecularly Imprinted Polymers for Trace Tetracycline Detection in Milk. University Chemistry, 2024, 39(5): 251-257. doi: 10.3866/PKU.DXHX202311084
13. [13]
  Jinwang Wu , Qijing Xie , Chengliang Zhang , Haifeng Shi . Rationally Designed ZnFe_1.2Co_0.8O₄/BiVO₄ S-Scheme Heterojunction with Spin-Polarization for the Elimination of Antibiotic. Acta Physico-Chimica Sinica, 2025, 41(5): 100050-0. doi: 10.1016/j.actphy.2025.100050
14. [14]
  Jiahong WANG , Zekun XU , Tianjiao LU , Jinming HUANG . Performance of N, Mn doped semi-coke activated carbon catalyzed ozone oxidation for the degradation of tetracycline hydrochloride in water. Chinese Journal of Inorganic Chemistry, 2025, 41(12): 2549-2560. doi: 10.11862/CJIC.20250120
15. [15]
  Rui LIU , Xinjun ZHOU , Tao WANG . Photocatalytic degradation performance of tetracycline by MOF-74-Mn/g-C₃N₄ Z-type heterojunction. Chinese Journal of Inorganic Chemistry, 2025, 41(9): 1796-1804. doi: 10.11862/CJIC.20250033
16. [16]
  Zhuoya WANG , Le HE , Zhiquan LIN , Yingxi WANG , Ling LI . Multifunctional nanozyme Prussian blue modified copper peroxide: Synthesis and photothermal enhanced catalytic therapy of self-provided hydrogen peroxide. Chinese Journal of Inorganic Chemistry, 2024, 40(12): 2445-2454. doi: 10.11862/CJIC.20240194
17. [17]
  Yang Li , Jiachen Li , Daidi Fan . 二硫化钼纳米片的制备及其纳米酶性能探究——介绍一个大学化学综合实验. University Chemistry, 2025, 40(8): 233-240. doi: 10.12461/PKU.DXHX202410016
18. [18]
  Hong LI , Xiaoying DING , Cihang LIU , Jinghan ZHANG , Yanying RAO . Detection of iron and copper ions based on gold nanorod etching colorimetry. Chinese Journal of Inorganic Chemistry, 2024, 40(5): 953-962. doi: 10.11862/CJIC.20230370
19. [19]
  Shumin Zhang , Yaqi Wang , Zelin Wang , Libo Wang , Changsheng An , Difa Xu . Ultrafast electron transfer at the ZIS_1−x/UCN S-scheme interface enables efficient H₂O₂ photosynthesis coupled with tetracycline degradation. Acta Physico-Chimica Sinica, 2025, 41(11): 100136-0. doi: 10.1016/j.actphy.2025.100136
20. [20]
  Yaling Chen . Basic Theory and Competitive Exam Analysis of Dynamic Isotope Effect. University Chemistry, 2024, 39(8): 403-410. doi: 10.3866/PKU.DXHX202311093

Get Citation

PDF

XML

Metrics

PDF Downloads(13)
Abstract views(1039)
HTML views(215)

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142