基于牛血清白蛋白稳定的金纳米簇的荧光免疫分析方法检测孔雀石绿

引用本文: 魏春豪, 杨光昕, 迟海, 陶乐仁, 孔聪, 蔡友琼. 基于牛血清白蛋白稳定的金纳米簇的荧光免疫分析方法检测孔雀石绿[J]. 分析化学, 2022, 50(1): 73-81. doi: 10.19756/j.issn.0253-3820.201750 shu

Citation: WEI Chun-Hao, YANG Guang-Xin, CHI Hai, TAO Le-Ren, KONG Cong, CAI You-Qiong. Inner Filter Effect-based Fluorescence Immunoassay with GoldNanoclusters for Malachite Green Detection[J]. Chinese Journal of Analytical Chemistry, 2022, 50(1): 73-81. doi: 10.19756/j.issn.0253-3820.201750 shu

基于牛血清白蛋白稳定的金纳米簇的荧光免疫分析方法检测孔雀石绿

魏春豪 ^1,2, ,
杨光昕 ^{1,
,} ,
迟海 ^{1,2,
,} ,
陶乐仁 ^2, ,
孔聪 ^1, ,
蔡友琼 ^1,

1.
中国水产科学研究院东海水产研究所, 上海 200090;
2.
上海理工大学医疗器械与食品学院, 上海 200093

通讯作者: 杨光昕,E-mail:yang_sh33@163.com; 迟海,E-mail:andychihai@126.com

基金项目:
中央级公益性科研院所基本科研业务费专项项目（No.2016T09）资助。

摘要: 建立了一种基于金纳米簇（BSA-AuNCs）和荧光内滤效应（IFE）检测孔雀石绿（MG）的免疫分析方法。以牛血清蛋白（BSA）为稳定剂，快速合成了荧光性能好、超小尺寸的双发射峰BSA-AuNCs，合成过程仅需7 min，其最大激发波长（390 nm）与对硝基苯酚（PNP）的吸收峰（400 nm）重叠，两者之间产生IFE效应，使BSA-AuNCs的荧光被强烈猝灭。基于此，以BSA-AuNCs为荧光探针，以碱性磷酸酶（ALP）替代传统ELISA中的辣根过氧化物酶（HRP），通过ALP分解对硝基苯磷酸二钠（PNPP）产生的PNP猝灭荧光信号，根据BSA-AuNCs的荧光强度变化实现MG的间接检测。优化了包被原、抗体浓度、二抗浓度、底物浓度、反应温度和反应时间等因素。在最佳实验条件下，本方法检测MG的线性范围为0.1~100 ng/mL，线性回归方程为y=181.53lgC_MG+241.3（R²=0.995），检出限为0.089 ng/mL，回收率为84.0%~110.2%，相对标准偏差为4.6%~11.2%。本方法具有较高的灵敏度和准确度，可用于实际环境样品中痕量MG的快速高通量检测。

关键词:

English

Inner Filter Effect-based Fluorescence Immunoassay with GoldNanoclusters for Malachite Green Detection

1.
East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China;
2.
Schoolof Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China

Corresponding author: YANG Guang-Xin, ; CHI Hai,

Received Date: 11 December 2020
Revised Date: 09 June 2021
Fund Project:
Supported by Central Public-interest Scientific Institution Based Research Fund (No.2016T09)

Abstract: Malachite green (MG), an effective insecticide and preservative, is commonly used in the aquaculture industry. However, MG has acute toxicity to terrestrial and aquatic animals, which can lead to mutagenicity, carcinogenicity, and teratogenicity on animals and human bodies. Therefore, it is necessary to explore a reliable and sensitive method for the rapid detection of MG. Herein, a novel and sensitive fluorescence-linked immunosorbent assay (FLISA) was constructed based on glod nanoclusters (BSA-AuNCs) for MG detection. The assay signal was originated from inner filter effect (IFE) between BSA-AuNCs and p-nitrophenol (PNP). Firstly, a double emission peak fluorescent gold nanocluster with good light stability and ultra-small size was quickly synthesized in 7 minutes, which was used as fluorescence probe in this immunoassay. Secondary, alkaline phosphatase labeled secondary antibody (IgG-ALP) was used to replace IgG-HRP in the immune reaction, which could generate PNP with the substrate of p-nitrophenyl phosphate (PNPP). As the maximum excitation wavelength of BSA-AuNCs (390 nm) overlapped with the absorption peak of PNP (400 nm), the fluorescence of BSA-AuNCs could be strongly quenched by PNP. Thus, with the absence of MG in the indirect immunoassay, much more IgG-ALP would be adsorbed in the 96-well plates, more PNP would be produced, then with the fluorescence quenched more strongly. The fluorescence signal could be used for MG detection indirectly. In this immunoassay, several factors including coating antigen, antibody concentration, secondary antibody concentration, substrate concentration, reaction temperature and reaction time were optimized. Under the optimized conditions, the linear range of this method was 0.1-100 ng/mL, linear equation was y=181.53lgC_MG+241.3 (R²=0.995), and the limit of detection (LOD) was 0.089 ng/mL. The sensitivity of FLISA was 30 times that of ic-ELISA. Furthermore, the proposed method was successfully applied to determination of MG in spiked aquaculture water and tap water. The recoveries of MG were in the range of 84.0%-110.2%, and the coefficient of variation was 4.6%-11.2%. The new immunoassay could be used as a promising tool for rapid detection of MG in real environmental samples.

Key words:

1. [1]
  WU L, LIN Z Z, ZHONG H P, PENG A H, CHEN X M, HUANG Z Y. Food Chem., 2017, 229:847-853.WU L, LIN Z Z, ZHONG H P, PENG A H, CHEN X M, HUANG Z Y. Food Chem., 2017, 229:847-853.
2. [2]
  SAHRAEI R, FARMANY A, MORTAZAVI S S, NOORIZADEH H. Environ. Monit. Assess., 2013, 185:5817-5822.SAHRAEI R, FARMANY A, MORTAZAVI S S, NOORIZADEH H. Environ. Monit. Assess., 2013, 185:5817-5822.
3. [3]
  ZHANG Y Y, HUANG Y Q, ZHAI F L, DU R, LIU Y D, LAI K Q. Food Chem., 2012, 135(2):845-850.ZHANG Y Y, HUANG Y Q, ZHAI F L, DU R, LIU Y D, LAI K Q. Food Chem., 2012, 135(2):845-850.
4. [4]
  ZHANG K, SONG G, YANG L X. Anal. Methods, 2012, 4(12):4257-4263.ZHANG K, SONG G, YANG L X. Anal. Methods, 2012, 4(12):4257-4263.
5. [5]
  LIN Z Z, ZHANG H Y, PENG A H, LIN Y D, LI L, HUANG Y. Food Chem., 2016, 200:32-37.LIN Z Z, ZHANG H Y, PENG A H, LIN Y D, LI L, HUANG Y. Food Chem., 2016, 200:32-37.
6. [6]
  JIA F, YANG X D, LI Z Y. RSC Adv., 2016, 6:92723-92728.JIA F, YANG X D, LI Z Y. RSC Adv., 2016, 6:92723-92728.
7. [7]
  XIE J, PENG T, CHEN D D, ZHANG Q J, WANG G M, WANG X, QI G, JIANG F, CHEN D, DENG J. J. Chromatogr. B, 2013, 913:123-128.XIE J, PENG T, CHEN D D, ZHANG Q J, WANG G M, WANG X, QI G, JIANG F, CHEN D, DENG J. J. Chromatogr. B, 2013, 913:123-128.
8. [8]
  WANG Li-Bo, SU Li-Qiang, HAN Chao-Nan, LYU Yan-Rong. J. Anal. Sci., 2018, 34(2):229-233. 王丽博, 苏立强, 韩超男, 吕艳荣. 分析科学学报, 2018, 34(2):229-233.
9. [9]
  CHEN Bai-Shen, WANG Xi-Yue, ZHU Bo, LIAN Li-Li, LOU Da-Wei. J. Instrum. Anal., 2020, 39(8):993-999. 陈柏森, 王希越, 祝波, 连丽丽, 娄大伟. 分析测试学报, 2020, 39(8):993-999.
10. [10]
  GONG Xiao-Ming, HUA Meng-Meng, WANG Hong-Tao, WANG Bing-Jun, MA Rong-Hui. J. Instr. Anal., 2017, 36(7):897-901. 宫小明, 华萌萌, 王洪涛, 王炳军, 马荣桧. 分析测试学报, 2017, 36(7):897-901.
11. [11]
  GUO Y R, ZOU R B, SI F F, LIANG W L, ZHANG T Y, CHANG Y Y, QIAO X S, ZHAO J H. Food Chem., 2020, 335:127609-127617.GUO Y R, ZOU R B, SI F F, LIANG W L, ZHANG T Y, CHANG Y Y, QIAO X S, ZHAO J H. Food Chem., 2020, 335:127609-127617.
12. [12]
  ZHAO M T, LI X L, ZHANG Y L, WANG Y W, WANG B, ZHENG L L, ZHANG D W, ZHUANG S L. Food Chem., 2020, 339:127857-127864.ZHAO M T, LI X L, ZHANG Y L, WANG Y W, WANG B, ZHENG L L, ZHANG D W, ZHUANG S L. Food Chem., 2020, 339:127857-127864.
13. [13]
  ZHANG C, JIANG Z J, JIN M J, DU P F, CHEN G, CUI X Y, ZHANG Y D, QIN G X, YAN F Y, EL-ATY A M A, HACIMVFTVOǦLU A, WANG J. Food Chem., 2020, 326:126813-126820.ZHANG C, JIANG Z J, JIN M J, DU P F, CHEN G, CUI X Y, ZHANG Y D, QIN G X, YAN F Y, EL-ATY A M A, HACIMVFTVOǦLU A, WANG J. Food Chem., 2020, 326:126813-126820.
14. [14]
  CHEN S, YU Y L, WANG J H. Anal. Chim. Acta, 2018, 999:13-26.CHEN S, YU Y L, WANG J H. Anal. Chim. Acta, 2018, 999:13-26.
15. [15]
  DONG B L, LI H F, MARI G M, YU X Z, YU W B, WEN K, KE Y B, SHEN J Z, WANG Z H. Food Chem., 2019, 294:347-354.DONG B L, LI H F, MARI G M, YU X Z, YU W B, WEN K, KE Y B, SHEN J Z, WANG Z H. Food Chem., 2019, 294:347-354.
16. [16]
  LUO L, SONG Y, ZHU C Z, FU S F, SHI Q R, SUN Y M, JIA B Z, DU D, XU Z L, LIN Y H. Sens. Actuators, B, 2018, 255(3):2742-2749.LUO L, SONG Y, ZHU C Z, FU S F, SHI Q R, SUN Y M, JIA B Z, DU D, XU Z L, LIN Y H. Sens. Actuators, B, 2018, 255(3):2742-2749.
17. [17]
  CUI M L, YUAN Z, SONG Q J. TrAC-Trends Anal. Chem., 2014, 57:73-82.CUI M L, YUAN Z, SONG Q J. TrAC-Trends Anal. Chem., 2014, 57:73-82.
18. [18]
  PENG Tao, WANG Jian-Yi, XIE San-Lei, YAO Kai, SUN Shu-Juan, ZENG Yu-Yang, JIANG Hai-Yang. Chin. J. Anal. Chem., 2018, 46(3):373-378. 彭涛, 王见一, 谢三磊, 姚凯, 孙淑娟, 曾于洋, 江海洋. 分析化学, 2018, 46(3):373-378.
19. [19]
  XIE J P, ZHENG Y G, YING J Y. J. Am. Chem. Soc., 2009, 131(3):888-889.XIE J P, ZHENG Y G, YING J Y. J. Am. Chem. Soc., 2009, 131(3):888-889.
20. [20]
  LI H F, WEN K, DONG B L, ZHANG J, BAI Y C, LIU M G, LI P P, MUJTABA G M, YU X Z, YU W B,KE Y B, SHEN J Z, WANG Z H. Sens. Actuators, B, 2019, 297:126787-126796.LI H F, WEN K, DONG B L, ZHANG J, BAI Y C, LIU M G, LI P P, MUJTABA G M, YU X Z, YU W B,KE Y B, SHEN J Z, WANG Z H. Sens. Actuators, B, 2019, 297:126787-126796.
21. [21]
  WANG Xue-Chuan, BAI Peng-Xia, LUO Xiao-Min, LI Ji. Spectrosc. Spectral Anal., 2016, 44(9):1385-1393. 王学川, 白鹏霞, 罗晓民, 李季. 光谱学与光谱分析, 2019, 39(4):1154-1161.
22. [22]
  JU Y J, LI N, LIU S G, HAN L, XIAO N, LUO H Q, LI N B. Sens. Actuators, B, 2018, 275:244-250.JU Y J, LI N, LIU S G, HAN L, XIAO N, LUO H Q, LI N B. Sens. Actuators, B, 2018, 275:244-250.
23. [23]
  LIU H Y, ZHANG X, WU X M, JIANG L P, BURDA C, ZHU J J. Chem. Commun., 2011, 47(14):4237-4239.LIU H Y, ZHANG X, WU X M, JIANG L P, BURDA C, ZHU J J. Chem. Commun., 2011, 47(14):4237-4239.
24. [24]
  KONG Y F, CHEN J, GAO F, BRYDSON R, JOHNSON B, HEATH G, ZHANG Y, WU L, ZHOU D J. Nanoscale, 2013, 5(3):1009-1017.KONG Y F, CHEN J, GAO F, BRYDSON R, JOHNSON B, HEATH G, ZHANG Y, WU L, ZHOU D J. Nanoscale, 2013, 5(3):1009-1017.
25. [25]
  HSUA N Y, LIN Y W. New J. Chem., 2016, 40(2):1155-1161.HSUA N Y, LIN Y W. New J. Chem., 2016, 40(2):1155-1161.
26. [26]
  WANG Yu, YANG Jin-Yi, XU Zhen-Lin, QI Ping, SHEN Yu-Dong. Chin. J. Anal. Chem., 2016, 44(9):1385-1393. 王宇, 杨金易, 徐振林, 戚平, 沈玉栋. 分析化学, 2016, 44(9):1385-1393.
27. [27]
  SUN Wen-Fang, HUANG Wei, LIU Xiang-Ping. J. Environ. Health, 2018, 35(8):733-735. 孙文芳, 黄薇, 刘祥萍. 环境与健康杂志, 2018, 35(8):733-735.

扫一扫看文章

计量

PDF下载量: 12
文章访问数: 1129
HTML全文浏览量: 202

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

基于牛血清白蛋白稳定的金纳米簇的荧光免疫分析方法检测孔雀石绿

通讯作者: 杨光昕,E-mail:yang_sh33@163.com; 迟海,E-mail:andychihai@126.com

English

Inner Filter Effect-based Fluorescence Immunoassay with GoldNanoclusters for Malachite Green Detection

Corresponding author: YANG Guang-Xin, ; CHI Hai,

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？

计量

通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于牛血清白蛋白稳定的金纳米簇的荧光免疫分析方法检测孔雀石绿

通讯作者: 杨光昕,E-mail:yang_sh33@163.com; 迟海,E-mail:andychihai@126.com

English

Inner Filter Effect-based Fluorescence Immunoassay with GoldNanoclusters for Malachite Green Detection

Corresponding author: YANG Guang-Xin, ; CHI Hai,

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？

计量

文章相关

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