基于氧化还原反应界面可视化定量检测辣根过氧化物酶

doi:10.3724/SP.J.1123.2019.04030

色谱 ›› 2020, Vol. 38 ›› Issue (2): 177-182.DOI: 10.3724/SP.J.1123.2019.04030

基于氧化还原反应界面可视化定量检测辣根过氧化物酶

孔昊¹^,², 张强²^,³, 张薇², 刘伟文³, 曹成喜³^,^*(), 樊柳荫¹^,^*()

¹ 上海交通大学学生创新中心, 上海 200240
² 上海交通大学生命科学技术学院, 上海 200240
³ 上海交通大学电子信息与电气工程学院, 上海 200240

收稿日期:2019-04-16 出版日期:2020-02-08 发布日期:2020-12-10
通讯作者: 曹成喜,樊柳荫
作者简介:樊柳荫.Tel:(021)34205820, E-mail:lyfan@sjtu.edu.cn
曹成喜.E-mail:cxcao@sjtu.edu.cn;
基金资助:
国家自然科学基金项目(21675067);国家自然科学基金项目(31727801);国家自然科学基金项目(21605101);上海市科学技术委员会项目(15142200300)

Visualized and quantitative detection of horseradish peroxidase based on redox reaction boundary

KONG Hao¹^,², ZHANG Qiang²^,³, ZHANG Wei², LIU Weiwen³, CAO Chengxi³^,^*(), FAN Liuyin¹^,^*()

¹ Student Innovation Center, Shanghai Jiao Tong University, Shanghai 200240, China
² School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
³ School of Electronic Information & Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2019-04-16 Online:2020-02-08 Published:2020-12-10
Contact: CAO Chengxi,FAN Liuyin
Supported by:
National Natural Science Foundation of China(21675067);National Natural Science Foundation of China(31727801);National Natural Science Foundation of China(21605101);Foundation of Shanghai Science and Technology Committee(15142200300)

摘要/Abstract

摘要：

该文建立了一种可视化的、基于氧化还原反应界面移动距离定量检测辣根过氧化物酶（HRP）的方法。比较了隐色结晶紫显色体系和3，3'，5，5'-四甲基联苯胺显色体系对HRP的显色效率，并构建了基于3，3'，5，5'-四甲基联苯胺显色体系的氧化还原反应电泳滴定模型。同时，文中还设计了适用于该模型的小型化、便携式滴定检测芯片，并对滴定通道凝胶中组分进行了优化。结果表明，界面移动距离与HRP浓度存在对数线性关系，检测灵敏度可达0.002 mg/L，且可在10 min内完成HRP的裸眼检测。该方法不需要配备信号读取装置，用户只需要读取有色界面移动的距离即可实现对待测物的可视化定量检测，对于即时检测具有潜在的应用价值。

关键词: 氧化还原反应, 电泳滴定, 即时检测, 可视化, 辣根过氧化物酶

Abstract:

A new portable method for the detection of horseradish peroxidase (HRP) is reported in this paper. Visualized and quantitative detection of HRP was achieved based on the theory of electrophoresis titration (ET) and redox reaction boundary (RB). The ET-RB model was built on the 3, 3', 5, 5'-tetramethyl benzidine chromogenic system because it significantly increases the efficiency of the chromogenic reaction and can react with L-ascorbic acid. A series of experiments were performed on a tiny and portable titration chip based on the material of polymethyl methacrylate designed for the developed model. The composition of the titration gel was optimized to ensure higher detection sensitivity and stable detection performance. The experimental results manifested a log-linear relationship between the moving distance of the RB and the HRP concentration. The dynamic range of the developed method ranged from 0.002 to 0.073 mg/L, and the detection could be accomplished within 10 min. Visualized and quantitative detection could be realized by reading the moving distance of the colored boundary with the naked eyes instead of using any signal-reading device or analyzing instrument. Thus, this method has great potential for further application to the development of a real-time detection method for various target substances based on the detection of peroxidase activity.

Key words: redox reaction, electrophoresis titration (ET), real-time detection, visualization, horseradish peroxidase (HRP)

孔昊, 张强, 张薇, 刘伟文, 曹成喜, 樊柳荫. 基于氧化还原反应界面可视化定量检测辣根过氧化物酶[J]. 色谱, 2020, 38(2): 177-182.

KONG Hao, ZHANG Qiang, ZHANG Wei, LIU Weiwen, CAO Chengxi, FAN Liuyin. Visualized and quantitative detection of horseradish peroxidase based on redox reaction boundary[J]. Chinese Journal of Chromatography, 2020, 38(2): 177-182.

图/表 7

图1 电泳滴定芯片结构

Fig. 1 Structure of electrophoresis titration (ET) chip 1. anode well; 2. bridge channel; 3. sample well; 4. titration channel; 5. cathode well.

图2 基于(a)LCV显色体系和(b)TMB显色体系的电泳滴定模型

Fig. 2 ET models based on (a) leucocrystal violet (LCV) chromogenic system and (b) 3, 3′, 5, 5′-tetramethyl-benzidine (TMB) chromogenic system CV+: crystal violet cation; CV-OH: crystal violet carbinol; TMB·+: 3, 3′, 5, 5′-tetramethyl benzidine radical cation; TMB: 3, 3′, 5, 5′-tetramethyl benzidine; AA: L-ascorbic acid; AA·-: L-ascorbic acid radical anion. The symbols "+" and "-" indicate anode and cathode, respectively.

图3 LCV、TMB、EL-TMB显色液在HRP催化反应中显色效率比较

Fig. 3 Comparison of the chromogenic efficiencies between LCV, TMB, and EL-TMB chromogenic solutions under horseradish peroxidase (HRP) catalysis Experimental conditions: chromogenic reaction for 4 min at 25 ℃.

图4 基于TMB显色体系的电泳滴定界面迁移照片

Fig. 4 Images of boundary motion during ET based on TMB chromogenic system Experimental conditions: 0.1 mmol/L AA and 40 mmol/L KCl in 1% (1 g/100 mL) agarose gel-filled titration channel; 40 mmol/L KCl in 1% (1 g/100 mL) agarose gel-filled bridge channel; 40 mmol/L NaOH as anolyte; 0.1 mmol/L AA and 40 mmol/L HCl as catholyte; 0.073 mg/L HRP in chromogenic solution; operating voltage as 36 V; ET running for 4 min. Photos were acquired before ET running (t0), and after ET running for 2 min (t1) and 4 min (t2).

图5 滴定凝胶中(a)电解质浓度与(b)AA浓度对界面迁移的影响

Fig. 5 Influence of (a) electrolyte concentration and (b) AA concentration in gel on the boundary motion Experimental conditions: a. 10, 40, and 60 mmol/L KCl in titration and bridge channel, 0.073 mg/L HRP in chromogenic solution; b. 0.4, 0.2, 0.1, and 0.05 mmol/L AA in titration channel, 0.009 mg/L HRP in chromogenic solution. Concentrations of NaOH in anolyte and HCl in catholyte were the same as KCl concentration in titration channel. Other conditions were the same as those in Fig. 4. The arrow indicates the formation of precipitation.

图6 电泳滴定检测HRP的(a)界面迁移距离和(b)标准曲线(n=3)

Fig. 6 (a) Boundary motion distance and (b) calibration curve of HRP detection with developed ET method (n=3) Experimental conditions: 0.002, 0.004, 0.009, 0.018, 0.036, and 0.073 mg/L HRP in chromogenic solution; photos were acquired after ET running for 4 min. Other conditions were the same as those in Fig. 4. V: moving velocity of the RB in titration channel (mm/min); ρ: mass concentration of horseradish peroxidase detected in reaction system (mg/L); R2: correlation coefficient.

表1 氧化还原反应界面-电泳滴定检测方法与数种已报道的HRP检测方法的比较

Table 1 Comparison of ET-redox reaction boundary (RB) method with several reported detection methods for HRP assay

Theory and material	Detection method	Dynamic range/(mg/L)	Portability	Reference
Glassy carbon electrode	electrochemical detection	0.0005-0.01	no	[6]
Disposable screen-printed carbon electrode		0.003-0.1	no	[7]
Capillary zone electrophoresis		0.00096-0.96	no	[8]
Biomicroelectro-mechanical system	chemiluminescent detection	0.082-0.82	no	[15]
Amorphous silicon photodiode		0.1-10	no	[16]
InP/ZnS quantum dot	fluorescent detection	0.04-1.2	no	[17]
Catalyzation	ET-RB	0.002-0.073	yes	this work

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基于氧化还原反应界面可视化定量检测辣根过氧化物酶

Visualized and quantitative detection of horseradish peroxidase based on redox reaction boundary

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