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

doi:10.3724/SP.J.1123.2019.04030

Abstract

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)

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.

Figures/Tables 7

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

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.

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 ℃.

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).

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.

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.

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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