Triplex DNA-based Bioanalytical Platform for Highly Sensitive Homogeneous Electrochemical Detection of Melamine

Abstract

Melamine detection has attracted much attention since the discovery of the damage of melamine to human health. Herein, we have developed a sensitive homogeneous electroanalytical platform for melamine detection, which is relied on the formation of triplex molecular beacon integrated with exonuclease III (Exo III)-mediated signal amplification. The formation of triplex molecular beacon was triggered by the recognition and incorporation of melamine to the abasic (AP) site contained in the triplex stem. The stem of the triplex molecular beacon was designed to have a protruding double-strand DNA, which can be recognized and hydrolyzed by Exo III for releasing methylene blue (MB)-labeled mononucleotide. These released MB molecules exhibit high diffusivity toward indium tin oxide electrode with negative charge, thus producing a significantly increased electrochemical response. Taking advantages of the high binding affinity of the DNA triplex structure containing AP sites towards melamine and the unique features of Exo III, this sensing platform is capable for sensitive and selective melamine assay with a detection limit as low as 8.7 nM. Furthermore, this strategy shows good applicability for melamine assay in real samples. Therefore, this strategy broadens the application of triplex DNA and presents a new method for sensitive detection of melamine.

Figure 1

Principle of the homogeneous electroanalytical strategy for melamine assay.

Figure 2

Differential pulse voltammograms (DPV) of the sensing system in the presence of: (a) melamine; (b) HP, MB-DNA, and melamine; (c) MB-DNA only; (d) MB-DNA and Exo III; (e) HP, MB-DNA, and Exo III; (f) HP, MB-DNA, melamine, and Exo III.

Figure 3

DPV peak current observed under (A) different pH values: 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0; (B) different Exo III concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1, and 1.25 U/μL; (C) different reaction time of Exo III: 0, 20, 40, 60, 80, and 100 min; (D) different reaction time of melamine: 0, 1, 3, 5, 7, and 9 h. The concentration of melamine was 50 μM. The error bars represent the standard deviation of three measurements.

Figure 4

(A) DPV currents of the sensing system after adding melamine with different concentrations: (a–k) 0, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, and 1,000 μM, respectively. (B) Calibration curve corresponding to the peak current versus melamine concentration ranging from 50 nM to 1,000 μM. Inset: the linear relationship between DPV peak current and melamine concentration ranging from 50 nM to 500 μM. The error bars represent the standard deviation of three measurements.

Figure 5

Comparison of DPV peak currents in the presence of melamine, cytidine, guanosine, methionine, glycine, lactose, vitamin C, vitamin B₂, Zn²⁺, and K⁺. The concentrations of melamine are 50 μM and other substances are 100 μΜ. The error bars represent the standard deviation of three measurements.