Simple and Label-Free Fluorescent Detection of Melamine Based on Melamine⁻Thymine Recognition

Abstract

In the past few years, melamine has been illegally added into dairy products to increase the apparent crude protein levels. If humans or animals drink the milk adulteration of melamine, it can form insoluble melamine⁻cyanurate crystals in their kidneys which causes kidney damage or even death. In the present work, we constructed a simple and label-free fluorescent method for melamine detection based on melamine-thymine recognition. SYBR Green I was utilized as a reporter for this method as it did not require any modification or expensive equipment. In the absence of melamine, polythymine DNA was digested by Exo I, which caused a decrease in the fluorescence signal. In the presence of melamine, the polythymine DNA was able to fold into a double chain structure, however this was done with the help of T-melamine-T mismatches to prevent degradation. Then, the SYBR Green I combined with the double-stranded DNA to result in an intense fluorescence signal. The limit of detection in this method was 1.58 μM, which satisfied the FDA standards. This method also had a good linear relationship within the range of 10⁻200 μM. In addition, this new method has a good selectivity to distinguish melamine from the component of milk. As a result, we developed a simple and highly selectivity method for melamine detection.

Keywords: SYBR Green I; exonuclease I; melamine detection; melamine-thymine recognition.

Figure 1

Schematic illustration of the simple and label-free fluorescent method for melamine based on melamine–thymine recognition.

Figure 2

The fluorescence signal change (F/F₀) under different length polythmine DNA. where F₀ and F were the area under curve of the emission spectra in the absence and presence of 500 μM melamine, respectively. Error bars were estimated from at least three independent measurements.

Figure 3

(a) The fluorescence emission spectra in the presence of difference melamine concentrations. (b) The fluorescence intensity signal change F/F₀ for melamine concentrations ranging from 6 to 500 μM, which matched the dose-response curve. Inset: the signal change matched a linear relationship at concentrations of 10–200 μM. F₀ and F are the area under curve of the emission spectra in the absence and presence of melamine, respectively. Error bars were estimated from at least three independent measurements.

Figure 4

The selectivity of our method was evaluated in the presence 40 μM melamine and 400 μM interfering materials. Error bars are estimated from at least three independent measurements.