The Study And Application On Aptamer Optical Sensors For The Detection Of Melamine

Melamine is an ordinary chemical compound widely used in the manufacture of plastics, laminates, adhesines, flame retardants, fertilizer and so on. Because it contains a large amount of nitrogen(66 % nitrogen by weight), it has been illegally added into foodstuff to boost the apparent protein. Although the toxicity of melamine is low, most animal studies demonstrated effects on health following sub-acute or chronic melamine exposure. Ingestion of melamine may even cause reproductive damage and kidney stones, which can eventually lead to bladder cancer. Currently, many methods have been reported for the detection of melamine, such as liquid chromatography, GC- MS、 Raman spectrometry and so on. Aptamers, a length of sequences synthesized by single-stranded DNA or RNA, usually created via a process known as SELEX, or systematic evolution of ligands via exponential enrichment. Aptamers have many advantages, such as high affinity, selectivity and was widely used in biosensor field. In this article, two simple, sensitive and rapid optical sensors based on aptamers and CS-Au NPs, aptamers and G-quadruplex for detection melamine in practical samples were with the special properties of aptamers.1. A rapid and facile visual method for detecting melamine in raw milk was developed by using T-rich single-stranded DNA(ss DNA) aptamers as the recognition element and cysteamine-stabilized gold nanoparticles(CS-Au NPs) as the color indicator. The electrostatic interaction between the negatively-charged melamine-binding aptamers and the positively-charged CS-Au NPs induced the aggregation of the CS-Au NPs and a remarkable change in color from wine red to blue. The specific recognition between melamine and T-rich aptamers could cause the formation of melamine-aptamer complex through hydrogen bonding. Compared with the electrostatic interaction between CS-Au NPs and aptamers, the strongly specific recognition between melamine and aptamers played a dominant role, thus the presence of melamine could prevent the aptamers-induced aggregation and color change of CS-Au NPs. As a result of this, using CS-Au NPs as the colorimetric probe, a simple and sensitive aptasensor for melamine determination is developed. Under the optimum conditions, the absorbance change(ΔA526) of CS-Au NPs was linearly proportional to melamine concentration in the range of 1- 24 n M, with the detection limit of 0.389 n M. The proposed method is a promising approach for rapid screening of melamine in raw milk and the results were in full accordance with the results from HPLC, providing a simple method for the analysis of melamine.2. A novel method for detecting melamine based on a single labled fluorescence probe which was designed based on the efficient quenching ability of an aptamer-based G-quadruplex has been developed. The special sequence which is labeled with 6-carboxyfluorescencein(FAM) is constituted by two parts: melamine-binding aptamer and a G-rich sequence which can form a G-quadruples structure. At first, FAM became strongly fluorescence upon binding to aptamer-based G-quadruplex. However, with the addition of melamine, because of the specific recognition between T-rich aptamers and melamine, the melamine-binding aptamer is folded into a hairpin structure which leads to the G-quadruplex closing to the FAM, and eventually caused the fluorescence quenching. And the resulting in decreased fluorescent signal was inversely related to melamine concentration, which can demonstrate that it is possible to detect melamine by fluorescence spectroscopy. Under the optimum conditions, the degree of fluorescence quenching in the assay is proportional to the concentration of melamine. The detection limit in raw milk was calculated to be 6.32 n M and the recoveries were in the range of 97.15 %- 101.92 %, which are in full accordance with the results from HPLC. In summary, the proposed method provides advantages such as rapidity, simple operation procedure, reduced detection time, and practical application, making it promising for the rapid screening of melamine residues.