Visual Detection Of Melamine In Milk Powder Based On Methanobactin-induced Gold Nanoparticles Formation

Methanobactin is a small chromopeptide showing high affnity for copper that released by methanotrophic bacteria. It has a molecular mass of1200Da and is composed of seven amino acids and two chromophoric residues. Methanobactin coordinates with Cu(I) by two hydroxyimidazolates, each contributing with one N and one S atom to c-opper binding. Methanobactin can also bind to Au(III) and reduce it to Au(0). In the presence of hydroquinone as a electron donator, Mb can catalyze Au(III) reduce to Au(0)and yield gold nanoparticles continuously. Surface plasmon resonance of gold nanop-articles makes its aqueous solution has a typical absorption peak between500and600nm, and a unique color (wine-red or purple) that can be observed by naked eyes. The molar absorption coefficient of gold nanoparticles is hundreds even thousands of times than that of other compounds. The properties of gold nanoparticles were widely used in the analysis and detection.In recent years, melamine was illegally and unethically adulterated in proteinrich ingredients to increase the apparent protein content.Therefore, there is an increasing need for rapid and reliable methods to detect melamine in milk and other food products. A safety limit of melamine (2.5mg·kg-1) in milk and milk based products has set by Food and Drug Administration (FDA) of USA and European Union. The maximum residue levels of melamine in infant formula and other dairy products are legally regulated at1and2.5mg·kg-respectively, by Chinese government. But it always happens that adding melamine illegally, so the detection of melamine becomes a key link for solving the problem of food safety. Except for the HPLC, GC-MS/MS, researchers have detected melamine using metal nanoparticles, such as gold and silver nanoparticles. Melamine can make these two kinds of nanoparticles from dispersed state into aggregated state with a color change which can achieve detection by naked eyes, but it’s difficult to handle which needs a series of complex process as synthesis, storage, modify.Visual detection of melamine in milk powder based on methanobactin-induced gold nanoparticles formation is the study focus of this topic. Different concentrations of melamine in milk powder have been detected. The interference of several kinds of ingredients in milk powder have been investigated. The detection limit and the recovery rate has been calculated, either. The study results have been obtained as below:1. The presence of hydroquinone promots the synthesis of nanogolds induced by Mb apparently. The optimal conditions for synthesizing AuNPs has been obtained as below:at room temperature, pH=5.3, the molar concentration ratio of Mb:Au(Ⅲ)is0.5:1, HQ:Au(Ⅲ)is0.4:1. The reagent order is500μL Mb of10-3M,100μL H AuCl4·4H2O of10.2M,800μL HQ of 5×10-4M. AuNPs will increase as the extension of time until the reaction finished completely. The nanogolds were characterized by UV-visible absorption spectra, TEM and FT-IR spectra. The results indicated that the wine-red AuNPs aqueous has a higher, narrower resonance absorption peak, and a shift of the surface plasmon band to shorter wavelength, and a better symmetry of the peak, and had a smaller, more uniform diameter, mostly particles had a spherical shape, the average diameter was13nm, the latter one, however, particles presented triangle, pentagonal, hexagon shape except spherical, the average diameter was32nm. The presence of vibration bands of C=O, C-H, N-H, O-H implied clearly the presence of Mb molecules on the nanoparticle surface.2. UV-visible spectrophotometer showed that it will inhibit the synthesis of gold nanoparticles after adding lppm of melamine into solution of Mb.The optimal condition for detecting melamine is:at room temperature, pH=5.3, the molar ratio of Mb to Au(III) is0.7:1, the molar ratio of HQ to Au(III) is0.25:1. The reagent order is700μL Mb of10"3M,100μL HAuCl4·4H2O of10-2M,500μL HQ of5x10-4M. It can detect O.lppm (0.79μmol/L)of melamine. Characterized the nanogolds synthesized in the presence of0.01ppm(0.079μmol/L),0.05ppm(0.4μmol/L),0.08ppm(0.63μmol/L) of melamine by UV-visible spectra and TEM, the result shows that gold particles in solution aggregates more and more seriously as the increasing concentration of melamine, there is no single gold particles exists in the solution until0.08ppm when it blocks the synthesis of gold nanoparticles preliminarily.3. The optimal condition to detect the melamine in milk powder is follow:700μL Mb of10-3M,100μL HAuCl4·4H2O of10-2M,500μL HQ of5×10-4. The result indicates that the concentration of melamine shows a good liner relationship with the absorption of gold nanoparticles, the macroscopic qualitative detection limit is0.7ppm (5.55μmol/L), between0.05ppm (0.4μmol/L) and0.5ppm(3.96μmol/L) can achieve quantitative detection by UV-visible spectrophotometer. The detection limit (D.L) of this method is0.03ppm (0.26μM), the recovery rate is between97%and103%. We found that Ca2+, Cl-,Fe3+, K+, Mg2+, Na+, glucose, lacose, sucrose, Arg, Glu, Phe, Ala, Tyr, Pro, glycerol, cyanuric acid and uracil showed no interference except for Vc and Vb12which have a little effect.