A Fluorescence Detection Method for Melanin Content in Black-Bone Silky Fowl Melanocyte Based on H2O2 Oxidation
CHEN Lu-lu1, TIAN Ying-gang1,2*
1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
2. Engineering Research Center of Biomass Conversion, Ministry of Eduction, Nanchang University, Nanchang 330047, China
Abstract：We established a simple, accurate and highly sensitive fluorescence analysis method for detecting the melanin content of Black-bone Silky Fowl (BSF) melanocytes for the first time. Melanins did not fluoresce but, once they were subjected to oxidative conditions (heating in alkaline hydrogen peroxide solution), they acquired strong fluorescence. Melanin content of melanocytes was determined by measuring its relative fluorescence intensity. In this paper, the excitation wavelength and emission wavelength of BSF melanin were determined by fluorescence spectrophotometer with a bandwidth of 5 nm, scanning speed of 2 400 nm·min-1 and scanning pitch of 1 nm. The optimal oxidation conditions of BSF melanin in the range of 10～100 μg·mL-1 were screened by a single factor experiment. The linear range, detection limit, instrument precision and repeatability of the method were analyzed and evaluated. 25, 40 and 80 μg·mL-1 melanin standards were added to samples of A375 cells (melanoma cells without melanin), and the melanin content in the cell samples was determined after that the reaction was carried out under optimal oxidation conditions. Calculate the relative error value of the measured value and the theoretical value for exploring the effect of heterologous proteins and lipids in cells on the quantitative results of melanin; After that 5.00, 7.50 and 10.00 μg melanin standards were added to BSF melanocyte samples, the total melanin content in the cell samples was determined under optimal oxidation conditions and then the spiked recovery was calculated. The results showed that the BSF melanin had an excitation wavelength of 354 nm and an emission wavelength of 453 nm; The optimal oxidation conditions for BSF melanin were as follows: the PH value was 8, oxidation temperature was 55 ℃, hydrogen peroxide concentration ranged from 24% to 26% and oxidation time was 2 h; The concentration of melanin in the range of 10～100 μg·mL-1 showed a good linear relationship with the relative fluorescence intensity. The correlation coefficient was 0.997 4 and the linear regression equation was y=0.014 7x+0.313 8. The detection limit of the method of fluorescence analysis was 0.30 μg·mL-1 and the detection limit of ultraviolet spectrophotometry was 3.68 μg·mL-1. The RSD value of the precision experiment was 1.87%, and the RSD value of the repeat experiment was 4.59%. After adding melanin standards of different quality to A375 cells, the relative errors of measured values and theoretical values were 2.78%, 3.53%, and 0.25%, respectively; After adding different melanin standards to BSF melanocytes, the measured recovery rates were 95.94%, 92.14%, 99.83% and the RSD value was 4.00%. In summary, the method was simple, accurate, stable, and low detection limit. In addition, the measurement result was not interfered by intracellular heteroprotein and lipid. So the method was more suitable for determining the content of melanin in BSF melanocytes.
