Determination of protein with methylene blue-sodium tetraphenylborate phosphorescence probe solid substrate-room temperature phosphorimetry

A new sensitive method for the determination of protein has been established based on methylene blue-sodium tetraphenylborate phosphorescence probe solid substrate-room temperature phosphorescence (SS-RTP) sensitization. Methylene blue (MB) can not only exist as nitrogen quinine cation structure in the acidity agent but also associate with anion of [B(C6H5)4]−. According to the facts above, ionic association of [MB]+[B(C6H5)4]− can emit strong and stable SS-RTP on acetyl cellulose membrane. It is found that the SS-RTP intensity of luminescence system is increased sharply in the presence of bovine serum albumin (BSA). Under the optimum condition, the linear dynamic range of this method is 0.044-17.6 ag spot−1 (corresponding concentration: 0.11-44.0 fg ml−1, the sample volume: 0.4 μl spot−1), and the regression equation can be expressed as ΔIp = 20.80 + 9.702 mBSA (ag spot−1) with the correlation coefficient r = 0.9995 (n = 6). The detection limit of this method is 13.3 zg spot−1 (corresponding concentration: 33 ag ml−1). This sensitive, accurate, selective and repeatable method has been applied to determine the content of protein in real samples with satisfactory results. The reaction mechanism for determination of protein by methylene blue-sodium tetraphenylborate phosphorescence probe SS-RTP sensitization has also been discussed.