Ruan Chuanmin, Wang Wei, Gu Baohua
Oak Ridge Institute for Science and Education, Oak Ridge, TN 37831, USA.
Anal Chem. 2006 May 15;78(10):3379-84. doi: 10.1021/ac0522106.
A new approach was developed to detect the activity of alkaline phosphatase (ALP) enzyme at ultralow concentrations using a surface-enhanced Raman scattering (SERS) technique. The approach is based on the use of gold nanoparticles as a SERS material whereas 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used as a substrate of ALP. The enzymatic hydrolysis of BCIP led to the formation of indigo dye derivatives, which were found to be highly SERS active. For the first time, we were able to detect ALP at a concentration of approximately 4 x 10(-15) M or at single-molecule levels when ALP was incubated with BCIP for 1 h in the Tris-HCl buffer. The same technique also was successfully employed to detect surface-immobilized avidin, and a detection limit of 10 ng/mL was achieved. This new technique allows the detection of both free and labeled ALP as a Raman probe in enzyme immunoassays, immunoblotting, and DNA hybridization assays at ultralow concentrations.
开发了一种新方法，利用表面增强拉曼散射（SERS）技术检测超低浓度碱性磷酸酶（ALP）的活性。该方法基于使用金纳米颗粒作为SERS材料，而5-溴-4-氯-3-吲哚基磷酸酯（BCIP）用作ALP的底物。BCIP的酶促水解导致靛蓝染料衍生物的形成，发现其具有高度的SERS活性。首次，当ALP与BCIP在Tris-HCl缓冲液中孵育1小时时，我们能够检测到浓度约为4×10⁻¹⁵ M的ALP或单分子水平的ALP。相同的技术也成功用于检测表面固定的抗生物素蛋白，并实现了10 ng/mL的检测限。这种新技术允许在酶免疫测定、免疫印迹和DNA杂交测定中以超低浓度检测游离和标记的ALP作为拉曼探针。
