碱性磷酸酶标记的表面增强拉曼散射活性夹心免疫分析检测大肠杆菌。

Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli.

机构信息

Department of Food Engineering, Faculty of Engineering, Hacettepe University, Beytepe, 06800 Ankara, Turkey.

Department of Analytical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey.

出版信息

Spectrochim Acta A Mol Biomol Spectrosc. 2018 Apr 5;194:8-13. doi: 10.1016/j.saa.2017.12.057. Epub 2017 Dec 21.

DOI:10.1016/j.saa.2017.12.057

PMID:29306060

Abstract

In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R=0.992) was established between the specific SERS intensity of BCI at 600cm and logarithmic E. coli concentration in the range of 1.7×10-1.7×10cfumL. LOD and LOQ values were also calculated and found to be 10cfumL and 30cfumL, respectively.

摘要

本研究利用表面增强拉曼光谱（SERS），开发了一种基于碱性磷酸酶（ALP）在 5-溴-4-氯-3-吲哚磷酸盐（BCIP）上的酶活性用于检测大肠杆菌（E. coli）的三明治免疫测定方法。为此，合成并修饰了球形磁性金涂核壳纳米粒子（MNPs-Au）和棒状金纳米粒子（Au-NRs），用于从溶液中免疫磁分离（IMS）大肠杆菌。为了将开发的方法具体指定到 ALP 活性，用该酶标记了 Au-NRs。成功构建免疫测定法后，添加 BCIP 底物以产生 SERS 活性产物；5-溴-4-氯-3-吲哚（BCI）。在 600cm 处 BCI 的特定 SERS 强度与 1.7×10-1.7×10cfumL 范围内大肠杆菌浓度的对数之间建立了良好的线性关系（R=0.992）。还计算并确定了 LOD 和 LOQ 值，分别为 10cfumL 和 30cfumL。