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

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 (R²=0.992) was established between the specific SERS intensity of BCI at 600cm^-1 and logarithmic E. coli concentration in the range of 1.7×10¹-1.7×10⁶cfumL^-1. LOD and LOQ values were also calculated and found to be 10cfumL^-1 and 30cfumL^-1, respectively.

Keywords: Alkaline phosphatase; Escherichia coli detection; Sandwich immunoassay; Surface enhanced Raman spectroscopy.