Bacillus thuringiensis Cells Selectively Captured by Phages and Identified by Surface Enhanced Raman Spectroscopy Technique.

Salvatore Almaviva,Alessandro Rufoloni,Eleonora Aruffo,Antonia Lai,Antonio Palucci

doi:10.3390/mi12020100

Abstract

In this work, the results on the detection and identification of Bacillus thuringiensis (Bt) cells by using surface-enhanced Raman spectroscopy (SERS) are presented. Bt has been chosen as a harmless surrogate of the pathogen Bacillus anthracis (Ba) responsible for the deadly Anthrax disease, because of their genetic similarities. Drops of 200 μL of Bt suspensions, with concentrations 102 CFU/mL, 104 CFU/mL, 106 CFU/mL, were deposited on a SERS chip and sampled after water evaporation. To minimize the contribution to the SERS data given by naturally occurring interferents present in a real scenario, the SERS chip was functionalized with specific phage receptors BtCS33, that bind Bt (or Ba) cells to the SERS surface and allow to rinse the chip removing unwanted contaminants. Different chemometric approaches were applied to the SERS data to classify spectra from Bt-contaminated and uncontaminated areas of the chip: Principal Component Regression (PCR), Partial Least Squares Regression (PLSR) and Data Driven Soft Independent Modeling of Class Analogy (DD-SIMCA). The first two was tested and trained by using data from both contaminated and un-contaminated chips, the last was trained by using data from un-contaminated chips only and tested with all the available data. All of them were able to correctly classify the SERS spectra with great accuracy, the last being suitable for an automated recognition procedure.

Highlights

IntroductionFor public health, industrial processes and in the case of a bio-terroristic attack
Nowadays the rapid detection and identification of microorganisms in the environment, including aerosol, water and food, is a major issue to prevent large numbers of infections [1]for public health, industrial processes and in the case of a bio-terroristic attack.For this purpose, fast analytical techniques, not requiring time-demanding samples preparation and suitable to be applied in a continuously running device, are strongly desired.In this context Surface-Enhanced Raman Spectroscopy (SERS) [2] has been successfully used as a label-free bio-sensing optical technique for rapid identification of bacteria and related spores [3], thanks to its ability to identify molecules from their intrinsic vibrational modes [2,3]
The fluorescence background subtraction was performed with an iterative procedure, similar to the one reported in [28], replacing the standard high-order polynomial function with a new fitting function obtained from an iterative data processing algorithm

Summary

Introduction

For public health, industrial processes and in the case of a bio-terroristic attack For this purpose, fast analytical techniques, not requiring time-demanding samples preparation and suitable to be applied in a continuously running device, are strongly desired. Fast analytical techniques, not requiring time-demanding samples preparation and suitable to be applied in a continuously running device, are strongly desired In this context Surface-Enhanced Raman Spectroscopy (SERS) [2] has been successfully used as a label-free bio-sensing optical technique for rapid identification of bacteria and related spores [3], thanks to its ability to identify molecules from their intrinsic vibrational modes [2,3]. EmSERS originates from the excitation of surface plasmons at the surface of the SERS substrate, when illuminated with laser light

Results

Discussion

Conclusion