Functionalized polymeric magnetic nanoparticle assisted SERS immunosensor for the sensitive detection of S. typhimurium.

The quest for detecting bacteria has gained momentum in food and beverage industry for preventing spoilage of products to maintain requisite quality. The present paper describes the development of a SERS immunosensor for the detection of model pathogen, S. typhimurium using strategically synthesized functionalized polymeric magnetic nanoparticles (FPMNPs) as effective capture probe and immunomagnetic separator. The synthesized probe contains surface diketonic functionalities which covalently link with amino groups of antibodies against Salmonella common structural antigen (CSA-1-Ab) and hence specifically captured the target bacteria. Magnetic core of nanoparticles facilitated easy separation of target bacteria from the milieu of non-specific molecules. Gold nanoparticles (GNPs) modified with CSA-1-Ab and external Raman reporter molecules (RRM) were used as signal probes. We compared the signalling attributes of 4-mercapto benzoic acid (MBA) and 5,5'-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB) as RRMs. Capture and signal probes sandwich the target bacteria upon its addition, generating Raman signal from the 'hot-spots' created by signal probe. Under optimal conditions, the SERS intensities of MBA and DSNB at 1588 and 1336 cm-1 respectively were used to measure the concentration of the pathogen in the range of 101-107 cells mL-1. Limit of detection (LOD) of MBA and DSNB based immunosensor was measured as 100 cells mL-1, and 10 cells mL-1 respectively. Moreover, appreciable recovery (82-114%) was recorded for sensing method for different spiked food products. Thus, the developed magnetically assisted SERS immunosensor is sensitive, specific and has strong potential to be used for detecting contamination in food samples in field conditions.

Functionalized polymeric magnetic nanoconstructs for selective capturing and sensitive detection of Salmonella typhimurium.

Rapid detection and enumeration of pathogens is essential for monitoring contamination and spoilage of food products to ensure improved quality control management. Functionalized polymeric magnetic nanoconstructs (FPMNCs) were developed as an effective immunomagnetic separator and sensing platform for the selective capturing of Salmonella typhimurium. Novel FPMNCs were prepared in three stages involving synthesis of iron oxide (IO) dispersion, capping with sodium oleate and encapsulation of preformed IO nanoparticles by in-situ free radical emulsion polymerization of styrene (St), methyl methacrylate (MMA) and acetoacetoxy ethylmethacrylate (AAEM). PMMA improves the stability of FPMNCs by bridging extremely hydrophobic PS and hydrophilic PAAEM. Core-shell morphology of hydrophobic core of IO, PS & PMMA and hydrophilic shell of PAAEM was demonstrated by SEM, TEM and FTIR studies. FPMNCs with surface functionalized acetoacetoxy groups were covalently attached with polyclonal antibodies against Salmonella common structural antigen (CSA-1-Ab) without using any linker and catalyst. Colorimetric readout signal was acquired using CSA-1-Ab-HRP as secondary antibody after formation of sandwich immunocomplex with bacteria where the optical density of the samples were recorded using ELISA plate reader at 450 nm. The developed immunoassay was specific and selective which captures only targeted S. typhimurium with a detection limit of 10 cells/mL lower than infectious dose of salmonellosis infection. Minimal interference of food matrix with high signal to noise ratio was shown by various food samples. In addition, the performance of developed FPMNC based immunoassay was superior to commercially available immunomagnetic microbeads demonstrating undisputed advantage for capturing and detecting specific bacteria without any pre-enrichment of sample.