Biosensor Development: Optimizing Immunomagnetic Separation of Bacteria

Abstract

For years, there have been several millions of deaths around the world from diseases such as Tuberculosis, Meningitis, Pneumonia, Cholera, etc. which are caused by different types of bacteria. In addition, a large number of food industries face a challenge of bacterial contamination that further results in human death and wastage of food resources. With the recent advancements in nanotechnology, many methods have been demonstrated to separate and detect bacteria from a given sample using magnetic nanoparticles (ex- Fe3O4) of small size (1-100 nm). However, there is still no optimization reported for use of Fe3O4 nanoparticles for immuno-magnetic separation of bacteria. To construct a suitable biosensor technology and a small microfluidic device, we have optimized the immuno-magnetic separation of bacteria using very small ~7 nm iron oxide nanoparticles (NPs). In this work, we have synthesized ~7 nm Fe3O4 nanoparticles using organic phase synthesis and further tuned their surface chemistry to disperse them in water. Polyclonal antibodies targeting E. coli K-12 were then immobilized over Fe3O4 nanoparticles dispersed in water. We then tested different ratio of NPs : E. coli cells to – (a) evaluate toxicity of NPs towards E. coli cells, (b) find the optimal number of nanoparticles required for efficient capture, and (c) derive capture efficiency for different concentrations of E. coli K-12. The optimization for the use of ~7nm Fe3O4 NPs for capture of bacteria has provided us with results that led to a conclusion for using 106:1 ratio of NP: E. coli cells, meanwhile minimally affecting cell viability. The same ratio provided us with maximum capture efficiency of more than 60% with average capture of more than 50% for all different concentrations of E. coli cells. In addition, we have successfully demonstrated the proof of concept and specificity of our assay using transmission electron and fluorescence microscopy imaging. Our results have successfully optimized bacterial capture using ~7nm Fe3O4 NPs (the smallest to be reported in literature) which will be further used for the construction of a suitable biosensor and hand-held microfluidic device for detection of pathogenic bacteria at very low concentrations (<100 cfu/ml)