Poster Y4 - Layer-by-Layer film Coated Self-Floating Hollow Glass Microspheres for Rapid Isolation and Recovery of Salmonella

Introduction: Isolation, recovery and detection of Salmonella from food samples and bodily fluids is essential for elimination or significant reduction of such infections. Currently used traditional isolation techniques such as culture-based isolation are time consuming and take 4-5 days for Salmonella confirmation. Newly developed rapid isolation techniques such as microfluidics and immunomagnetic nanoparticles mediated isolation need sophisticated lab equipment, tedious microfabrication or may impact cell viability, function and phenotype due to nanoparticle interaction and magnetic forces. These limitations of the established Salmonella isolation techniques make them inaccessible in resource limited areas. Here, we present a rapid yet simple method for specific Salmonella isolation and recovery from mixed bacterial populations using self-floating hollow glass microspheres (HGMS), coated with biodegradable layer-by-layer (LbL) films and Salmonella specific antibodies. This method bypasses the limitations posed by established isolation techniques mentioned earlier as it does not require any sophisticated laboratory equipment, trained personnel or external force (electric, magnetic or optical).
In this work, we demonstrate a proof-of-concept study of isolation, recovery, and detection of Salmonella Typhimurium (S.T.) from various food and body fluid matrices. However, we suggest that this method can be extended for detection of various bacterial populations. We were able to achieve maximum recovery of 22% and a minimum detection limit of 100 CFU/ml. Owing to its easy, efficient, rapid and reliable operation technique, coupled a with rapid growth rate of Salmonella, this functionalized-HGMS mediated isolation and recovery method is a promising alternative to conventional isolation method, especially in resource limited areas.

Materials and

Methods: Materials - Alginate (ALG), Polydiallyldimethylammonium chloride (PDDA) , Biotin hydrazide, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), sulfo-N-hydroxysuccinimide (sulfo-NHS) (24510), neutravidin (Mw = 60,000 Da), neutravidin Texas Red conjugate (A-2665) (Mw = 200 000–350 000), ampicillin, salmonella polyclonal antibody, biotin (PA1-73022), Gentamicin, Difco® nutrient agar and Difco® pseudomonas isolation agar, LB broth, hollow glass microspheres (HGMS, density 0.6 g/mL and particle size 18µm), Blood from healthy donors. Biotin conjugated ALG (BALG) was prepared following a previously published method.

Layer-by-layer nanofilm assembly on Hollow Glass Microspheres – BALG and PDDA were dissolved in deionized water (pH 7) to a concentration of 2 mg/ml and subsequently pH adjusted to 5. HGMS were sterilized with ethanol and washed with deionized water. The deposition of polymers via layer-by-layer method involved incubating HGMS with PDDA and BALG polymers for 10 minutes for the first bilayer and 5 minutes for each subsequent bilayer, totaling 5 bilayers. A washing step with deionized water was conducted in between each polymer deposition. After LbL deposition neutravidin and biotinylated antibodies were introduced subsequently for functionalization.

Simulation of motion of HGMS in tubes on rotating mixer – Simulation of motion of HGMS during rotational mixing was performed using laminar flow and particle tracing packages (COMSOL Multiphysics 5.5, COMSOL).

Bacteria Culture and Counting –

Cell Isolation and Recovery –

Quartz Crystal Microbalance with Dissipation Monitoring -

Atomic Force Microscopy -

Fluorescence Microscopy –

Scanning Electron Microscopy –

Results, Conclusions, and Discussions: Results and discussion –

Preparation and characterization of LbL coated HGMS –

Hollow glass microspheres (HGMS) were chosen for capture and isolation of Salmonella since they have a density (0.6 g/ml) lower than aqueous mediums, hence float to the top when mixed with aqueous medium and can be easily separated. In addition, glass provides a stable substrate with an inherent strong negative charge which makes it easy to coat it with alternate positive and negatively charged layer-by-layer (LbL) polymeric films.

Simulation of HGMS motion in tube on rotating mixer –
HGMS covered 72.05% of the total tube area after 1 rotation (t = 6 s), while after 10 rotations (t = 60 s) coverage was 84.09% with a steady particle velocity of around 0.1 m/s. From these results it is clear that HGMS move at low velocities and cover majority of the microtube area throughout the capture process.

Capture, release and recovery capability of the functionalized HGMS –
These studies established the capability of HGMS platform in isolation and recovery of specific bacterial population, at concentrations as low as 100 CFU/ml in PBS and 200 CFU/ml in spiked matrices such as beef, cantaloupe, and lysed blood with a maximum capture efficiency of 22%. AFM and QCM-D results also confirmed successful film formation, bacteria deposition, and release.

Conclusion –
In this study we proposed the use of layer-by-layer nanofilm and specific antibodies coated self-floating hollow glass microspheres for rapid isolation and recovery of bacteria using Salmonella Typhimurium GFP as the model organism. The whole process took 2 h for isolation and recovery and we achieved a detection limit of 100 CFU/ml. Moreover, we also demonstrated the application of our approach in spiked food and blood samples, which indicates that it could be translated to clinical applications. The entire process requires no sophisticated lab equipment, trained personnel, external power, magnetic or optical source and hence, can be used in remote resource deficient areas for point-of-care diagnostic. In addition, our method can be readily integrated with conventional techniques such as PCR, ELISA, MALDI-TOF, fluorescence detection, SERS based detection, etc. for isolate confirmation.

Acknowledgements (Optional):