Experimental Section/Methods
Bacteria preparation: Salmonella Typhimurium VNP20009 cheY+ \cite{Broadway_2017} from a single colony was cultured overnight in MSB medium (10 g/L tryptone, and 5 g/L yeast extract, 2 mM MgSO4, 2mM CaCl2, pH 7.0) at 37°C and 100 rpm (Excella E24 Incubator Shaker Series, New Brunswick Scientific). The overnight bacterial culture was diluted in MSB to 1% (v/v) and incubated at 37°C and 100 rpm until an optical density at 600 nm (OD600) of 1.0 was reached. The bacterial culture was centrifuged at 1,700 ×g for 5 minutes twice and re-suspended in motility buffer (6.4 mM K2HPO4, 3.5 mM KH2PO4, 1 μM L-methionine, 10 mM Sodium DL-lactate, 2 mM MgSO4, 2 mM CaCl2, pH 7.0) to a final OD600 of 1.0.
PLGA Nanoparticle Synthesis: 100 mg of Pluronic® F127 and 20 mL of deionized water were added to a glass vial. The vial was placed in a water bath sonicator (Branson 2510 Ultrasonic Cleaner, 100 W) for 30 minutes to dissolve the Pluronic. Then, a magnetic stir bar was added, and the solution was stirred at 600 rpm. Acid-terminated PLGA (Mw: 25,000 g mol−1, 50:50 lactic acid:glycolic acid, acid end‐capped, Akina Inc. PolySciTech, West Lafayette, IN) was dissolved with dimethylformamide (DMF, Sigma‐Aldrich, St. Louis, MO) to a final concentration of 22.22 mg/mL; the solution was sonicated for 30 minutes to ensure molecular dissolution. While the PLGA solution was sonicating, 6,13‐bis(triisopropylsilylethynyl) pentacene (TIPS, Sigma‐Aldrich, St. Louis, MO) was dissolved in tetrahydrofuran (THF, anhydrous and uninhibited, >99.9%, Sigma‐Aldrich, St. Louis, MO) to a final concentration of 3.05 mg/mL. After the PLGA solution was fully dissolved, TIPS solution was added to the PLGA solution to achieve a 1:10 THF:DMF volume ratio. The mixture was vortexed for ~5 seconds before it was loaded into a 5 mL glass syringe with a 21-gauge needle attached. Care was taken to remove all macroscopic air bubbles from the syringe. The TIPS:PLGA mixture (1 mL) was added dropwise (0.5 mL/min, NE-1000, New Era Pump Systems Inc.) to the stirring (600 rpm) Pluronic F127 solution. The resulting nanoparticle suspension was allowed to stir for 5 hours at 600 rpm. The suspension was protected from light to prevent degradation of the fluorophore. After 5 hours, the suspension was centrifuged at 22,789 ×g for 30 minutes at 4ºC (Sorvall Legend X1R, Thermofisher Scientific). The supernatant was discarded. The pellet was resuspended in 20 mL of 1× PBS by vortex mixing for 2 minutes and sonicating the suspension for 30 minutes. The final dispersed suspension was passed through a nitrocellulose syringe filter (0.45 µm pore size) to remove any remaining aggregates. The filtrate was stored in a foil-wrapped vial at room temperature.
Streptavidin Functionalization of PLGA Nanoparticles: Microcentrifuge tubes were filled with 700 µL of PLGA NP suspension. The tubes were centrifuged at 16,060 ×g for 10 minutes at room temperature (accuSpin Micro, Fisher Scientific). The pellets were resuspended in 800 µL of EDAC coupling solution (20 mg/mL EDAC, 5 µg/mL streptavidin-Cy3, pH 5.2 50 mM MES buffer). The streptavidin coupling reaction took place on a vortex mixer (500 rpm, Fisher Digital Vortex 120V, Fisher Scientific) for 3 hours. Following streptavidin coupling, the microcentrifuge tubes were centrifuged at 16,060 ×g for 10 minutes at room temperature. The pellets were resuspended in 100 µL of motility buffer.
Dynamic Light Scattering (DLS) Measurements: DLS measurements were performed using a Zetasizer Nano ZS (Malvern Instruments) operating with Zetasizer Software v7.12. Disposable polystyrene cuvettes were filled with 1 mL of the final aqueous nanoparticle suspensions. Measurements were performed at room temperature, and the results are shown in Figure S6 and Table S1.
Zeta Potential Measurements: The final aqueous nanoparticle suspensions were loaded into disposable polystyrene capillary cells. Zeta potential measurements were performed using a Zetasizer Nano ZS at room temperature. The results are shown in Figure S6 and Table S1.
Nanoparticle Tracking Analysis (NTA): Dilutions (10× and 100×) of the functionalized nanoparticle suspensions were analyzed via nanoparticle tracking analysis using a NanoSight NS500 (Malvern Instruments) operating with NanoSight NTA v3.4. All measurements were performed at room temperature. Five 1-minute videos were taken for each sample, and the nanoparticle scattering cones were tracked to determine the number concentrations of the nanoparticle suspensions before incubating the nanoparticles with bacteria to form NanoBEADS.
PLGA NanoBEADS assembly: To prepare the ABS NanoBEADS, the prepared bacterial suspension in motility buffer was incubated with 10 µg/mL biotinylated Salmonella polyclonal antibody (Thermo Scientific, Waltham, MA, USA) on a vortex mixer at 500 rpm at room temperature for 1 hr. The antibody-coated bacteria suspension was then centrifuged at 1,700 ×g for 5 minutes to remove free antibody and suspended in motility buffer to an OD600 of 2.0 (Cary 60 UV-Vis, Agilent Technologies). Next, the suspension of biotinylated antibody-coated bacteria was mixed with the streptavidin-coated nanoparticle at a bacteria to particles ratio of 1:100 in a volume of 100 µL or 800 µL (as described in Table 1) and incubated on a vortex mixer at 500 rpm, a Belly Dancer® mixer (IBI Scientific) at 100 rpm, or an end-over-end mixer (Fisher Scientific) at 15 rpm for 30 minutes, 60 minutes or 90 minutes to facilitate the assembly of nanoparticles onto the bacteria. For BS NanoBEADS, the bacterial suspension in the motility buffer was incubated with 0.4 mg/mL biotin (Fisher BioReagents, Fair Lawn, NJ) on a vortex mixer at 500 rpm at room temperature for 30 minutes to physisorb biotin onto bacteria. Subsequently, the suspension was centrifuged at 1,700 ×g for 5 minutes to remove free biotin and resuspended the biotin-coated bacteria in motility buffer to an OD600 of 2.0. Next, the suspension of bacteria coated with biotin was mixed with the streptavidin-coated nanoparticles at a bacteria to particles ratio of 1:100 in a volume of 800 µL and incubated on a belly dancer mixer at 100 rpm or an end over end mixer at 15 rpm for 60 minutes. After the assembly process, the suspension of NanoBEADS was transferred to a centrifugal filter unit with a 0.8 µm pore size high-flux polyethersulphone membrane (Sartorius Vivaclear, Elk Grove, IL) and centrifuged at 1,700 ×g for 30 seconds to remove free nanoparticles. The NanoBEADS were suspended in motility buffer to an OD600 of 1.0 for NP areal density characterization experiments or in McCoy's 5A for the growth and motility assays.
NanoBEADS samples preparation for field emission scanning electron microscope (FE-SEM): To quantify the number of nanoparticles attached to the outer membrane of bacteria, SEM images of NanoBEADS were taken using FE-SEM. A 10 µL aliquot of the NanoBEADS suspension was deposited on 0.005% (w/v) poly-L-lysine (PLL) treated glass slides and incubated at room temperature for 5 minutes to allow for attachment. Afterward, the slide was rinsed in DI water to remove the loosely attached nanoparticles and NanoBEADS. Then, the slide was covered with 4% glutaraldehyde for 2 hours at 4°C to fix the attached NanoBEADS. Next, the slide was soaked in 0.1 M Phosphate-buffered saline (PBS) for 20 minutes twice. Finally, the same soaking process was repeated with deionized water. After air-drying overnight, the slide was sputter-coated with 7 nm Pt/Pd prior to imaging (Leica ACE600 sputter). High-resolution images were obtained utilizing a LEO (Zeiss) 1550 FE-SEM at an accelerating voltage of 5 kV and working distances of <8.6 mm. To determine the average number of attached nanoparticles for each NanoBEADS experiment, the particle numbers on ~ 50 bacteria were counted for each replicate. A minimum of two independent experiments were carried out for each case.
Viability assay: The filtered NanoBEADS were diluted in motility buffer to an OD600 of 0.05. To this 1 mL diluted NanoBEADS solution, 1.5 µL aliquots of the 3.34 mM SYTO 9 nucleic acid stain and 20 mM propidium iodide (LIVE/DEAD® BacLight™ Bacterial Viability Kit, Thermo Fisher, Eugene, Oregon, USA) were added, followed by incubation in the dark at room temperature for 15 minutes as per the manufacturer's instructions. The fluorescence microscopy images of NanoBEADS were taken using a Zeiss AxioObserver Z1 inverted microscope equipped with an AxioCam mRM camera at 40× objective. Live cells with an intact membrane stained green only, where cells with a damaged membrane or dead cells also stained red.
Growth rate measurement: The NanoBEADS suspensions were diluted in 3 mL of McCoy's 5A medium supplemented with 10% FBS to a final OD600 of 0.001. The diluted NanoBEADS suspension was incubated at 37°C with shaking at 100 rpm for 10 hours. A 100 µL sample was taken every hour, diluted, and plated on triplicate 1.5% LB agar plates. Following overnight incubation at 37°C, the bacteria colonies on the agar plates were counted to determine the NanoBEADS growth rate. A minimum of two independent experiments were carried out for each case.
Swimming speed measurement: The bacterial and NanoBEADS suspensions were diluted in 3 mL of McCoy's 5A medium supplemented with 10% FBS to a final OD600 of 0.001. The diluted suspensions were incubated at 37°C with shaking at 100 rpm for 2 hours. A 10 µL sample was taken, diluted, and placed on the glass coverslip. The videos of the bacteria movement were taken with a Zeiss AxioObserver Z1 inverted microscope equipped with an AxioCam Hsm camera and 63× oil immersion objective. The videos were analyzed in ImageJ using the MTrackJ plug-in tool. The average swimming speed was calculated by averaging the instantaneous speed, which is the traveled distance in each unit of time divided by the time unit. For each experiment, about 50 bacteria or NanoBEADS were tracked in the 15 s videos acquired at 32.8 fps. A minimum of two independent experiments were carried out for each case.