2.?Experimental Section2.1. MaterialsN-isopropylacrylamide (NIPAM, more 99%), N,N��-methylenebisacrylamide (MBA, 99%, crosslinker), potassium persulphate (KPS), TEMED (99.9%), isopropyl alcohol (IPA, 99.9%), urografin and phosphate-buffered saline (PBS) solution were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. PDMS (Sylgard 184, Dow Corning, Midland, MI, USA) and SU-8 photoresist (Microchem, Newton, MA, USA) were used for the fabrication of PDMS microfluidic device. Refined grape-seed oil (G-oil, Beksul, Seoul, Korea) and Abil EM90 (Degussa, Essen, Germany) were mixed and used as a continuous flow solution.2.2. Preparation of Bacterial SporesAll bacterial strains and plasmids used in this study were reported previously [19].
We cultured BT subspecies israelensis 4Q7 harboring expression vector for displaying proteins in CDSM media [20] at 37 ��C with 250 rpm for 48�C60 h. Pellet was obtained from 100 mL of culture by centrifugation (10,000 �� g, 10 min) and suspend the pellet in 1 mL of 20% (wt/vol) urografin. This suspension was gently layered over 10 mL of 50% (wt/vol) urografin in a 15 mL centrifuge tube, and then centrifuged for 4 ��C, 10 min at 16,000 �� g. The collected pellets containing only free spores were stored at ?20 ��C [20,21].2.3. Fluorescence AnalysisThe purified EGFP-displaying BT spores by the urografin gradient method [20] were washed and resuspended at ~1.0 �� 108 CFU/mL in PBS. Fluorescence assay was performed using a multi-plate reader, SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA).
Flow cytometry data was obtained using a FACSCalibur? flow cytometer and the Cell Quest Pro? software (BD Bioscience, San Jose, CA, USA). Spores displaying EGFP was analyzed for relative fluorescence intensity using an FL1 green fluorescence detector with a 530/30 nm bandpass filter. The mean value (M) indicates the mean fluorescence intensities obtained by FL1 detectors.2.4. Imaging of EGFP-Displayed SporesThe purified EGFP-displaying spores were mounted on poly-L-lysine-coated glass slides (Cel & Associates, Pearland, TX, USA) and analyzed under an LSM 510 confocal laser scanning microscope (Carl Zeiss, G?ttingen, Germany). Samples were excited at 488 nm with an argon laser, and the images were filtered with a longpass 505 nm filter. All final images were generated from 4�C5 serial images made by automatic optical sectioning.
2.5. Fabrication of PDMS Microfluidic DevicesPDMS/PDMS bonded microfluidic channel designs were fabricated by soft lithography and PDMS molding technique. The silicon master was coated with SU-8 photoresist by spin-coating and transferred the design onto the wafer using the mask and UV light exposure. Microfluidic devices were Entinostat obtained inhibitor licensed with PDMS using silicon master with SU-8 pattern. A mixture of PDMS prepolymer and curing agent (10:1 Sylgard184, Dow Corning) was stirred and degassed in a vacuum oven at 70 ��C.