11 elite

For separation of the magnetic HRP-bacteria, both polyvinyl pyrrolidone and polyethylene oxide were purchased from Sigma Aldrich (St. Louis, MO, USA) and prepared in the deionized water (1%, w/v, 18.2 MΩ·cm, Advantage 10, Millipore, Billerica, MA, USA) as the viscoelastic solution. For parameter optimization, the fluorescent polystyrene (PS) microspheres with the sizes of both 2.2 µm and 100 nm purchased from VDO Biotech (Suzhou, China) were prepared in the PVP solution with the concentration of 0.05% (w/v) for simulating the magnetic HRP-bacteria and the unbound MNPs. After the magnetic HRP-bacteria were formed, they were resuspended in 50 µL of the PVP solution. To prevent the particles from aggregating during the separation, Tween-20 (0.5%, v/v, Amresco, Solon, OH, USA) was added into both the sample flow and the sheath flow.
The sample flow and the sheath flow were injected into the microchannel using two precision syringe pumps (11Elite, Harvard Apparatus, FL, USA) with different flow rates. The separation of the fluorescent PS microspheres was observed using the fluorescent inverted microscope. The fluorescent trajectories were recorded using long exposure time (up to 600 ms). The experimental results were analyzed using the microscope’s software NIS-Elements AR 2.30.

A 200-μm wide and 40-μm high polydimethylsiloxane (PDMS) microchannel was bonded to the groove-patterned glass substrate by plasma treatment (Plasma Cleaner CY-P2L-B), forming an enclosed microfluidic device (see Figure 1c). Before the bonding, the glass substrate was placed into tap water for 10-min ultrasonic cleaning to remove the surface debris that was produced due to the laser engraving. Then, the glass substrate was dried by nitrogen blast. Additionally, a control group was established using a glass substrate without groove patterns. The particle samples were injected into the channel via the inlet using a syringe pump (Harvard Apparatus 11 Elite). Particle positions across the channel width direction were recorded under an inverted microscope (Zeiss Axiovert 100 Microscope).

The bacterial cells were lysed by applying a voltage of 12 V on this self-developed eddy heater. The temperature was measured using both a temperature probe (NR81530, LiHuaDa, Shenzhen, China) and a smartphone infrared thermal imager (HT-102, XinTai, Dongguan, China). First, the microfluidic chip was placed on the eddy heater to preheat the iron rod for 30 s, making its temperature reach ~150 °C. Then, 1 mL bacterial sample was centrifuged at 10,000 rpm for 5 min, mixed with 100 µL DNA extraction buffer and injected into the microfluidic chip at different flow rates using a syringe pump (11Elite, Harvard Apparatus, FL, USA). Finally, the lysate was mixed with 15 μL MSBs to form the MSB-DNA complexes, followed by magnetic separation for 2 min, cleaning with washing buffer for 4 times and elution with 50 μL eluent to obtain the purified DNA, which was determined using quantitative PCR to evaluate the performance of DNA lysis. The sequences for the primer set were listed in Table S2 and PCR reaction was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s [31 (link)]. All the samples were tested in triplicates using the same protocol.

A microfluidic device for particle focusing consists of two layers: a polydimethylsiloxane (PDMS) layer having a straight rectangular microchannel, one inlet and one outlet, and a glass substrate with laser engraved grooves (Fig. 1b). The PDMS microfluidic channel of 100 μm in width and 20 μm in height was bonded to the laser engraved glass substrate after plasma treatment (Plasma Cleaner CY-P2L-B). Briefly, the channel embedded in PDMS was partially bonded to the glass substrate to reduce movements between them. Then the irradiated glass and the PDMS channel were manually aligned under an optical microscope carefully. The samples containing polystyrene particles or live cells were infused into the microfluidic device using a 250-μL Hamilton syringe by a syringe pump (Harvard Apparatus 11 Elite). The distributions of lateral positions of particles across microchannel width before and after passing the arrays of grooves were monitored and recorded under an inverted microscope (Axiovert 135, Carl Zeiss, Germany).

Wild type magnetotactic bacteria of the strain Magnetospirillum gryphiswaldense (MSR-1) were grown following standard protocols^{41 (link)}. The bacteria were immobilized by exposure to 75 °C for 15 minutes to avoid active swimming in the microfluidic channel, diluted in PBS buffer containing 10% glycerol and fluorescently labeled with DAPI (Sigma-Aldrich, Germany). For this experiment a chip with reduced dimensions was used (Supplementary Fig. S3). Bacterial cells were injected from a 0.5 ml syringe (Hamilton, Sigma-Aldrich, Germany) at 0.1 µl/min while the buffer PBS was injected 0.5 µl/min at the second inlet in order to adjust for broadening of the flow during the experiment, using a syringe pump (Elite 11, Harvard Apparatus, USA). The flow of cells was observed at the center of the sorting channel on an inverted microscope (Axiovert 200 M, Zeiss, Germany) and imaged with a fluorescence camera (Flea3, Point Gray, Canada) at 60 fps and an exposure time of 16 ms. In order to ensure laminar flow in the system prior to sorting, the flow was observed also in the absence of the external magnets, which were placed multiple times with one placement defining a trial. Data sets were discarded if laminar flow conditions were not observed without the magnets, ensuring observation of real sorting events.

Linear, purified peptide Agaphelin (1–58) was dissolved in 6 M guanidin•HCl/200 mM PBS at a concentration of 2 mmol, and the solution was introduced by a Harvard Apparatus “Elite 11” through a syringe, to a 30 times larger volume of the stirred solution of degassed, 50 mM Tris/1 mM EDTA, pH∼8, containing reduced glutathione and oxidized glutathione at concentrations of 1.6 mM and 0.2 mM, respectively. The progress of folding was monitored by HPLC using a gradient of acetonitrile/water with UV monitoring at 215 nm. After 3 h, the reaction mixture was acidified with 2% trifluoroacetic acid to pH 5. The folded peptide was isolated by preparative reverse-phase HPLC and its purity checked by HPLC analyses and mass spectrometry using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer Axima CFR+ (Shimadzu Scientific Instruments). Pure fractions were combined, frozen, and lyophilized to afford pure, folded peptide. Agaphelin molecular mass is 6273 Da (58 amino acids, mature form) with an estimated pI 5.09. Extinction coefficient at 280 nm is 3355; A280 nm/cm 0.1% (1 mg/ml), 0.535. Agaphelin was diluted in PBS (1–1.5 mM) and frozen at −80°C.