The assembly of separation channels was the same as previous work [3
]. Channel was assembled using three layers of cut-out Mylar sandwiched between two plastic sheets. Plastic screws were used beside the splitter edge to seal the Mylar layers and avoid carrier leakage at high flow-rates. Four sets of channel length and channel breadth [(70 mm, 5 mm), (140 mm, 5 mm), (70 mm, 10 mm), and (140 mm, 10 mm)] were used. All experiments of susceptibility determinations used 70 mm of channel length, 5 mm and 10 mm of channel breadths. All channels used 0.25 mm of thickness.
Magnetic fields were generated by assembling one pair of rare-earth magnets [neodymium-iron-boron (Nd-Fe-B)] and soft ion pole pieces, which conducted the magnetic fluxes to the interpolar gap. The maximum energy products of the Nd-Fe-B magnets were 3.5 × 107G-Oe. Magnetic field intensities were generated from 5 mm and 10 mm of gap widths with 70 mm and 140 mm of gap lengths. Magnetic field was measured using a Gaussmeter and a Hall-effect probe (Model 5080, F. W. Bell, Orlando, FL, USA). The saturated magnetic field intensities (Bo) were 1.20 Tesla and 0.72 Tesla for 5 mm and 10 mm of gapwidths, respectively. Microstages were used to adjust magnetic field intensities.
The carrier of study was phosphate buffered saline (PBS) with pH of 7.02 and viscosity of 1.0 × 10-2 g cm-1s-1. Samples and carriers were delivered into the separation channel using a microtubing pump (Eyela Mp-3, Rikakikai, Tokyo, Japan) and LC pump (SSI series II, State College, PA, USA), respectively. Sample verification was done using light microscopy (Olympus BX-50, Tokyo, Japan). Sample counting was done using a hemacytometer (cell-counting chamber with microscalar grids having fixed volume). Trypan blue, manganese sulfate and iron nitrate were purchased from Sigma Chemical (St. Louis, MO, USA). Copper chloride was purchased from the Aldrich Chemicals (St. Louis, MO, USA). Erbium chloride was purchased from Strem (Newburyport, MA, USA). Dynabeads M-450 (4.5 μm) were uniform particles doped with iron oxides. Yeasts were purchased from local bakery of Taichung. Blood cells were obtained from the Jen-Ai Hospital in Dali (Taichung County, Taiwan).
Fresh blood from hospital was centrifuged at 50 g for 5 min to remove plasma. The cells were then washed with PBS and centrifuged three times before labeling. Various concentrations of labeling ion were obtained by diluting 100 mM of prepared stock solutions. Various ion-labeled RBC were prepared by mixing 1 mL of labeling ions at fixed concentrations with 9 mL of solutions containing 4.0 × 105 RBC and incubating in ice for 30 min with shaking every 10 min. All ion-labeled cells were washed three times with PBS solution before use to remove unlabeled ions. The labeling ions were Er3+, Fe3+, Mn2+, and Cu2+. Dye exclusion testing was carried out using trypan blue stain and a hemacytometer. This method was based on the assumption that viable cells did not take up dyes, whereas nonviable cells did. For viability testing, 0.5 mL containing 1.0 × 106cell suspensions were mixed thoroughly with 0.5 mL of 0.4% (w/v) trypan blue solution for 5 min before counting.
Fractional retrieval of samples at outlet a (Fa) was calculated using the following equation:
where Na and Nb were the number of particles exiting at outlets a and b, respectively. The sum of Fa and Fb was equal to one. The recovery percentage of RBC was calculated by adding the total number of RBC exiting at both outlets and dividing by the total number of RBC entering at the inlets. A minimum of 200 cells was counted in each retrieval experiment.
Pulsed sample injections were used for susceptibility determination in analytical application and optimization of separation conditions for continuous particle separation. The loop volume of pulsed sample injection was 0.7 ml. Reference measurements of magnetic susceptibility were made using an MPMS5 model superconducting quantum interference device (SQUID) magnetometer from Quantum Design (San Diego, CA, USA). Magnetic field intensities from 1.0 × 104 to 2.0 × 104 gauss were used for susceptibility measurements in SQUID. The cgs system and volume magnetic susceptibility, χ, are used throughout this study for convenient calculation unless otherwise indicated.
In preparative application, samples were continuously introduced into one inlet and fractionated RBC and yeasts were collected at two outlets. Fractionated RBC and yeasts were verified using microscopy for size and shape, and verified by permanent magnets for susceptibility at the end of each hour run during continuous separation.