3.2. Six consecutive serial two-fold dilutions for crystal
violet solution
To prove the serial dilution capability of the centrifugal microfluidic
system, we first fabricated the disc capable of conducting six
consecutive two-told serial dilutions and applied it for the serial
dilution of crystal violet solution using DW as a diluent. We used 256
μM solution of the crystal violet dye as an input 1 X concentration. To
start the dilution process, 100 μL of 1 X crystal violet solution and
150 μL of DW (300 μL for two chambers) were loaded to the dilution and
diluent chambers, respectively. To fully accommodate the solutions
during the dilution procedure, the three types of chambers were
fabricated to be slightly larger than the volumes actually needed by the
solutions (Figure 3(a)).
As presented in Figure 3(b), a single diluent chamber is supposed to be
radially occupied by the three equivalent 50 μL volumes of the 150 μL DW
diluent. As explained earlier, the six outlet channels from the diluent
chambers were initially all closed, which are serially opened to release
each 50 μL of the diluent from the diluent chamber to the dilution
chamber. The dilution chamber was configured to elute half the volume
(50 μL) of the initial solution or the diluted solutions by opening and
closing the appropriate ferrowax microvalves within the connecting
channels, followed by rotating the disc, while retaining the rest half
the volume (50 μL) in the dilution chamber. After the elution of the
initial 1 X sample solution, the first two-fold dilution can be
performed by the consecutive process of the diluent addition, mixing,
and elution. By repeating these dilution procedures six times (Figure
3(c)), we completed the six consecutive serial dilutions for the crystal
violet solution. Actual images of the initial and final disc are shown
in Figure 4, and step-by-step images of this process are provided in
Figure S5. A video is also available in the supporting information
(Movie S1).
After the completion of the serial dilutions, we collected the diluted
crystal violet solutions (50 μL each) from the final seven product
chambers, which range from 1 X (256 μM) to 2-6 X (4
μM), and analyzed them by spectrophotometer. Based on the spectrum from
the samples, (Figure 4(c)), the absorbances at 588 nm were obtained and
plotted against the concentrations of the diluted samples (Figure 4(d),
red data) using the least square method. As a result, the curve showed
excellent linearity (R2 ≥ 0.9910), quite comparable
with that from the manual pipetting (Figure 4(d), black data), clearly
verifying the accurate dilution capability of this centrifugal
microfluidic system.