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.