3.3. Six consecutive serial ten-fold dilutions for bacterial sample
To further verify the versatile capability of the system for the dilution in a wide dynamic range, we next fabricated the centrifugal microfluidic disc capable of accomplishing six consecutive ten-fold serial dilutions and applied it for the dilutions of the biological sample containing Escherichia coli O157:H7. As presented in Figure 5(a), the overall structural elements were all the same with the two-fold dilution system, and the same top layer used for two-fold dilution was used for ten-fold dilution. The principal design of the diluent chamber in the bottom layer was also almost same with that for two-fold dilution, and the each diluent chamber was designed to be radially occupied by three one third the volumes of the diluent (Figure 5(b)). However, the volumetric structures of the chambers in the bottom layer were modified to suit ten-fold dilutions. More specifically, the serially added diluent volume was changed to 90 µL from 50 µL in two-fold dilution, and the volume of the diluent chamber was accordingly larger to accommodate the larger volume of the diluent. The volumetric design of the dilution chamber was also quite modified, such that 90 % of the diluted samples (90 µL) were eluted to the product chambers while leaving only the rest 10 % of the samples (10 µL).
In this manner, the dilution ratio could be manipulated simply by changing the serially added diluent volume from the diluent chamber and the remaining analyte volume in the dilution chamber. To start the ten-fold dilution process, 100 µL of the target bacterial sample and 270 µL of Tris-HCl buffer diluent (540 μL for two chambers) were loaded into the respective dilution and diluent chambers. Again, to conveniently 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. Escherichia coli O157:H7 sample of 2.0 × 108 CFU/mL was set as a 1 X sample, and it was ten-fold serially diluted down to 2.0 × 102 CFU/mL following the same procedures for two-fold dilutions (Figure 5(c)). Actual images of the initial and final disc are shown in Figure 6, and step-by-step images of this process are provided in Figure S6.
After the completion of the six consecutive serial dilutions, the diluted bacterial samples were collected from the final seven product chambers, and mixed with iTPA reagent solution to prepare the final seven reaction solutions ranging from 1.0 × 102 to 1.0 × 108 CFU/mL. To validate the accuracy of the automated serial dilutions in such a wide dynamic range, we performed iTPA reactions for the seven samples and obtained the real-time fluorescence curves from the amplified bacterial DNAs (Figure 6(c)). Based on the curves, we determined threshold time (Tt), defined as the reaction time at which the fluorescence signal exceeds the threshold line (fluorescence intensity = 120) for the seven target concentrations, which was then plotted against the log of target DNA concentration (Figure 6(d), red data) using the least square method. As a result, the curve showed excellent linearity (R2 ≥ 0.9818) in a range from 1.0 × 102 to 1.0 × 107 CFU/mL, confirming the accurate dilution capability of the centrifugal microfluidic system, which is almost the same with that (R2 ≥ 0.9815) of the conventional manual pipetting (Figure 6(d), black data). Unfortunately, the Tt value from the sample of 1.0 × 108CFU/mL deviated from the line. We assume that the lysed cell debris and other inhibiting components from the bacterial sample at such a high concentration of 1.0 × 108 CFU/mL might have impeded the iTPA reaction because we conducted the iTPA reaction directly using the lysed samples without any prior purification of nucleic acids. However, this deviation of the initial sample is just due to the limitation of the dynamic range of the iTPA technique but not associated with dilution process. All these results confirm that the developed centrifugal microfluidic system is quite capable of accurately diluting the samples in a very wide dynamic range, up to six orders of magnitude.