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.