Chapter 4: Combinatorial Cell Assay Device on Glass with Membrane-Based Cell
4.2 Experimental
4.3.1 Combinatorial Mixer Characterization
A fabricated Parylene chip and a packaged device with three different colors of food coloring solutions injected are shown in Figure 4-9. The chip was able to withstand releasing in warm (35oC) acetone without any delamination of the SU8 or Parylene layers.
In contrast to the previous chip built on silicon, this version is based on glass substrate and is more compatible with microscopy. Both SU8 and Parylene C are transparent and
will allow the experiments to be observed from either side of the chip. The tubes will no longer obstruct the microscope objectives as the objectives can be used on the different side from the tubes. Fluid delivery follows the same straightforward procedure as previously described, using syringes and syringe pumps.
Figure 4-9. (a) Fabricated Parylene chip after sacrificial photoresist dissolution. (b) Experiment setup to deliver food coloring solutions into the chip. (c) Packaged device with various food coloring solutions injected.
Figure 4-10 shows the serial dilution experiment with sulforhodamine 101 by measuring the fluorescence intensity of sulforhodamine 101 at various concentrations on- chip. The linear fit of that data is included and has a coefficient of determination, r2, value of 0.9995. The result shows that sulforhodamine 101 has fluorescence intensity
(a)
(b) (c)
that varies linearly with the concentration at the concentration range we will work in and is suitable for our experiment.
Figure 4-10. Serial dilution of sulforhodamine 101 on-chip.
Figure 4-11 shows the concentration of the three input compounds at each chamber. The combinatorial mixer was able to generate the correct combinations of the compound solutions. The experiment was done by injecting red fluorescent sulforhodamine 101 solution into one of the combinatorial inlet while injecting DI water into the other inlets. Fluorescence image of each chamber was taken and the fluorescence level was measured with ImageJ. The same procedure was repeated for the other two combinatorial mixer inlets. While it is possible to use three fluorescent compounds with different excitation/emission spectra and injecting one compound into one separate inlet simultaneously, we avoided such procedure to prevent possible fluorescence crossover among the three fluorescent molecules. The concentration of the compounds can deviate from the design value because of dimensional inaccuracy in processing. Also, the sacrificial photoresist was baked during fabrication and this resulted in the edge of channel to be rounded and deviated from the rectangular profile
that was used when calculating fluidic resistance. A more precise control during the fabrication process can result in the concentration profile better matching the design values. This characterization allows us to know the concentrations of the input compounds at each chamber. Such information will assist us in deciding the input treatment compound concentration and let us know the treatment compound concentration at each chamber for the cell assay experiments. This method can be adopted for characterizing combinatorial mixer with higher inputs as choosing four or more fluorescent molecules with non-overlapping emission/excitation spectra can be difficult and add to the cost because four or more optical filters for the fluorophores have to be obtained.
Figure 4-11. Fluorescence characterization of the combinatorial mixer showing the concentration of the three input compounds at each chamber. Error bars are one standard deviation (SD). Number of experiments = 3.
The combinatorial mixing can be treated as matrix operation, shown as a linear equation, Ex = O. E represents the components of each input, x represents the
combinatorial function of the chip and O is the output components. For the current device with three-input combinatorial mixer (with one unmixed control), the matrix operation is
z z z
y y y
x x x
C B A
C B A
C B A
k k k k k
j j j j j
i i i i i
k k k
j j j
i i i
h g f e d
h g f e d
h g f e d
c b a
c b a
c b a
=
z z z z z
y y y y y
x x x x x
z z z
y y y
x x x
h g f e d
h g f e d
h g f e d c b a
c b a
c b a
. (4.7)
For the input matrix E, the components, A, B, and C represent the individual inputs and the component in each column represents the concentration of the individual input compounds in input A, input B, and input C. For example, if we inject solution I1 into input A, I2 into input B and I3 into input C, the input matrix E would be
E =
3 2 1
0 0
0 0
0 0
I I I
. (4.8)
The combinatorial mixer operator, x, governs how the inputs will be combined into the outputs. With the original combinatorial mixer design, the matrix operation is represented as
3 2 1
0 0
0 0
0 0
I I I
0 2 1
1 2 1 3 1
0 2 0
0 1 3 1
0 0 2 0
1 3 1
0 0 0
2 1 0 1
2 0 1 1
=
0 2 1
1 2 1 3 1
0 2 0
0 1 3 1
0 0 2 0
1 3 1
0 0 0
2 1 0 1
2 0 1 1
3 3 3 3
2 2
1 1
2 2 1 1
I I I I
I I
I I I I I I
. (4.9)
Each column in the output matrix, O, represents the compounds and concentrations in each chamber. Characterizing the combinatorial mixer allows us to know the values for the combinatorial mixer operation of the device and the equation becomes
3 2 1
0 0
0 0
0 0
I I I
0 1 64 . 0 49 . 0 25 . 0
0 0 32 . 0 0 26 . 0
0 0 0 54 . 0 5 . 0 0 0 0
1 5 . 0 0
0 55 . 0 1
=
0 1 64 . 0 49 . 0 25 . 0
0 0 32 . 0 0 26 . 0
0 0 0 54 . 0 5 . 0 0 0 0
1 5 . 0 0
0 55 . 0 1
3 3 3 3
2 2
1 1 2 2 1 1
I I I I
I I
I I I I
I
I . (4.10)
Knowing the values in the combinatorial mixer operator allows other output combinations to be designed using the same chip. As the values in the matrix x have been determined, one can choose the desired output and solved for E in the matrix operation, Ex = O.
For example, if the output
0 1 832 . 0 652 . 0 556 . 0
0 0 16 . 0 324 . 0 43 . 0
0 0 096 . 0 216 . 0 278 . 0 6 . 0 465 . 0 3 . 0
5 . 0 58 . 0 6 . 0
3 . 0 37 . 0 4 . 0
3 3 3
3
2 2
2
1 1
1
3 3 3
2 2
2
1 1
1
I I I
I
I I
I
I I
I I
I I
I I
I
I I
I
is
desired, one can solve for the necessary input matrix to generate such output. The input
would be
3 3 3
2 2
1 1
6 . 0 3 . 0
0 5 . 0 6 . 0
0 3 . 0 4 . 0
I I I
I I
I I
, and the whole matrix operation is
3 3 3
2 2
1 1
6 . 0 3 . 0
0 5 . 0 6 . 0
0 3 . 0 4 . 0
I I I
I I
I I
0 1 64 . 0 49 . 0 25 . 0
0 0 32 . 0 0 26 . 0
0 0 0 54 . 0 5 . 0 0 0 0
1 5 . 0 0
0 55 . 0 1
=
0 1 832 . 0 652 . 0 556 . 0
0 0 16 . 0 324 . 0 43 . 0
0 0 096 . 0 216 . 0 278 . 0 6 . 0 465 . 0 3 . 0
5 . 0 58 . 0 6 . 0
3 . 0 37 . 0 4 . 0
3 3 3
3
2 2
2
1 1
1
3 3 3
2 2
2
1 1
1
I I I
I
I I
I
I I
I I
I I
I I
I
I I
I
. (4.11)
It is noted that not all possible output can be generated because certain output combination would require inputs to have negative concentration. By characterizing the combinatorial mixer and transforming the combinatorial mixing into a matrix operation, we can expand the possible combinations of experiments that can be done using the current chip and also lay the foundation for easily designing and characterizing future devices with more inputs and outputs.
4.3.2 Device Packaging with Integrated Membrane and Microfluidic Simulation