Microfluidics Procedure
Engineering 1282H
Spring, 2015
Group Y1
Mahnoor Naqvi
Tony Satroplus
Spandan Shah
Matthew Viens
DMG
3:00 PM
Date of Submission: 03/27/15
1. Introduction
This purpose of this document was to provide the experimental procedure and rationale for
our microfluidics lab-on-a-chip experiment.
The first section details the Experimental Methodology, the second section details the
Hypotheses for the experiment, the third section provides the Schedule for assignments, and
the fourth section describes the Experimental Purpose and Analysis.
2. Experimental Purpose and Analysis
The key goal of each data point collection is to establish the threshold pressure value at
which the yeast that have been placed in the channel will shear off 50%. This establishes a
baseline for constant point for the dependent variable, cell shearing, so the relationship from
changing the pH of the incubation fluid. Each pH value recorded will have two trials of what
pressure achieves the critical value of shear before a different pH value is considered. The
analysis of these values are simply that a pressure value of the fluid has achieved above the
critical value of 50% shear as compared to a previous trial that had below that critical value
of shear percentage. These points can then be graphed and if applicable, see addendum to
the alternative hypothesis, a function therein determined.
3. Experimental Methodology
Pursuant to the cell adhesion and shear methods originally introduced in the Mercer-Bonin
Paper, the microfluidics variable that is being analyzed is the effect of pH variations of the
yeast incubating water on the pressure required to shear the yeast. The controlled
variables include using the same chip (barring some unforeseen circumstances, the same
channel), the pressures that are applied using the water column, length of incubation time,
the pH meter used to measure pH, and the types of yeast.
4. Hypotheses
Null Hypothesis: The pH of the incubating water solution, so long as the yeast itself is not
killed by the pH, does not affect the adhesion of cells.
Alternative Hypothesis: The pH of the incubating water solution, up to the bounds where the
pH kills the yeast, will have an effect on the adhesion of the cells. Using an alpha level of 0.05
in a one tailed t test, the team will find if the lower pH has a significant effect on the adhesion
of the yeast cells compared to tap water. A one-tailed t test will be used, since the team is
interested in if the adhesion decreases.
Addendum to the Alternative Hypothesis: If the alternative hypothesis is held as valid, then
a function should be able developed to model the relationship between incubating pH and
the pressure needed to achieve the 50% shear percentage.
5. Schedule and Outlook
5.1.
Schedule
3/27, 3/30, 4/1, 4/3, 4/6, 4/8, 4/10, 4/13, 4/15, 4/17, 4/20 are class days so these are
guaranteed work days.
Usually we meet on Tuesdays and Thursdays on open lab for a few hours so 3/31, 4/2, 4/6,
4/7, 4/9, 4/14, 4/16, and 4/21.
3/26 - Project Report Outline, Micro Experimental Procedure
3/27 - Chip Calibration
3/29 -Preliminary Design Nano, Project Report Outline
3/30 - Cell Adhesion Experiment (CAE)
3/31 - Draft 1, Part 1 Micro
4/1 - CAE
4/2 - Draft 1, Part 1 Micro
4/3 - CAE
4/5 - Draft 1 Nano
4/6 - CAE
4/7 - Draft 1 Nano
4/8 - CAE
4/9 - Oral Report Outlines
4/10 - CAE
4/12 - Draft 1 Part 2 - Micro
4/13 - CAE
4/14 - 3D Printing
4/15 - CAE
4/16 - Final Design
4/17 - CAE
4/19 - Oral Report, Final Submission 3D Printing
4/20 - Midterm 2 Review, Oral Report, Final Submission 3D Printing
4/21 - Oral Report, Final Submission 3D Printing
5.2.
Goals for Each Day and Week
Collecting data is estimated to take a maximum of 20 hours. The group is planning on
finishing the tap water trials on Sunday March 29th. The goal will be to finish the tap water
trials by Tuesday March 31st. The trials consisting of changing the pH will be done from April
1st to April 8th. A large portion of the data points with varying pH will be taken on Sunday
April 5th. Combining a large portion of data points per sitting will increase efficiency.
6. Experimental Notes
6.1.
Note on the Recording/Evaluation of pH Values
The pH variable that is being tested is specifically the water that the yeast is incubated in.
The water that is being used to perform the shearing experiments will have its pH recorded
but this will not be altered. The pH meter that is being used for the collection of data is a
commercially available pH meter with resolution of ± .05 in the pH readings from at least
accuracy region 2 – 12 on the pH scale. The pH meter that is being used for the trials will be
the same meter throughout and will not be used for any other purpose than the Y1
microfluidics experiments for spring 2015. The procedure for the initial calibration of the pH
meter will not be covered here as it comes directly from the instructions that came with the
meter at time of purchase and depend on which of the many pH meters commercially
available a group decides to use.
6.2.
Note on Changing pH
The pH variations will always be neutral or acidic, basic values will not be considered. To
achieve this the pH of the water used will be recorded and then acetic acid will be added with
the continued measurement of pH until the value that is being tested at that time is produced.
Water at this pH will then be siphoned off to provide the amount of water necessary to
incubate the yeast.
6.3.
Note on Channel Selection
The channel selection will depend on the measurement of the flow profiles of the chip. The
flow profiles will say if two channels are equivalent for the flow rate through them. An
analysis of the flow profiles equivalencies of the channels will be a separate document. At
this point all instructions will assume that the same channel will be used for all tests. It will
be referred to as the channel from here on out. If another channel needs to be used that will
be documented in the project notes and in the final report.
6.4.
Note on the Determination of Test Heights
Several data points will be taken at the unaltered pH of the available tap water. This data for
what heights of water for pressure is required to shear the critical value percentage of yeast
cells and then used to center the values used to evaluate those with altered pH.
6.5.
Note on Consistency and Placement of Measurements
For the preservation of consistency the general process of getting each data point will not
change with the pH being varied except for the preparation of the water-acetic acid solution,
see note on changing pH. Also the temperature of the incubating solution and the air
temperature will be recorded and every reasonable effort to have these values be constant
throughout will be made. With regards to the placement of measurements, 11 data points
will be collected total. 5 of these will be with the normal pH of the tap water to establish the
baseline. Three more pH values of the water will be tested with two data points at each value
recorded. The pH values will be 6.5, 6, 5.5.
6.6.
Low-Pressure Flushing
Flush with a column height of 14 cm. This produces 19571 dynes/cm of pressure in the
channel. This flush will be done for 2 spans of 2 minutes in order to flush and allow any cells
to seep back in and be flushed out once again.
6.7.
Significant Shearing
Significant shearing is determined as 30% or less of the original number of yeast cells still
adhered to the channel. The significant shearing will determine at which height the data
point is concluded.
6.8.
Heights as Constants
The heights we will be using will be 20 cm for the flush and 25.4 cm, 30 cm, and 40 cm for
the data points. The time for cup and channel incubation will be 20 minutes and 5 minutes
for the flush and two minutes at the different heights. The volumes will be dependent on the
heights and times used. The heights will stay at consistent intervals throughout the
experiment. The height should not be a novel variable to team is looking to test.
7. Timing of Procedure
7.1.

Time to Get Each Data Point
TOTAL TIME: ~ 2 hrs
o 10 min to set up
o 5 min to get yeast
o 5 min to stir
o 20 min for cup incubation
o 20 min for channel incubation
o 5 min to make sure everything to set up tubes
o 5 min low pressure flush
o 5 min to check under microscope
o 15 min at height 1

2 min for flush

5 min under microscope

8 min for delay
o 15 min at height 2
o 15 min at height 3
7.2.
Yeast Incubation

2.0 g of yeast cells

10 mL of 150 mM NaCl solution at approx 90 ºF

Stir in cup for 5 min

Incubation in cup for 20 min

Adhesion time for 20 min
8. Step-by-Step Procedure
1. LOC Set-up
o Assemble chip by placing chip between chip holder top and bottom and
tightening the screws to 14 IN-OZ of torque using a torque wrench.

Make sure the nuts are facing up and the head of the screws are on the
bottom of the LOC in order to ensure the chip is level when performing
experiments.
o Insert nozzle into entrance port and run water through the channel.

Make sure no water is leaking through the channels or spaces
surrounding the chip.
o Flip chip upside down to rid of excess water in the chip.

Yeast Incubation
o Add 2.0 grams of yeast to a clean, dry beaker. Record exactly how much yeast
was added.
o Add about 10 mL of 150 mM NaCl solution at 90˚ F to the beaker. Record exact
amount of solution added.
o Stir mixture of yeast and solution until the yeast is dissolved.
o Leave the yeast, undisturbed, for 20 minutes.
o Stir yeast again briefly to suspend the yeast in water. Siphon out 2 mL of yeast
solution making sure to flick the syringe to rid of bubbles.
o Insert the tip of the exit tube into the exit port to keep yeast from coming out
of this hole.
o Insert tip of syringe into entrance port and push a small amount through the
channel until the opposite basin is full without coming through the other side
of the tip. This should be done very slowly.
o Leave the yeast, undisturbed for 20 minutes.

Low Pressure Flush
o Dab both ports of the channel with Kim wipes to rid of any excess liquid.
o Weight empty cup used to collect exit water.
o Insert long tube connected to column syringe into exit port of channel. Insert
exit tube tip into entrance port of channel.
o Raise the column height of the syringe to 25 cm.
o Put the end of the drain tube at 8 cm above the table.
o Release the clamp on the column Tygon tube for 2 minutes. Let the chip sit for
1 minute. Repeat the flush for 2 minutes.
o Observe the observation section in the channel under the microscope,
allowing the fluid to settle for 2 minutes.
o Reweigh the cup and calculate the flow rate from the amount of fluid per unit
of time from that channel.

Shearing Yeast
o Set the height of the syringe to 30 cm.
o Weigh the cup before the yeast shearing flush is conducted.
o Mark the section on the channel where the microscope observations will be
made.
o Make an initial observation, and picture, of the observation region of the
channel.
o Run the flush of the channel with the column height port in the entrance port
of the channel and the exit port tip in the exit port of the channel. Run the flush
for 2 minutes.
o Let the channel sit for 2 minutes. Observe the cells in the observation region
and record by taking a picture.
o Weigh the cup.
o Refill syringe to 20 mL mark.
o Repeat for column heights of 35 and 40 cm, or until 70% of the yeast looks to
be sheared.

Cleanup and Iteration
o Flush the channels with a syringe of tap water, until no yeast is visible under
the microscope.
o Disassemble the chip and make sure chip parts are protected in a petri dish.
References
[1]
Lab3 – Chip Fabrication and Yeast Cell Adhesion. 2015, March 26th.
www.carmen.osu.edu.