Lab on a Chip
Team R
Joshua Epperson, Brooke Ott, Luisa Parish, and Benjamin Weisman
Engineering 1182.03 Section: 6388
April 21, 2015
Table of Contents
1.) List of Tables and Figures …………………………………………………………………………………………………………………
2.) Executive Summary……………………………………………………………………………………………………………………….
3.) Introduction ……………………………………………………………………………………………………………………………………
4.) Requirements, Constraints, and Information Needs………………………………………………………………………...
5.) 1st Chip Design………………………………….………………………………………………………………………………………………
6.) 2nd Chip Design…………………………………………………………………………………………………………………………………
7.) Circuit Analysis…………………………………………………………………………………………………………………………………
8.) Performance Analysis………………………………………………………………………………………………………………………
9.) Fabrication Issues……………………………………………………………………………………………………………………………
10.) Summary and Conclusion………………………………………………………………………………………………………………
11.) Appendix……………………………………………………………………………………………………………………………………….
List of Tables and Figures
1.) Figure 1: First Chip Design Drawing…………………………………………………………………………………………………
2.) Table 1: Calculations of Features of First Chip Design …………………………………………………………………….
3.) Figure 2: Second Chip Design Drawing ……………………………………………………………………………………………
4.) Figure 3: Circuit after DAD integration…………………..………………………………………………………………………...
5.) Figure 4: LED readout.…………………….………………………………………………………………………………………………
6.) Table 2: Data recorded from all Calibration and Testing labs ……………………………………………………………
Joshua Epperson, Luisa Parish, Brooke Ott, Ben Weisman
Group R – Instructor: Bruce Trott GTA: Mohini Dutt
Lab on a Chip
April 21, 2015
Executive Summary
The lab on a chip project consisted of constructing a small, portable, device that can be used to quickly
run tests on very small amounts of fluids. Lab on a chip has many practical applications such as detecting
varying fluorescein concentrations in tears to identify dry eye syndrome. Two chips were designed over
the course of the project. First, a generic chip was given to the group to run preliminary experiments on
and to familiarize the group with how the chip should function. Using the data and observations from
the generic chip, a new chip was designed and fabricated. Furthermore a circuit was configured. The
circuit used a Detection Alignment Device (DAD) along with the chip (filled properly with fluorescein) to
identify the concentration of fluorescein samples. This concentration was displayed as a binary number
by LEDs constructed on the circuit. After many tests on the first chip, a second chip was designed,
improving on many shortcomings of the first chip.
The final design of the first chip consisted of a circular base with a diameter of 5.08 cm. This design
contained four staging wells used to introduce fluid into the chip. Two of the staging wells were circular
and two elliptical. Each staging well was connected to a channel containing a check valve to prevent
fluid from flowing back into the staging well. Two of the channels contained two check valves, one close
to the staging well and one close to the well centered at the middle of the chip known as the detection
well. The detection well was an elliptical well where the fluorescein was held while being analyzed by
the DAD sensor. Lastly, a large elliptical waste well was placed at the bottom of the chip to catch any
excess fluorescein. The second chip designed was much simpler than the first. The second design only
consisted of two circular staging wells. Each staging well had one check valve in the corresponding
channel running to an elliptical detection well. This detection well was again attached to an elliptical
waste well.
The second chip design was used in the final test chip. The chip performed excellently. Three unknown
samples were tested. Sample A was the first sample tested. The sample was tested a total of three times
producing concentration readings of 328 ppm, 357 ppm, and 357 ppm respectively for each trial with an
average of 347 ppm. Overall, the chip produced only a 2% average deviation from the actual
concentration. Sample B was tested next. The chip produced concentration readings of 750 ppm for all
three trials. These readings, although precise, were not accurate, producing a 21% deviation. Lastly a
bonus sample was tested labeled sample X. All four trials for this test read 128 ppm, producing only a 9%
deviation.
Although the chip performed well, there was room for improvement. One possible way to improve the
chip would be to try a different check valve design; all check valves on both chips were circular. A
different design such as the fishbone structure could have been experimented with to obtain more
accurate readings. Another way to improve the chip would be to build a better circuit. The circuit used
did not always produce consistent readings. The LEDs would occasionally fluctuate between two
separate readings. Using new LEDs, a different DAD, or simply constructing a new circuit may have
remedied the LED fluctuation issue.
Introduction
The concentration of fluorescein detected when tears are collected from an eye after inserting
fluorescein can be used to detect whether a person has dry eye syndrome. The team set out to design a
Nano-chip that would detect the concentration of fluorescein in a patient’s eye to determine, if enough
tears were produced in the eye. Work in this field could lead to faster diagnoses for patients with dry
eye. Similar devices may be applied to other medical procedures in the future to identify conditions
such as HIV, diabetes, or even cancer. The future of nanotechnology has potential and could greatly
increase the efficiency and speed of medical testing along with many other applications.
Section 1 discusses the constraints, requirements and information needs after being given the product
request. Section 2 takes a look at the concept and performance of the first chip design. The second chip
design’s performance and design concepts were discussed in section 3. Section 4 of this report analyzes
the circuit used for detection and concentration identification. Section 5 analyzes the chips performance
during experimentation. Section 6 discusses issues in fabricating Nano-chip technology. Finally, section
7 summarizes the Nano-chip project as a whole.
Requirements, Constraints, and Information Needs
To create a successful chip many requirements had to be met. Most importantly, the chip had to be able
to pass fluorescein samples of varying concentrations to a detection well for testing. This requirement
was constrained by the size of the chip and its features. The chip itself had to fit within a 5.08 cm circle,
all features on the chip were to have a depth of 200 microns, channels had to be 300 microns to 400
microns in width, and the detection had to be designed to hold 3 microliters of fluid. Information
needed from these constrains include; what shape of features allow for most accurate readings, for
example circular vs. elliptical staging/detection wells. Information on channel shape and width was also
needed; for example, would straight channels or channels that have curves or sharp edges in allow for
more efficient sample transportation.
Secondly, the chip was required to be able to be cleaned quickly and effectively in between trials. This
requirement was constrained by the chip not being able to be removed from the chip holder for
cleaning between tests. Information needs that arouse from these constraints included; how to
effectively flush the chip of all sample from a previous test; what types of fluids should be passed
through the chip for proper cleaning as well as how to know that the chip was completely clean.
Next, the chip was required to be designed to fit in a specific chip holder. This requirement was
constrained by the type of chip holder; only three hole and four hole chip holders were available.
Information had to be gathered on the location of the holes in each chip holder to properly place staging
wells and waste wells.
Thirdly, the chip was required to be able to communicate the concentration of sample detected to the
operator. This requirement was constrained by the materials available; subsequently a circuit was
designed to give a binary reading corresponding to the concentration of sample tested. Information had
to be gathered on which binary readouts correspond to known concentrations of fluorescein. These
reading were used to create a calibration curve and equation for the chip.
Lastly, aside from being able to be cleaned between trials, the chip was required to be thoroughly
cleaned before chip testing began, and after all testing was completed. This requirement was
constrained by the fluids and equipment available for cleaning. Information was needed on how to use
an ultrasonic cleaner as well as which available fluids were best fit for cleaning.
1st Chip Design
The first chip was designed by the team to test the concentration of fluorescein on a micro level. The
chip was required to allow the flow of liquids into the detection well in order to correctly determine the
concentration of an unknown fluorescein sample. The team worked on an initial chip design that would
appropriately fit the constraints and requirements of the assignment and ideally perform the task of
properly identifying the concentration of fluorescein.
The first chip design consisted of a chip with four channels designed with slightly different features.
Figure 1 (below) displays the first chip design. As seen in figure 1, staging wells 1 and 4 were circular
while staging well 2 and 3 were elliptical. The variance of geometry was done in order to determine
which staging well shape was most effective in consistently filling the detection well. Another chip
component experimented with was location and number of capillary check valves. In staging wells 3 and
4, check valves were placed directly below the staging well; in staging wells 1 and 2 check valves were
placed below the staging wells as well as before the detection well. This variance in number and location
of check valves was attempted in order to see if the different concepts had any effect on decreasing the
backflow of fluid to the staging wells. At the time backflow appeared to be a major concern however, it
was later discovered that the amount of back flow did not greatly impact results.
3
4
2
1
Figure 1: First Chip Design Drawing
The team discovered that the staging well was not extruded properly, consequently changes were made
regarding the size of the staging well. Originally, the staging well was bigger than the constraints
allowed. This issue was corrected by completely redesigning and refining the chip using the same basic
design structure. Table 1 on the following page shows the calculations for the first chip design (Sample
calculations can be referenced in the Appendix).
Table 1: Calculations of Features of First Chip Design
Staging Wells
Volume of Circle Staging Wells
Volume= 4.42 µL
Volume of Ellipse Staging Wells
Volume= 4.34 µL
Capillary Check Valves
Check Valve Closest to Staging Well 1
Volume=0.198 µL
Check Valve Farthest from Staging Well 1
Volume= 0.298 µL
Check Valve Closest to Staging Well 2
Volume=0.309 µL
Check Valve Farthest From Staging Well 2
Volume=0.26 µL
Check Valve From Staging Well 3
Volume= 0.33 µL
Check Valve From Staging Well 4
Volume= 0.45 µL
Detection Well
Detection Well
Volume= 3.14 µL
Waste Well
Waste Well
Volume=44.6 µL
Channel Flow Length (including Capillary Check Valve
Channel Connecting Staging Well 1
Distance = 11314 µm
Channel Connecting Staging Well 2
Distance= 14675 µm
Channel Connecting Staging Well 3
Distance= 14435 µm
Channel Connecting Staging Well 4
Distance= 11440 µm
Many features from the first chip were analyzed and changed for future designs. To minimize air
bubbles, the number of staging wells was reduced. It was decided that capillary check valves
immediately above the detection well were not necessary and were removed. Lastly, the channels were
connected before they released into the detection well to minimize air bubbles.
The second chip’s concept was to be simple and effective. This goal was accomplished by including only
two circular staging wells. Circular wells allowed for a higher volume of fluorescein to enter the staging
well within the same amount of space. Two staging wells gave the team fewer places to insert fluid but,
fewer places for fluid backflow and air to enter into the staging wells. The capillary check valves were
reduced to one on each channel in order to allow smoother fluid flow into the detection well. All of
these changes were implemented in order to simplify the design and to improve consistency and results.
2nd Chip Design
After testing the first chip and noting its shortcoming and successes the second chip was designed
and fabricated.
Figure 2: Second Chip Design Drawing
The second chip design (shown above in Figure 2) only had two staging wells compared to the first
chip design which had four. The second chip was 50900 µm in diameter. Both staging wells were
circular with volumes of 5.03 µL (Please refer to appendix A for sample calculations for finding the
volumes of the features).
The closest edge of the chip to a feature on the chip was 2700 µm which was the distance
between the staging wells and the edge of the chip. The channels are straight because testing on
previous chips showed that the straight channels worked the best to effectively transports liquid
to the detection well. Each channel has a check valve to prevent back flow immediately following
the staging well. The two channels meet right before the detection well to avoid air bubble
formation, an improvement from the first chip. An elliptical detection well was placed at the
center of the chip. An ellipse was used because it allows the most amount of light from the DAD
sensor to excite the liquid. The volume the detection well is 3.02 µL. From the bottom of the
ellipse a channel leads to a waste well which is the largest feature on the chip having a volume of
24.3 µL. The entire design was created in SolidWorks and the chip was fabricated using a milling
machine.
The second chip design differed slightly from the first chip design. The first chip had four staging wells,
two of which were circles and the others ellipses. In the first chip two of the channels had two check
valves which did not significantly decrease back flow and actually made pumping fluid to the detection
well more difficult. Therefore, the second check valve was removed from the channels in the second
design. The purpose of the check valve is to stop the back-flow into other staging wells. This back flow
stoppage occurs because the flow speed in the check valve is slower than in the channel. This difference
in flow rate is due to a larger width and volume in the channel at the location of the check valves. The
amount of time to get the same amount of liquid through the check valve is longer than that of the
channel because it is larger. Fishbone check valves were considered, however this idea was scrapped
because the milling machine could not cut the small sharp corners required for the fishbone design.
Circuit Analysis
At the beginning of the design process, the team designed a circuit that would be used for all
preliminary and final testing. The circuit was designed to determine the amount of fluorescein present in
a given sample. To test the samples the circuit was first connected to a power source. The electricity
from the outlet flowed through a 47 Ohm resistor and then into the DAD detector. There were two
cords from the DAD: one with three prongs, the other with two. The two-pronged wire powered the
light.
Figure 3: The circuit after the DAD integration
The prong farthest to the left, marked with a silver line (seen in figure 3 above), was connected to
ground, and the other prong powered the blue light of the DAD. The three-pronged cord also powered
the photodetector. The prong farthest to the left (marked with a silver mark) was connected to ground,
the middle prong sent electricity to the photodetector, and the prong to the farthest right (not marked
with a silver line) was used to return the analog signal to the binary voltmeter.
Figure 4: LED readout
The binary voltmeter changed the signal from analog to digital, and subsequently lit up 8 LEDs in a
pattern that corresponded to the analog signal (as seen in figure 4 above). Using the eight LEDs as a
binary reading the team was able to extract the decimal value which corresponded to the amount of
fluorescein in the sample.
One suggestion for an alternative circuit was based upon the reading of the chip. In a mass-production
use fluid would enter through a compartment of the device and be directed through channels on a chip
to a detection well. To more accurately simulate this form of detection, the chip holder and DAD should
be one unit. Many of the team’s errors arose from dirt, soap or air bubbles present in between either
the holder and the PDMS or the chip and the holder because the chips and the holder would be
separated for cleaning and storage purposes. To avoid these errors while still maintaining the integrity
of the experiments a permanent holder should be created to house both the DAD and the base. The
holder would be circular and have a slight ridge around half of the circumference of the chip so that the
chip could simply slide into place perfectly centered above the photodetector. The use of a permanent
chip holder would ensure that it does not dirty as long as the chips are thoroughly cleaned before and
after each experiment and the holder is cleaned to remove dust and other particulates accumulate on
the holder during storage.
While this circuit worked well for the team’s purpose, there are precautionary measures that could have
been taken to avoid discrepancies in the testing. One thing the team noticed was that the prongs on
both of the cords for the DAD were rusted from years of use. Rust hinders the movement of electrons,
which is necessary for accurate signal conversion. To avoid discrepancies the prongs should be cleaned
with sandpaper. This would allow unimpeded electron movement and more accurate readings.
Another adjustment that could be made would be to adjust the schematic used to create the circuit
board. In the schematic the wires that lead to the three- and two-pronged cords are separated by 1
wire space. This close range can lead to confusion as to where each cord is placed. To adjust for this
discrepancy the wires leading to their respective cords should be placed farther apart. The team’s
recommendation would be to place the three-pronged wires closer to the binary voltmeter to show a
better difference in functions of the cords.
Performance Analysis
This section gives an overview of the performance of each of the Team’s chip designs as well as the
effectiveness of the system in producing accurate results.
Fluorescin Concentration (ppm)
0
125
250
500
750
7A
12.00
37.00
52.00
74.00
85.00
7B
13.33
24.00
34.67
43.33
48.00
7C
14.00
35.33
52.33
74.00
84.67
7D
17.33
36.67
51.67
64.00
77.33
8A
10.00
38.33
50.67
66.67
79.67
7F
9.70
26.00
41.00
55.00
66.67
7J
12.00
35.00
63.67
87.00
93.67
7I
11.33
41.00
63.66
83.33
91.67
7G
9.00
40.33
55.33
75.33
87.00
7H
9.00
35.67
64.33
94.67
99.00
Table 2: data recorded from all of the calibration and testing labs. Labs 7A-7F used the first chip design, Labs 7J-7H utilized the
second design.
The results of all preliminary testing can be seen above in table 2. The system that the team used for the
duration of the labs remained unchanged from one lab to the next. However, the second chip design
led to values within a closer range than that of the first chip design—the standard deviation for the first
chip ranged from 2-14 while the standard deviation of the second chip ranged from 1.5-8. This narrower
range and lower average—4.4 for the second chip compared to 8.5 for the first—led the team to believe
that the second chip design would be better for the final testing since the accuracy of the chip was
better. The first chip’s design included 4 staging wells with each channel connecting directly to the
detection well. Often times during testing before the detection well was fully filled the fluid would backflow into the other channels. This often led to air bubbles in the detection well. To counteract this
problem, the second chip design had 2 staging wells and a connection of the channels prior to reaching
the detection well. Since there were fewer channels and the channels were more parallel the fluid flow
back-flow did not occur as often and less air bubbles were observed in the detection well.
Discrepancies in readings and testing came from the cleanliness of the chip—slight soap residue, a small
bubble in the detection well, dust, etc.—back-flow of the liquid, or air bubbles in the detection well.
When identifying how to counteract the latter two of these difficulties the team decided to change the
number and orientation of the channels. The team decided that the majority of the air bubbles arose
from the back-flow of the liquid, thus less fluid entering the detection well to fill it completely. The
second chip design addressed these issues by creating straighter and more parallel channels. This led to
less backflow, and thus less air bubbles in the detection well. The second chip was much more accurate
in the readings. A fluorescein concentration of 750 ppm had the widest range of values, which can be
seen above in table 2. For the first chip design the values ranged from 48-85—a range of 37—
meanwhile the second chip design ranged from 87-99—a range of 12. While the values for the second
chip were generally higher the team reasoned that this was because of either a cleaner chip or more
accurate readings from the detection well. Evidently, the fewer staging wells and more parallel channels
created a positive outcome on the accuracy of the fluorescein readings.
Fabrication Issues
Nano-manufacturing has been of great interest in the past few years due to its potential medical and
consumer product applications. However, in order for nano-manufacturing to be success and prevalent
in the future the equipment and facilities needed to create nano-products needs to become less
expensive and more accessible. Companies and education institutions are investing in the R&D and
infrastructure for the production of nano-scale products. The nano-scale is where classical mechanics
meets quantum physics and it is in that gray area where properties of materials can change from one to
the other. An example of this is gold. Gold at the macro-scale is yellow in color, but if it is cut to be 1
nm in width the color at the nanoscopic level is red. This unpredictability in the nano-scale makes it
hard to predict how materials will react on such a small scale and if the materials will retain their
properties at a macro-scale. Because of this, nano-technology is of much interest to the scientific
community.
If this chip was to be scaled down to nano scale there would be problems with accuracy. To be
considered nano-scaled the chip would have to three times smaller. To test the fluorescein samples at
the nano-scale, a machine would have to be used instead of an operator using a syringe. This machine
would be the best option to get an accurate prediction of the fluorescein concentration. Also the sensor
that would test the chip would also have to be extremely small. Factors such as dust, dirt, and air
bubbles would affect the results even more than the micro scale chip that was created for this report.
As stated before in the “Circuit Analysis” section if there was a way to have the chip and chip holder one
piece this would eliminate the potential of air bubbles and dust in between layers of the chip and
holder. Mass production of the chip at the nano-scale would not be plausible because of its price and as
well as how easily factors such as dirt and dust would affect the results. As stated earlier nano-scale
products are very expensive to create because of the facilities needed to produce such a product.
The chips were made of acrylic wafer and the features were created with a milling machine which is a
top-down manufacturing method. Due to the nature of the milling of the process there were some
limitations on the design of the chip. For example, the features could not have any sharp corners
because the bit that was used for the milling was circular. The edges had to have at least a 200 micron
radius and a channel’s opening could not be smaller than 300 microns. Also slopes could not be used in
the channels because the milling machine could not smoothly create this. When the chip was put under
the light box the lines that the milling machine made could be seen. Although this did not seem to
affect the performance of the chip, those uneven edges could reflect light back into the sensor on the
DAD and skew the results. If the team wanted smaller channels or a sloping channel this would be a
problem using this particular milling machine. Other options would have to be explored like getting a
smaller bit to do the milling or creating and using a mold.
If the chip was to be mass produced, at the micro-scale, a milling machine would not be the most
efficient means of production. A more efficient way to create a large volume of chips would be to have
a mold created and to use some sort of melted material. This material would then be allowed to cool
and then removed from the mold. This process would not only be more efficient but it would allow
sharp corners to be created as well as slopes in the channels. Even if the cooling time took longer than
the milling machine multiple molds would allow a large number of chips to be created at one time. Also
the molds would be reusable.
Conclusion
In conclusion, the second chip design performed more consistently. This report contains a look into the
requirements, first chip design, first chip’s analysis, second chip design, a circuit analysis, a performance
analysis, and fabrication issues. These explain the reasoning behind the chip designs as well as the final
performance. In the future, the design process could be improved by understanding the results
required before beginning to design the part. As a whole, the project could be improved by further
analyzing how to effectively use this technology on a smaller scale to detect results for a patient. The
team learned about the design process and the steps required in order to complete a design. The team
members would like to remember to keep the information up to date at all times for future projects.
Also in the future, the team would work to improve communication about meeting times.
Appendix
Sample Calculations
First Chip
Staging Well Volumes:
1
1
Equation= 𝑉 = 4 𝐴𝐵ℎ𝜋
𝑉 = 4 (6350𝑢𝑚)(4358𝑢𝑚)(200𝑢𝑚)𝜋 = 4.34𝑢𝐿
(A1)
Equation= 𝑉 =
𝑉=
= 4.42𝑢𝐿
(A2)
𝑉 = (1201𝑢𝑚)(1050𝑢𝑚)(200𝑢𝑚)𝜋 = .198𝑢𝐿
(A3)
1
𝐴𝐵ℎ𝜋
4
1
(6350𝑢𝑚)(4427𝑢𝑚)(200𝑢𝑚)𝜋
4
Capillary Check Valve Volumes
This is the first check valve reached when liquid flows from the well.
1
4
Equation= 𝑉 = 𝐴𝐵ℎ𝜋
1
4
This is the second check valve reached
1
Equation= 𝑉 = 4 𝐴𝐵ℎ𝜋
Equation= 𝑉 =
Equation= 𝑉 =
Equation= 𝑉 =
Equation= 𝑉 =
1
𝐴𝐵ℎ𝜋
4
1
𝐴𝐵ℎ𝜋
4
1
𝐴𝐵ℎ𝜋
4
1
𝐴𝐵ℎ𝜋
4
1
𝑉 = 4 (1192𝑢𝑚)(1594𝑢𝑚)(200𝑢𝑚)𝜋 = .298𝑢𝐿
1
𝑉 = 4 (1446𝑢𝑚)(1362𝑢𝑚)(200𝑢𝑚)𝜋 = .309𝑢𝐿
1
𝑉 = 4 (998𝑢𝑚)(1639𝑢𝑚)(200𝑢𝑚)𝜋 = .26𝑢𝐿
1
𝑉 = (1885𝑢𝑚)(1127𝑢𝑚)(200𝑢𝑚)𝜋 = .33𝑢𝐿
4
1
𝑉 = 4 (1398𝑢𝑚)(2067𝑢𝑚)(200𝑢𝑚)𝜋 = .45𝑢𝐿
(A4)
(A5)
(A6)
(A7)
(A8)
Detection Well Volume
1
𝑉 = 4 (3661𝑢𝑚)(5322𝑢𝑚)(200𝑢𝑚)𝜋 = 3.14 𝑢𝐿
1
4
𝑉 = (14897𝑢𝑚)(19052𝑢𝑚)(200𝑢𝑚)𝜋 = 44.6 𝑢𝐿
Equation= 𝑉 = 4 𝐴𝐵ℎ𝜋
1
(A9)
Waste Well Volume
Equation = 𝑉 = 𝐴𝐵ℎ𝜋
1
4
(A10)
Channel flow lengths (including capillary check valve)
Distance= 11314um
Distance= 14675um
Distance= 14435um
Distance=11440um
Second Chip:
Staging Well Volume:
Staging wells 1 and 2 are identical circles
Equation= 𝑉 = 𝜋𝑟 2ℎ
𝑉 = 𝜋(2.83𝑚𝑚)2 (0.20𝑚𝑚) = 5.03𝑢𝐿
(A11)
Capillary Check Valve Volume:
Although the check valves were created using a circle they will be treated as elliptical cylinders because
they are not closed circles.
1
4
Equation= 𝑉 = 𝐴𝐵ℎ𝜋
1
4
𝑉 = (1𝑚𝑚)(1𝑚𝑚)(200𝑢𝑚)𝜋 = .198𝑢𝐿
(A12)
Detection Well Volume:
1
𝑉 = 4 (5156𝑢𝑚)(3728𝑢𝑚)(200𝑢𝑚)𝜋 = 3.02 𝑢𝐿
1
𝑉 = 4 (14186𝑢𝑚)(10914𝑢𝑚)(200𝑢𝑚)𝜋 = 24.3 𝑢𝐿
Equation= 𝑉 = 4 𝐴𝐵ℎ𝜋
1
(A13)
Waste Well Volume:
Equation = 𝑉 = 4 𝐴𝐵ℎ𝜋
1
(A14)