Micro-fluidic Device for Antigen
Discovery
Khine Lwin
August 27, 2007
University of Maryland
Professor: Dr. Abraham Lee
Mentor: Armando Tovar
Table of Contents
Abstract ........................................................................................................................................... 2
Introduction ..................................................................................................................................... 3
Electric Double Layer ................................................................................................................. 4
Methods and Materials .................................................................................................................... 5
Photolithography ......................................................................................................................... 5
Polydimethylsiloxane (PDMS) Micro-channel........................................................................... 6
Protein Spotting .......................................................................................................................... 6
Fabrication of Entire Device ....................................................................................................... 7
Testing Procedures ...................................................................................................................... 7
Results ........................................................................................................................................... 10
Discussion ..................................................................................................................................... 13
Works Cited .................................................................................................................................. 14
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Abstract
When the body is infected by antigens, foreign molecules that trigger an immune response, the
body releases antibodies: proteins that attach to these antigens, tag them as foreign, and
neutralize their effects. Therefore, the presence of antigens and/or antibodies indicates a possible
disease contraction. A micro-fluidic device has been designed to trace antigen-antibody
interactions in order to detect diseases early in their developmental stages. This biosensor
consists of an array of titanium and gold-etched electrodes separated by a micro-channel that was
created using photolithography and polydimethylsiloxane (PDMS). Inside the channel, proteins
such as H3L, an antigen of the Vaccinia virus, are immobilized on the electrodes. Then, an
alternating current voltage is generated, flow is induced, and a liquid sample with primary and
secondary antibodies, which specifically bind to H3L proteins, is forced through the channel via
a syringe. Once the antibodies bind to the antigens, there is a change in the
impedance/admittance of flow in the channel, resulting in a voltage differential, which is
detected using a National Instrument data acquisition device, LabVIEW software, and/or
ExcelLink software. To further confirm the antigen-antibody binding, antibodies are
fluorescently tagged and the intensity of the fluorescence is used to verify that the antigens are
bound to the antibodies. Results show that the device is most sensitive around 3 to 5 kHz. More
importantly, the percent change in impedance once antibodies are pumped through the channel is
much greater when antigens are bound to the primary antibodies; suggesting that the device is
able to effectively detect antigen-antibody binding. This micro-fluidic device holds the potential
to save many lives by rapidly and efficiently identifying the presence of a disease in the human
body.
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Key Terms:
Immunoassay, Micro-fluidic device, H3L, Antigen-antibody binding
Introduction
Antigens are foreign molecules that trigger an immune response. As part of the immune
response, the body releases antibodies: proteins that attach to antigens, tag them as foreign, and
attempt to neutralize their effects. The antibody is a Y-shaped protein that is made up of four
polypeptides: two heavy and two light polypeptide chains (University of Arizona, 2000). These
light chains have a specific shape that allows it to only bind to certain antigens; therefore,
antigen-antibody binding is highly specific and the presence of certain antibodies indicates the
presence of specific antigens or diseases. One method that uses this concept to detect diseases is
called the enzyme-linked immunosorbent assay (ELISA). The ELISA technique involves plating
antibodies, adding serum which could potentially contain antigens, and using detectingantibodies as well as enzyme-linked antibodies to fluorescently verify bondage of antigens to
antibodies. Although this is a commonly-used method for detecting diseases, it requires a lot of
antibodies and time (approximately 24 hours). As of now, there are no quick, efficient ways to
detect antibody-antigen interactions. The objective of this project is to create a micro-fluidic
device for antigen discovery that will rapidly and efficiently detect diseases. The device consists
of a titanium and gold-etched electrode array which is separated by a PDMS micro-channel.
Proteins of the Vaccinia virus, H3L, are immobilized on the electrode tips that lie within the
PDMS channel. When a solution containing antibodies is pumped through the channel, the
antibodies bind to the antigens, causing impedance in flow to change. This change in impedance
is detected by the electrodes and the voltage differential created by the antigen-antibody binding
signifies the presence of a disease. One advantage of this device is that solutions such as urine
samples, serum concentrations, or liquid substances that pose a bioterrorist threat can be pumped
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through the micro-channel for rapid detection of harmful diseases. Also, the current design and
experimental procedure takes about 2 hours, requiring significantly less time than the ELISA
method. These advantages heighten the micro-fluidic device’s potential to quickly diagnose
diseases before they become lethal.
Electric Double Layer
As flow passes through the micro-channel, an electric double layer is formed. This layer is about
2 nm thick and consists of an immobile or Helmholtz layer and a diffusion layer (Huang, Greve,
Nguyen, & Domach, 2003). The antibodies and antigens bound to the electrodes form the
immobile layer, while the charged ions near the immobile layer make up the diffusion layer.
Both the immobile and diffusion layers collect electric charge near the surface of electrodes,
acting as capacitors and causing impedance in flow. The theory is that if antibody-containing
solution is pumped through the channel, the antibodies will bind to the antigens in the immobile
layer, causing impedance of the entire electric double layer to change, and this change will be
detected by the electrodes. However, the impedance in flow is also caused by the bulk solution
(Collins & Lee, 2004). Further away from the electrode tips, the bulk solution creates resistive
and capacitive impedance in flow. Since the device is supposed to only detect antigen-antibody
binding, the goal is to focus on the impedance changes caused only by the electric double layer.
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Figure 1: Equivalent Circuit of Channel
Methods and Materials
Photolithography
Several procedures are involved in the fabrication of micro-fluidic devices (Figure 3). First,
photolithography is used to etch micro-channels onto a microscope slide. Titanium (Ti) is etched
onto glass, and gold metal is deposited onto an adhesion layer of Ti. A layer of Shipley 1827
photoresist is then coated onto the gold metal layer. A thin-layered plastic mask with the
electrode array pattern is placed on top of the photoresist. The current electrode array design
consists of 1000 µm x 500 µm electrodes that have a 10 µm gap between them. To develop the
photoresist, the device is placed under ultra-violet light, resulting in the dissolution of photoresist
in areas not included in the pattern design. The remaining photoresist serves as a mask for the
gold and titanium etching process. Commercial etchants are used to etch the metal layers. GE8148, which etches at a rate of 50Å per second, is used to etch gold. TFT-Ti is used to etch
titanium at a rate of 25Å second. After the etching, the slide is chemically treated to help
immobilize proteins, which are spotted later in the process, and to help seal the PDMS onto the
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device. Lastly, the device is rinsed with acetone and de-ionized water to remove any remains of
the photoresist.
Polydimethylsiloxane (PDMS) Micro-channel
For a liquid sample to pass through the device, a 200 µm channel is fabricated into a polymer,
polydimethylsiloxane (PDMS). This PDMS channel completes the circuit as it is placed in the
gap between the electrode tips. To create a channel in the polymer, curing agent and aqueous
PDMS are mixed in an arbitrary container in a one-to-ten weight ratio. This container is then
placed into a vacuum for about 45 minutes. Later, the mixture is poured into a Petri dish with a
wafer containing the channel pattern. The Petri dish must be baked in a 65-71 °C oven for 3-4
hours or until the PDMS hardens.
Protein Spotting
The proteins are immobilized onto the electrode tips by using the
BioForce NanoEnabler. This machine consists of a surface
patterning tip (SPT), which places droplets of protein onto the
electrode. About 5 µL of H3L proteins are mixed with a buffer
solution and deposited into the reservoir of the surface patterning
tip. Using a program called, Nanoware and a laser, the SPT is
dragged along the surface of the electrodes, leaving minute
Figure 2: BioForce Nano eNabler
amounts of H3L proteins. For testing procedures, only one or
two electrodes are spotted with proteins while the non-spotted electrodes are used as control in
the experiments.
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Fabrication of Entire Device
The last bit of the device fabrication involves putting all the parts together. After the proteins are
spotted onto the electrodes, the glass slide is placed inside a plasma cleaning machine for two
minutes. Then, there is a period of about three to five minutes to seal the entire device with
PDMS. The channel in the PDMS is carefully aligned with the gap between the electrodes and
the PDMS is used to seal the micro-channel. These devices must be stored in the refrigerator to
protect the proteins from denaturing. At this point, the fabrication process is complete.
Figure 3: Device Fabrication Process
Testing Procedures
First, solution is passed through the micro-channel using a syringe and a PicoPlus pump. A
function generator is used to send an alternating current to the device that is connected to a 1
kilo-ohm resistor (Figure 4). The signal is amplified by a current amplifier and received by a
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digital multi-meter (DMM). The data is recorded by a National Instrument USB-6008 data
acquisition device (DAQ). Also, the device is placed in a Faradic cage to block out excess noise.
Figure 4: Flow Chart of System
The solutions that are pumped through the device are as follows:
Solutions
Flow Rate (µL/min)
Duration (min)
Data Collection
TTBS Washing Buffer
40
5 Washing
5 Measurements taken
Flow is turned on/off
for 50 seconds (repeat
once)
Protein Blocking
Buffer
10
12
TTBS Washing Buffer
40
5 Washing
5 Measurements taken
Primary Antibodies
Vaccinia Immune
Globulin (VIG)
5
20
40
5 Washing
5 Measurements taken
TTBS Washing Buffer
Secondary Antibodies
20
TTBS Washing Buffer
40
5 Washing
5 Measurements taken
De-ionized H20
--
Approximately 3
Flow is turned on/off
for 50 seconds (repeat
once)
Flow is turned on/off
for 50 seconds (repeat
once)
Flow is turned on/off
for 50 seconds (repeat
once)
Table 1. Protocol of Solutions Pumped through Micro-channel
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Air is pumped through the channel to rid the channel of any residue in between changing
solutions. Measurements are taken when washing buffer is flowing through the channel. Voltage
readings are taken using a digital multi-meter for 50 seconds when the flow is turned on, and also
when the flow has been turned off for 50 seconds. This process is repeated once for each
electrode being tested. After data is recorded for one electrode, the circuitry is changed to take
measurements on another. While the TTBS is pumped through, one set of measurements is taken
with the electrodes that have been spotted with proteins; then the circuit is switched to include
the electrodes that do not contain the proteins and another set of measurements is taken before
the solution is changed. In theory, there should be a greater voltage differential when flow is
turned on and off when antigen-antibody bonding has occurred. To further investigate antigenantibody binding, Cy3 dye is used to detect fluorescence once antigens are bound to antibodies.
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Results
Figure 5: Results of Frequency Optimization
Figure 5 shows the results of the frequency optimization tests. Frequency was continually
increased and voltage across the micro-fluidic device was recorded accordingly. This data was
collected with a 40 µL/min flow and also without any flow across the micro-channel.
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Figure 6: Voltage Differential of Flow On/Off
Figure 6 is a graph of the voltage differential that exists as flow is turned on and off. The data
alternates between the two electrodes: one is spotted with proteins and the other does not contain
any H3L proteins. The first curve is the baseline before any antibodies are pumped through the
channel.
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Figure 7: Percent Change in Impedance of Protein-Spotted Proteins and Control
Figure 7 represents the percent change in impedance before and after the primary antibodies,
VIG, and the secondary antibodies were pumped through the channel.
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Figure 8: Fluorescently-tagged antibodies along micro-channel
Figure 8 shows the electrodes along the micro-channel that contain fluorescently-tagged
antibodies.
Discussion
The results of the frequency optimization test show that as frequency increases, the impedance of
the channel approaches a steady state. This trend is independent of whether the flow is turned on
or off. The plateau exists at high frequencies because at high frequencies the resistance of the
bulk solution is dominant over the impedance created by the electric double layer (Wu & Chang,
2004). At frequencies lower than 10 kHz, the electric double layer impedance dominates the
impedance across the channel. To increase the sensitivity of the device, impedance of the double
layer must be kept to a minimum. Therefore, the peak frequency at which the device is most
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sensitive and is still able to detect the impedance of the electric double layer instead of the bulk
solution’s impedance is around three to five kHz.
Percent change in impedance was calculated in comparison to the previous measurements taken.
Figure 7 shows that there is a greater change in impedance when antibodies are pumped through
a channel that has protein-spotted electrodes (E1) relative to the electrodes without H3L proteins
(E2). However, the percent change in impedance did not significantly differ once secondary
antibodies were added. Antigen-antibody binding was further verified by examining the device
under a fluorescent microscope. The areas of fluorescence signify that the antigens are in fact
bound to antibodies, which coincides with the data represented in Figure 7. Conclusively, when
antibodies flow through the channel and bind to antigens, there is a greater percent change in
impedance; indicating the presence of a disease, which was successfully detected by the microfluidic device.
Works Cited
Collins, J., & Lee, A. P. (2004). Microfluidic Flow Transducer based on the Measurement of
Electrical Admittance. Lab on a Chip , 16.
Huang, X., Greve, D., Nguyen, D., & Domach, M. (2003). Modeling of Impedance of CellCovered Electrodes. Sensors , 5.
University of Arizona. (2000, June 12). Antibody Structure. Retrieved August 12, 2007, from
Antibody Structure:
http://www.biology.arizona.edu/immunology/tutorials.antibody/structure.html
Wu, J., & Chang, H.-C. (2004). Micro-electrical Impedance Spectroscopy for Particle Detection.
Second International Conference on Micro-channels and Minichannels (pp. 865-868).
Rochester, NY: ASME.
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