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Many mentors participate each year, but additional mentors become available each year. Projects from
previous years are briefly described below.
Program Year 2011
Stratton Haywood: (Mentor – Niel Crews)
Title: Performance of Two-Inlet PCR on a Chip
Introduction: Polymerase Chain Reaction (PCR) is an important method for DNA analysis. In
this method, a template DNA pattern is combined with the agents required for DNA synthesis.
Nucleotides are assembled onto the DNA pattern, after which the resulting double-stranded DNA
is heated to divide it into two single strands. Assembly begins anew on the single strands, and
the process is repeated. The number of strands doubles with each heating cycle. In the present
study, we use a microchannel system that includes two inlet ports, one for the synthesis
compounds and one for the template. The two-port system allows templates to be rapidly
changed, allowing rapid analysis of multiple genes.
Methods: The two-port system is shown in Figure 1. The inlets are on the left. The temperature
along the upper loops is high, allowing for DNA melting, and along the lower loops it is low to
promote synthesis. The template is injected to one inlet. The other inlet contains a mixture of
20% water, 10% buffer, 10% LCGreen, 10% left primers, 10% right primers, 10% DNTPs, 10%
Taq Polymerase, and 10% Tween. As the number of strands increases, the fluorescent light from
the LCGreen increases.
Figure 1: Two-port PCR system. The inlets are on the left, and the fluid temperature changes as
it passes along each loop.
Results: Figure 2 shows results on a graphical user interface that was designed for the system.
The line plot on the left side is the derivative of the image intensity along the green dashed line
in the image on the right. The peak of the line graph indicates a large gradient that corresponds
to a melt. The bright fluorescent signal at the bottom of the image indicates that DNA is being
synthesized within the system.
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Figure 2: Graphical user interface for the system. The peak of the line graph indicates the
location of a melt.
Conclusions: These experiments have demonstrated the feasibility of the two-port system for
PCR analysis.
Arnold Hwang and Daniel Verona: (Mentor – Steven A. Jones)
Title: The Combined Effects of Variations in L-Arginine, ADP and Shear Rate on Platelet
Adhesion
Introduction: Platelet activation and adhesion involves both positive and negative feedback
effects. ADP, a platelet activator, is important in the positive feedback mechanism, whereas
nitric oxide, a platelet inhibitor, is one of the negative feedback agents. The interaction between
the positive and negative feedback mechanisms is not well understood. An additional parameter
of interest is the shear rate of the flow, which alters both the rate of delivery of platelets to the
wall and the force tending to remove adhered platelets. A simple system for investigations into
platelet adhesion consists of multiple microchannels that are coated by a given adhesion protein
through layer-by-layer self assembly. This study was undertaken to determine whether Larginine, a precursor for nitric oxide, decreases platelet adhesion in this system, whether ADP
increases platelet adhesion, and whether shear rate affects the amount of adhesion. A recentlyreported method for dynamically layering fibrinogen and collagen on the channel surface was
used. Platelet-rich plasma was infused through the channels at controlled shear rates and with
different concentrations of L-arginine and ADP.
Methods: Fibrinogen was coated onto the microchannels with a dynamic layer by layer self
assembly method, in which the polyion and protein solutions were continually agitated during
the submersion of the surface. Whole blood was collected from bovines into enough sodium
citrate to create a 9:1 blood:citrate ratio. The blood was then centrifuged at 320 rcf for 35
minutes, and the plasma layer was carefully extracted. A tris buffer solution is added to the
plasma to bring the fluid level back to its original volume prior to the centrifuging. A syringe
pump was then used to inject the platelet-rich plasma through the microchannels with a know
flow rate. Shear rate at the bottom surface of the channel was estimated from the equation
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𝜏𝑤 =
3𝑄
,
𝑤ℎ2
Where 𝑄 is the flow rate of plasma, 𝑤 is the channel width, and ℎ is the channel height.
After injection, the channels were stained with acridine orange and imaged under a fluorescent
microscope. The images were analyzed with MATLAB program to give us the percent surface
area coated by platelets. These experiments were performed with different concentrations of Larginine (to inhibit platelet adhesion, i.e. negative feedback) and ATP (to stimulate platelet
adhesion, i.e. positive feedback).
Results: Figure 1 shows the percent surface coverage as a function of added L-arginine and
shear rate. The percent surface coverage decreased monotonically with the added L-arginine
concentration and increased with shear rate. For the lowest shear rate, a minimum was reached
at 15 L added L-arginine, which may represent the baseline noise level for surface coverage
detection.
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Surface Coverage (%)
3
Shear Rate 500/s
Shear Rate 1000/s
Shear Rate 1500/s
2
1
0
0
5
10
15
20
Added L-Arginine (L)
Figure 1: Combined effect of L-arginine and shear rate on platelet adhesion in the microchannel system.
Figure 2 shows the effect of ADP on adhesion. As ADP concentration increased, the amount of
adhesion increased. Preliminary experiments with ADP and L-arginine combined generally
demonstrated a proportional response, but more data need to be collected to obtain statistically
significant conclusions.
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Surface Coverage (%)
4
3
2
1
0
0
5
10
15
20
Added ADP (L)
Figure 2: Effect of ADP on adhesion in the microchannel system.
Conclusion: Increased concentrations of L-arginine and increased shear rates generally inhibited
platelet aggregation. ADP generally increased platelet adhesion. These results suggest that the
system provides a valid method for the study of platelet adhesion.
Kristopher Janiec: (Mentor – Long Que)
Title: Carbon Nanotube Film Optimization for CNT Film-Based Cantilevers Used for Light and
Thermal Energy Harvesting
Introduction: A piezoelectric cantilever with a carbon nanotube coating has been shown to
generate voltage and current in response to light and heat. The efficiency of the cantilever is
likely to depend on the processing methods involved in its construction. Therefore, experiments
were performed to examine the effects of various concentrations of
Methods: The cantilever geometry is shown in Figure 1. The piezoelectric material is coated on
both sides with nickel electrodes, and the voltage is measured across these electrodes. The
carbon nanotube coating is placed over the top electrode, and is exposed to light and/or heat to
generate electrical energy.
Figure 1: Geometry of the piezoelectric/carbon nanotube cantilever. (Not to scale)
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Two methods were attempted to generate the carbon nanotube sheet. In the first, long and short
single-walled carbon nanotubes were sonicated for 2 hours in a sonication medium of either IPA
or chloroform. The solution was then filtered, with the carbon nanotubes remaining on the filter
paper. The filter paper was then dissolved away. Because this method yielded inconsistent
results, a new method was devised in which the carbon nanotubes were transferred to doublesided tape, and then transferred from the tape directly to the nickel coating on the piezoelectric
material. This new process has the additional advantage of being acetone free. The nickel
electrodes were exposed to 95% purity sulfuric acid diluted in 35 mL of de-ionized, filtered
water, over which the carbon nanotube layer was placed.
Results: Figure 2 shows the voltage and current obtained with different sonicating conditions.
The highest voltage and current were obtained when 20 mg of long carbon nanotubes were
sonicated in chloroform without acid.
6.E-06
Chloroform-Acid-30mg(L-SWNT)
8.E-04
Chloroform-NOAcid-30mg(L-SWNT)
IPA-Acid-30mg(L-SWNT)10mg(S-SWNT)-Noacetone
5.E-06
IPA-NOAcid-30mg(L-SWNT)10mg(S-SWNT)-Noacetone
IPA-Noacid-30mg-noacetone
Chloro-Noacid-30mg-noacetone
4.E-06
Current (amps)
Voltage (Volts)
6.E-04
4.E-04
3.E-06
2.E-06
2.E-04
1.E-06
0.E+00
0.E+00
0
100
200
300
400
0
100
Time (sec)
200
300
400
Time (sec)
Figure 2: Voltage (left) and current (right) measured from the cantilever.
Conclusions: Sonication conditions affected the efficiency of the cantilever system. The
modified tape method was able to provide equivalent efficiencies to the best sonication method.
In addition, the combination of light and heat applied to the cantilever increased the amount of
electrical energy generated.
John Nguyen: (Mentor – C. O’Neal)
Title: Micromechanical Cleavage of Graphene using Photolithography methods
Introduction: Graphene is an allotrope of Carbon in which one carbon molecule is covalently
bound to 3 others by sp2 bonds. Each carbon in graphene is covalently bonded to 3 other
carbons, whereas Van der Waals forces contribute to the last carbon bond. The Van der Waals
forces provide a surface along which graphene can be cleaved. Graphene is unique as the
strongest material that has been tested, and it has high electron mobility in that it can conduct
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electricity faster than silicon at room Temperature. Proposed applications for graphene include
transistors and ultracapacitors. To develop these applications, it will be necessary to find a
simple method for cleavage of graphene. Novoselov et al. etched highly-oriented pyrolytic
graphite, with an oxygen plasma to produce patterned “mesas.” The mesas were stripped from
the graphene layers with a photoresist, and the photoresist, which had the graphene flakes stuck
to it, was dissolved, allowing the graphene to be transplanted onto a substrate [1].
Photolithography is an option for graphene production. Kumar et al. used chemical vapor
deposition to grow graphene onto a substrate and then used lithography to pattern it. A Ni
hardmask protected the patterned graphene, and a photoresist removed unwanted material [2].
This project applies a method similar to that of Nososelov et al. to obtain thin-layered graphene
samples.
Methods: Two samples were commercially purchased (from www.2spi.com). Stainless steel
shadow masks of dimensions 14 mm x 14 mm x .0254 mm were designed in AutoCAD. The
samples were etched in a reactive ion etcher, and surface roughness was measured with a
profilometer. The etching conditions are shown in Table 1. A CEE Model 100 spinner was used
to lay photoresist on a glass substrate. The etched sample was then applied to the photoresist and
the combination was soft-baked. The upper-layers of graphene were then cleaved from
photoresist, and the bottom layer was removed by placing it in contact with glass. Residual
acetone was evaporated with a hot plate.
Table 1: Etching conditions
Condition
Pressure (mTorr)
Power (Watt)
Time (min)
Trial 1
5
200
5
Trial 2
100
200
8
Trial 3
50
350
10
Results: Sample graphene flakes are shown in Figure 1.
Figure 1: Graphene flake samples
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Conclusion: These experiments demonstrated that graphene could be cleaved through
photolithography to produce high-quality samples. The samples were successfully transferred
from an acetone solution to glass and silicon wafers. However, because photoresist will crack
when it is dissolved in acetone, graphene flakes will be less continuous and smaller in size.
References:
1. Novoselov KS, Geim AK, Morozov SV, Jiang D, Zhang Y, Dubonos SV, Grigorieva IV,
and Firsov AA, “Electric field effect in atomically thin carbon films,” Science, 306, 666669, 2004.
2. Kumar S, Peltekis N, Lee K, Kim H-Y, and Duesberg GS, “Reliable processing of
graphene using metal etchmasks, Nanoscale Research Letters, 6, 390, 2011.
Casey Orndorff: (Mentor – D.P. O’Neal)
Title: Low-Cost Fluorescent Imaging System
Introduction: A potential treatment of cancer uses injected gold nanoparticles, which
accumulate in cancerous tissue and tumors and can then be activated by light at a specific
frequency that induces vibration, heating and destroying the cancerous tissue with minimal
damage to surrounding tissue. To study this technique, it is important to have an effective
imaging method. While imaging systems exist, they can be overly expensive for many research
laboratories. Web cameras have recently become dramatically more powerful, with CMOS
sensors surpassing CCD sensors in pixel count, allowing the utilization of extreme low light
imaging. We propose that a low-cost fluorescence imaging system, using a simple desktop USB
web camera, is a viable alternative to more expensive systems. We have therefore constructed
such a system and tested it as a detector of nanoparticles in a mouse model.
Methods: A Logitech Quickcam Pro 9000 was used as the camera. The sensor can support up to
2 megapixels and the typical size of each pixel for such a sensor is 2.8 microns. The infrared
filter was removed from the assembly. The original 6 foot USB cable was replaced with a
shorter 1 foot, thicker, shielded cable. The camera was inserted into a project box, and a fan,
powered by the USB cable, was added to the assembly. The removal of the IR filter disables the
automatic focus of the web camera. The camera was then placed in RAW image capture mode,
using the Bayer ON/OFF settings that native to the camera.
The experimental setup is shown in Figure 1. A 635 nm, 3 mW, laser diode was attached to a
collimating lens, and the collimated light was fed into a 1 mm fiber optic cable. An optical power
meter will be used to determine amount of power lost in the fiber cable. The specimen to be
imaged was placed at the focal distance. A 692 nm band pass lens was then be placed between
the camera and the cuvette.
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Figure 1: Experimental setup for imaging microspheres.
The fluorescent particles were Invitrogen fluorospheres (Carboxylate-modified microspheres)
with an excitation wavelength of 660 nm and emission wavelength of 680 nm (0,024µm size). A
635 nm laser diode was initially used as the light source. The fluorescence was imaged for a
period of 3 minutes. The procedure was repeated using a 650 nm 3 mW laser. The mass
attenuation coefficient of the fluorospheres in a cuvette was obtained from the equation
𝐼 = 𝐼0 𝑒 −𝜇𝑙 ,
Where 𝐼0 is the intensity of incident light on the sample, 𝐼 is the intensity of the laser light
transmitted through the sample, 𝑙 is the path length through the sample, and 𝜇 is the attenuation
coefficient. To produce and record an in vivo signal, a mouse was injected with 100 µL of
fluorospheres and placed on its side after being put under mild anesthesia using isoflurane. A
custom virtual instrument created in LabView was used to process the image. The processing
included a particle filter, a Fourier transform, a threshold to remove dark current noise, and a
convolution to sharpen the edges.
Results: Figure 2 shows the light power transmitted through the cuvette as a function of
fluorophore concentration. The transmitted light is reduced, with increased fluorophore
concentration, by scattering. Figure 3 shows the emitted light as a function of fluorophore
concentration. The emitted light power initially increases because the number of emitters is
increasing, but then decreases as the emitted light becomes scattered through interaction with the
large number of particles.
8
3.5
0.0007
0.0006
0.0005
0.0004
0.0003
0.0002
0.0001
0
3
Power (µW)
Power (W)
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2.5
2
1.5
1
0.5
0
0
0.05
0.1
0.15
Fluorophore Concentration (Moles/L)
0
0.05
0.1
0.15
Fluorophore Concentration (Moles/L)
Figure 2: Power transmitted through a cuvette as the fluorosphere concentration was increased.
Figure 3 shows images obtained from the mouse spleen and tail, at two exposure times,
immediately after injection of the fluorophores. The images were enhanced to emphasize the
fluorophore signal. Residual fluorophores are retained by the tail and are taken up by the spleen.
Figure 3 (Left) Image of fluorescing mouse spleen and tail at 50 seconds laser light exposure. (Right)
Image of fluorescing mouse spleen and tail at 100 seconds laser light exposure. PSNR Improved by
48.1dB at the higher exposure time. These images were enhanced to improve contrast.
Figure 4 shows images obtained 7 days after fluorophore injection. The fluorophore
accumulation in the liver and spleen can be readily detected.
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Figure 4: (Left) Fluorescence images (not enhanced for contrast) taken 7 days after initial fluosphere
injection. (Right) Image of the mouse and the position relative to the fluorescence.
Conclusions: The low cost system developed here was able to image fluorophores within the tail
and spleen region of the mouse.
Maimuna Secka: (Mentor – Eric Guilbeau)
Title: Comparison of Responses to Alternative DNA Templates in a Thermopile-Based DNA
Sequencer
Introduction: A new method for the sequencing of DNA uses thermopiles in a microchannel
system that generate a voltage in response to the heat of reaction imposed by the insertion of a
DNA base. This system is particularly useful for the screening for single nucleotide
polymorphisms that are associated with specific diseases. The DNA template to be tested is
immobilized on a glass slide. Studies performed with glass slides purchased with preimmobilized template have yielded different reaction heats from more recent studies with inhouse coated slides. Therefore, a series of controlled studies was performed in which the inhouse and commercial templates were compared directly.
Methods: The sequencing system is shown schematically in Figure 1. A buffer is injected into
Inlet 1, and the materials required for base insertion (neucleotide triphospate and polymerase) are
injected into Inlet 2. The combined flows from the two inlets create a hydrodynamically focused
stream, which flows to the site of immobilized DNA and causes base insertion if the appropriate
base is present from Inlet 2. The signal is detected by the thermopile measuring junctions.
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Figure 1: Top view (upper figure) and side view (lower figure) of the sequencing system.
2.E-07
2.E-07
-1.E-20
0.E+00
-2.E-07
-2.E-07
Voltage (V)
Voltage (V)
Results: Typical traces of voltage as a function of time are shown in Figure 2. The commercial
slides (left panel) demonstrated a longer duration of signal than did the in-house slides (right
panel). The area under the curve, in this case, is about double for the commercially purchased
slides.
-4.E-07
-4.E-07
-6.E-07
-6.E-07
-8.E-07
-8.E-07
-1.E-06
100
150
200
250
Time (s)
300
350
400
-1.E-06
100
200
300
400
Time (s)
Figure 2: Thermopile voltages as a function of time for commercial (left) and in-house (right) slides. The
thin line is an estimate of the noise baseline. The area between the thick line and the thin line is the heat
signal.
Conclusions: The commercially-produced slides produced heats approximately double those of
the in-house slides. The difference may be caused by the amount of streptavidin on the slide.
With more streptavidin on the glass, more of the enzyme binds to the nucleotide.
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Sundeep Sharma: (Mentor – Mark DeCoster)
Title: The Neurobiological Interaction of Calcium ion, Glutamate and Glial Cells
Introduction: Glutamate causes an increase in influx of calcium ion into the cell through an ion
channel pore in the cell membrane. Excessive calcium within neural cells causes neurotoxicity.
To decrease neurotoxicity and maintain homeostasis, glial cells uptake excess glutamate and
convert it into glutamine for neurons. This neurobiological phenomenon has been studied in vivo
and in vitro. However, a computational model of this system would assist in the interpretation of
such experiments and allow predictions of the outcomes of specific therapies. Therefore, a 3compartment model predator prey simulation has been developed that includes glutamate,
calcium ion, and glial cells.
Methods: The predator-prey software originally describes the relationships among grass, sheep
and wolves. In the neural simulation, calcium is analogous to sheep, glutamate is analogous to
grass, and glial cells are analogous to wolves. Thus, just as sheep depend on grass and are
consumed by wolves, calcium depends on glutamate and is consumed by glial cells. For the
static cases, the initial energy of Ca++ was kept at 100 and the initial energy of glial cells was set
at 200. The reproduction rates of glutamate, Ca++, and glial cells were set to 0.5, 1, and 0.5,
respectively. The feeding rates of Ca++ and glial cells were kept at 0.5 and 0.75, respectively. The
energies gained for Ca++ and glial cells were 10 and 20, respectively. The simulation was
performed and every fifth frame was recorded. For the low glutamate case, the initial population
of glutamate, Ca++, and glial cells was set to a 1:10:10 ratio. For high glutamate, the ratios were
10:1:1. In addition, a dynamic case was simulated. Reproduction rate of glutamate, Ca++ and
glial cells were kept at 0.1, 0.05, and 0.025, respectively. Initial energy, feeding rate and energygained values remained constant, as in low and high glutamate conditions. Initial population of
grass, sheep, and wolf were kept at 500, 100, and 50, respectively. One step in the simulation
represented one second and every fifth image was recorded.
Images were analyzed with ImageJ Version 1.44 because the program easily imports stack
images. Each image was imported and converted to 8-bit gray scale images. Ca++ was considered
as a foreground and other components were considered background. The brightness and contrast
of each image was adjusted so that the foreground became whiter and all background became
black. Threshold and masked applications were applied to the stacks so the signal to noise ratio
was increased and the error of the measurement of calcium was reduced. Regions of interest
were chosen based on calcium fluctuation within the stack, as shown in Figure 1.
Figure 1: Color Image from frame capture (left) was converted into gray scale image (right) and
four regions of interest were selected
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Results: Figure 2 shows the area representing Ca++ for high and low glutamate. For high
glutamate, the Ca++ initially increases strongly, reaching 3 to 5 times its original value. Within
about 15 seconds, it has reached a near zero value. For low glutamate, Ca++ increases slightly to
approximately 1.2 times its initial value, and reaches zero within 10 seconds.
Figure 2: Image area representing Ca++ for high glutamate (left) and low glutamate (right).
Figure 3 shows the Ca2+ response for the oscillatory case. While Ca++ initially increases with
increased glutamate, an increase in calcium results in neurotoxicity, leading to cell death. The
calcium oscillations observed are typical of normal brain function.
Figure 3: Calcium response for the oscillatory case within four regions of interest.
Discussion: Our predator prey simulations produced dynamic relationships among glutamate,
calcium, and glial cells that were similar to previously published measurements. As shown in
Figure 2, increased glutamate increases the amplitude of calcium, which supports the abstract of
Sattler et al. (2000), and previous experiments performed by McNamara et al. (2009).
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Glial cells uptake excess glutamate and convert into glutamine, in order to reduce the
neurotoxicity of the cell (Bouvier and Szatkowski, 1992). Therefore, the cycle of calcium and
glutamate for normal cells act as a negative feedback system oscillations as shown in Figure 3.
Conclusion: Since the predator prey model supports the neurobiological relationship of
glutamate, calcium ion, and glial cells, a biocomputational model can be generated which can
help in tissue engineering and also studying the cell activities like apoptosis.
References:
Sattler R and Tymianski M, "Molecular mechanisms of calcium-dependent excitotoxicity."
Journal of Molecular Medicine 78(1): 3-13, 2000.
McNamara J, Cotton K, Masvekar R, and DeCoster MA, “Delay of Staurosporine-Induced
Apoptosis by Glutamate in Brain Tumor Glia and Normal Astrocytes.” Society for Neuroscience
2009, Poster, Chicago, IL; 547.9/W18
Bouvier M, Szatkowski M, et al. "The glial cell glutamate uptake carrier counter-transports pHchanging anions." Nature 360(6403): 471-474, 1992.
Stephen Sholden (Mentor – David K. Mills)
Title: Gentamicin-Loaded Halloycyte as a Bandage Antibacterial
Introduction: While standard bandages protect a wound from external damage and invasion by
bacteria, they can be improved through the addition of bacteriocidal agents, which kill harmful
bacteria, and growth factors, which promote the healing process. A carrier is needed that can
allow these agents to be released along a controlled timeline throughout the healing process.
Halloycyte (Figure 1), a naturally occurring clay nanoparticle, is a good candidate for such a
carrier because it is biocompatible, inexpensive, and strong. In addition, its multi-layered
structure allows it to contain multiple agents. Experiments were therefore performed to load
these nanotubes with gentamicin and test their ability to reduce the population of E. coli bacteria.
Figure 1: Structure of halloycyte nanotubes.
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Methods: Halloycyte nanotubes were incubated in gentamicin solution and then incorporated
into a 10% by weight solution of polycaprolactone in chloroform and allowed to set for 12 hours.
The mixture was then injected through a 22 gauge needle onto a plate placed 15 cm away. A
voltage of 18 kV was applied between the needle and the plate, and the syringe pump ejected the
solution at 10 L/min. E. coli cells were then seeded onto this scaffold. A live-dead assay was
used to image the cells.
Results: Figure 2 shows the live-dead assay. These images demonstrate the ability to image the
cells and show some dependence on the depth within the scaffold. More experiments are
necessary to reach a statistically significant conclusion about the effectiveness of the halloycyte
particles.
Figure 2: Live-dead assay of E. coli bacteria from the scaffold surface (left) and a deeper layer (right).
Conclusions: These experiments demonstrated the feasibility of loading halloycite with
gentamicin and incorporating the loaded particles into an electrospun scaffold.
Lauren Uffelman: (Mentor – Long Que)
Title: Wind Energy Harvesting of CNF-PZT Cantilever Device
Introduction: A lead zirconate titanate (PZT) cantilever that is plated with nickel leads and
coated with carbon nanotubes has been shown to produce energy in response to light and upon
bending. The device has been studied under exposure to light and heat, but not under exposure
to mechanical waves, particularly in the form of wind. The ability of the device to harvest
multiple energy sources would provide additional flexibility when one or more of the energy
sources is absent. To investigate the device’s voltage response as a function of wind, the
mechanical aspects of the device were studied.
Methods: To make a nanotube film of approximately 30 μm thickness, 10 mg of carbon
nanotubes are mixed with 100 mL of isopropyl alcohol. The mixture is sonicated for a minimum
of 3 hours to ensure even distribution of the tubes within the solution. After sonication, the
mixture is vacuum filtrated. Once the bulk of the solution has been filtered away, the film is
washed several times with small volumes of distilled water followed by more IPA. Vacuum
filtration is continued for another hour, and the film is allowed to dry overnight. The paper is
then transferred to a silicon wafer and submersed in an acetone bath for 15 minutes. The wafer
is taken out of the bath and allowed to dry. Excess filter paper is cleared away with a cotton
swab. The process is repeated a minimum of 3 times, each time using fresh acetone. To transfer
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the film to the nickel-coated PZT, it is first removed from the silicon wafer with double-sided
tape, which is then cut to the dimensions of the beam and taped to the beam with the carbon
nanotube side up. Two wires are then attached, one to the carbon nanotube film, and the other to
the nickel electrode on the opposite side.
The device’s response to simultaneous exposure to wind, light, and thermal energies was studied.
Two wind sources were used. One was a Massey high velocity fan, and the other was a airblowing port of a Kemore vacuum. An Olympus TL2 lamp was used as the light source
Results: Figure 1 shows the response of the device to various stimuli. The lamp along generated
a voltage with an initial peak of 10.8 Volts, which decreased over time. The fan, with a 4.47 m/s
wind speed, generated a much smaller peak voltage of 0.028 Volts. The vacuum, which blows
warmer air and has a 10.9 m/s wind speed, produced a larger voltage than the fan. When the
lamp and fan were combined, the maximum voltage was larger than the fan alone, but smaller
than the lamp alone.
Figure 2: Response of the cantilever to the lamp (upper left), the fan (upper right), the vacuum (lower
left), and the combined fan and light (lower right).
Discussion: The smaller voltage found when the wind and light were applied, as opposed to the
lamp only, may be caused by convective cooling induced by the wind. The cooling overcomes
the additional energy caused by the wind. The device works most efficiently when the energy
applied to it oscillates. For wind harvesting, therefore, the ideal condition would be to match the
natural frequency of the beam to the vortex shedding frequency of the given wind speed. To
obtain a direct measurement of the beam oscillation frequency, an impulse was applied to the
beam, and the frequency was read from the voltage signal on the oscilloscope. The value of
2700 Hz compared favorably to a theoretical value (based on the beam’s material properties) of
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3000 Hz. The vortex shedding frequency for the beam, assuming that the Strouhal number is
0.2, is 𝑓 = St 𝑣/𝑑, whe𝑑re 𝑣 is wind speed and 𝑑 is the beam diameter. The values for the fan
and vacuum are 112 Hz and 272 Hz, respectively. Thus, it was not possible in these experiments
to reach the oscillation frequency of the beam.
Conclusions: The imposition of wind energy on the beam generates a voltage. However, the
combination of wind energy and light energy did not provide as much electrical energy as light
alone. It is expected that a cantilever design with a lower resonant frequency would be needed to
take full advantage of wind energy.
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Program Year 2009
(Mentor – David K. Mills)
Title: Cell Culture and Tissue Engineering the Temporomandibular Joint (TMJ) Disc
Introduction: The temporomandibular joint (TMJ) is a complex, sensitive, and highly mobile
joint in the jaw. Millions of people in the United States suffer from temporomandibular disorders
(TMD) which are painful and affect the ability to chew. The TMJ connects the mandible or
lower jaw to the skull and regulates the movement of the jaw. It is a bi-condylar joint in which
the condyles, located at the two ends of the mandible, function at the same time. The movable
round upper end of the jaw is called the condyle and the socket is called the articular fossa.
Between the condyle and the fossa is a disc made of fibrocartilage that acts as a cushion to
absorb stress and allows the condyle to move easily when the mouth opens and closes. This
study examined the feasibility of tissue engineering a temporomandibular disk.
Methods: Osteoblast cells were cultured onto a nanoscaffold that was electrospun from 9% PCL
solution. A standard cell culture dish was used as the control. In one case the nanoscaffold was
supplemented with collagen, and in another both collagen and growth factor were added. The
DNA content of the cells was assayed with picogreen fluorescent dye.
Results: The picogreen results are shown in Figure 1. The control showed the least amount of
DNA (related to cell proliferation) while the scaffold+collagen+growth factor had the highest
amount of DNA.
Figure 1: Picogreen assay on four substrates, which suggest that the PCL scaffold improves cell
proliferation, while the addition of collagen and growth factor yielded additional improvement.
Conclusion: The results presented here are preliminary, but they suggest that the PCL scaffold
favors cellular proliferation and may be a good substrate for osteoblast growth. Further tests are
needed to examine the phenotype and protein expression of the cells.
(Mentor – Sven Eklund)
Title: Calcium Biosensor
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Introduction: Calcium is an important ion in multiple signaling mechanisms throughout the
human body, and consequently a wide variety of diseases are linked to irregularities in calcium.
A small, reliable and low-cost sensor that can easily measure calcium concentrations would
therefore be valuable for fundamental physiological research. The immediate goal of the current
research is to implement the basic design as an oxygen sensor for later implementation as a
calcium censor. The proposed design is a
fluorescent biosensor for measurement of
extracellular calcium concentration.
Methods: A fiberoptic bundle, seen at the top of
the device shown in Figure 2, sends light through
the center fiber to a dye that fluoresces in
response to the target agent, which is oxygen for
this initial study. The dye is coated as a film on a
well-cup. The fluorescent signal is collected by
the six fiberoptic fibers that surround the
transmitting fiber, and spectroscopy is used to
detect the shift in the wavelength of light.
Results: To test the sensor, oxygenated distilled
water was loaded into one flow channel, while
Figure 2: Light transmitter and detector
deoxygenated water was loaded into another. The for the biosensor.
sensor was positioned to interrogate a common
channel whose source alternated between the deoxygenated and oxygenated water channels.
Figure 3 shows the response of the sensor to the alternating flow. The sensor output increased
slowly in response to the deoxygenated water and decreased slowly in response to the
oxygenated water. A similar, but weaker, response was found in response to cells that were
alternately exposed to flow and no-flow conditions. These results indicate that the sensor
functions, but that further work is needed to enhance signal-to-noise level and to improve
response time.
Figure 3: Response of the optical sensor to oxygenated and deoxygenated water.
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(Mentor – Niel Crews)
Title: Sample Preparation Device for use in continuous flow PCR
Introduction: DNA profiling requires
fast polymerase chain reaction (PCR)
Hot
DNA analysis using the 13 different
primer sets established by the FBI’s
combined DNA index system. Dr.
Cool
Crews’ group is developing a continousflow PCR microdevice in which a sample
moves through zig-zag channels
alternately through high and low
temperature regions to accomplish the
heating and cooling necessary for PCR
(Figure 4). Continuous-flow PCR,
particularly in conjunction with spatial
DNA melting analysis, provides fast
testing of DNA samples using a single
primer, but is difficult to use with
multiple primer sets. This work presents
Figure 4: Microdevice for continuous-flow
a sample preparation device that can
PCR. As the sample passes through the
interface with a continuous-flow PCR
channels, it moves alternately from hot to cold
device to provide it with serial droplets
temperatures.
of the same DNA sample wherein each droplet contains a different set of PCR primers.
Methods: The design requirements to be examined were: (1) Ability of the input section to
generate separate droplets of sample, (2) Ability of dried sample to provide correct amplification
and (3) Ability of the input section to pump the sample into the PCR device.
The sample preparation device consists of an inlet for a mixture of the DNA sample and water,
microfluidic channels to divide up the sample, wells containing pre-applied dehydrated primers,
reagents, and catalysts necessary for PCR, novel "flat" microfluidic channels which function as
valves, and a receiving channel with a continuous flow of oil that enters the PCR device and
accepts each sample as it is expelled from the wells. To provide alternating droplets of the PCR
sample, the aqueous sample was injected through a T-junction branch that had a flattened crosssection and oil was injected through a branch that had a square cross-section.
To test the ability of dried sample to support amplification, aPCR mixture of 1 part forward
primer, 1 part reverse primer, 1 part dNTPs, 1 part Taq with bovine serum albumin (BSA), 1 part
LC Green, 1 part buffer, 1 part DNA template (i.e. the sample), and 3 parts water was made.
Combinations of these components were placed on PDMS and allowed to evaporate. The dried
reagents were then further exposed to ionized air in the plasma cleaner and heat in the oven.
Then the parts of the PCR mixture that had not been dried were placed on the dehydrated
reagents. This mixture was allowed to sit for two minutes, and then it was inserted into a
capillary and run through the Light Scanner 32. A positive and negative control made up at the
same time as the sample were also run. This experiment was repeated for several combinations
of parts of the mixture, but only the last experiment results will be reported.
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The pumping action was tested for a configuration with an inlet channel, a well, and an outlet
channel. Alternating pressure applied to the well activates the pumping.
Results: Good droplet separation was found for a
T-junction with a 0.9 mm regular channel and a
0.525 mm flat channel (Figure 5). The droplet
lengths were approximately 2 mm.
Fluorescent intensities confirmed amplification
for the dried and undried samples (Figure 6).
Some amplification was also found for the
negative control, which is likely caused by some
contamination of that sample.
Figure 5: Droplets formed with the
T-junction configuration.
The pumping tests confirmed that the input
device was able to provide the needed flow
input.
Conclusion: The concept developed for
sample handling for continuous-flow PCR is
feasible. Samples as small as 20 µL can be
inserted into the well, although the issue of
complete well filling must be addressed before
the device will be functional, as incomplete
Figure 6: Amplification signals for the
filling introduces too many bubbles for the
rehydrated sample.
thermal gradient PCR to function properly.
Reagents can be dried and stored in the well during the fabrication process and remain viable.
They mix with the sample and can be pumped out of the well by simple mechanical force. The
sample predictably enters the oil flow as a droplet and can be funneled into the PCR channel and
achieve robust amplification.
(Mentor – D. Patrick O’Neal)
Title: Analysis of a Gold Substrate Prototype Using Surface-Enhanced Raman Spectroscopy and
Other Techniques
Introduction: In many microenvironments such as cell culture, small changes in the
concentration of certain substances can severely impact balance of the environment. The ability
to monitor these concentrations in real-time would allow for better management of the
microenvironments. Surface-enhanced Raman spectroscopy (SERS) can register concentrations
in the micro- to nanomolar levels. Placing a reusable surface for SERS in the microenvironment
to measure concentrations of certain substances would fill this need for better management.
The ability to rely on and reuse the GSP is the center of our ongoing research. Reusability hinges
on the ability to remove the substance adsorbed onto the surface of the GSP so it can again
measure concentrations without doing harm to the GSP. Two methods of obtaining reusability
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are looked at in this paper: heat modulated desorption and electrochemical desorption using
cyclic voltammetry.
Methods: This paper looks at two separate ways to remove adsorbed substances from a gold
substrate prototype (GSP) so that it can be reused to find real time concentrations down into the
nanomolar range. The first way is to use heat to remove the material adsorbed on the surface of
the GSP. The second way is to use cyclic voltammograms (CVs) to reverse the polarity of the
GSP to repel or desorb the material. Both methods were confirmed using surface enhanced
Raman spectroscopy (SERS). The material that was adsorbed onto the surface for heat tests was
pMA and the material used for the CVs was malachite green (MG). In addition differences in the
GSPs due to changes in the gold salt solution used to make them were qualified using SEM
images and physical appearance.
Results: An important finding that was not part of the initial study was that the age of the gold
salt solution has a direct effect on the physical properties of the GSPs. The solution that is used
immediately after it is made creates GSPs that are orange-matted in color and seem to have
colloid particles attached to its surface. If the solution is more than 24 hours old, however, the
GSPs appear shiny and gold.
Heat was able to remove pMA
from the surface of a GSP, and
the signal returns when new
pMA is applied. However, the
severe decrease in intensity from
almost 1800 counts to only 200
counts is problematic (Figure 7).
To achieve reliable reusability
the signal intensity cannot
depend on the number of
desorption cycles a GSP has
gone through. Either all the GSPs
Figure 7: Changes in intensity due to cycles of heat
must go through an initial
modulated desorption
desorption to bring the signal
intensity down to 200 counts or new substrates must be used each time. Also, after only 4 cycles
in the heat (225°C) the GSPs became so brittle that they snapped into many pieces even when
handled lightly by tweezers. 4 mediocre uses of a GSP do not qualify as quality reusability.
Desorption through electrochemistry could not be accomplished because the GSP was not
sufficiently conductive. Enhancement factor calculations showed that the GSP is an effective
SERS active surface.
Conclusion: Overall the GSP is an effective and reliable way to measure SERS spectra. The
GSPs give significant enhancement and can be reused to a certain extent. With more research
into electrochemistry their reusability may increase and allow the GSPs to be used in
microenvironments to monitor real-time concentrations of several substances important to the
growth or balance of organisms living in the microenvironment.
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(Mentor – Sidney Sit)
Title: Drug Release of FITC-loaded Hydrogel in Macro Patterned and Micro Patterned
Arrangements
Introduction: Successful treatment of disease depends on constant administration of drugs to an
affected area of the body. Researchers have examined the use of biocompatible molecular “nets”
that trap the drug and adhere to the affected site, where the drug is released. The drug is thus
released only in the targeted area and does not cause adverse effects at other locations.
Polyethylene Glycol (PEG) is a biocompatible polymeric hydrogel that can be used to contain various
drugs. An array of drug-loaded hydrogel spots can be applied to the surface of the human skin and
release the drug transdermally. However, little is known about how the patterning of the hydrogel
material effects drug absorption into the body. The more contact surface area to volume ratio, the faster
and more efficiently the drug should absorb into contact fluid. So this experiment will use different
hydrogel printing techniques to alter the surface area to volume ratio, or size, of spherical hydrogel spots.
Macrospots and microspots will be examined. The macrospots, with an average spot diameter of
0.2 centimeters, will be printed on a surface using a hydrogel filled micropipette. The
microspots, with an average diameter of about 12 microns, will be printed with a microspot
printing device called a NanoEnabler.
It is hypothesized that because the surface area to volume ratio of the microspots is much greater
than that of the macrospots, the drug will be released much more quickly than in the latter. Also,
it is hypothesized that the percentage of drug release from the microspots will be higher because
of their small size; not as much drug will remain trapped inside of the spot.
Methods: Standard 3x1 in. glass slides were cleaned with methanol, ethanol, and chloroform for
seven minute intervals each. Twenty milliliters of chloroform were then mixed with one
milliliter of octadecyltrichlorosilane. Slides are immersed in this mixture for thirty minutes and
then rinsed with pure chloroform. The silane compound chemically modifies the surface of the
slide and makes it hydrophobic. The solution to be patterned was a mixture of 0.6 ml of PEGDA stock solution with 0.134 ml of FITC stock solution. Three of the slides were pattered with a
size 20 RAININ micropipette. Each spot volume was 0.002 (Figure 8).
Figure 8: Left: macropatterned slide. Right: Micropatterned surface (viewed through the NanoEnabler
software interface).
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The patterned slides were then placed in Petri dish and covered with 1.5 ml of 1X PBS solution.
The solution was removed and replaced after 30, 60, 90, 120, 180, 300, 420, 540, and 1440
minutes. The fluorescent intensity of the removed solution was then measured and translated to
drug concentration with the aid of a standard curve.
Results: Figure 9 shows the release of the FITC for the macro and microspot patterning. The
rate of drug release and the total amount of drug released after 24 hours were both greater for the
microspotted slide. However, because the tip of the NanoEnabler is only 30 micrometers in
diameter, any small obstruction can clot the tip and reduce the flow of hydrogel. FITC Dextran
and FITC BSA drugs were both used and if they were present in concentrations greater than 12.5
mg/ml, the microspot size was limited to approximately 7 microns on average. To get larger spot
sizes, pure FITC dye was used in these experiments. Since the FITC dye has a lower molecular
weight than the FITC Dextran or BSA, 12 microns diameter spots could be patterned.
Figure 9: Drug release over time for the micro and macro patterning.
Conclusions: The results support the hypothesis that decreased spot sizes lead to larger ratios of
surface area to volume and therefore to greater drug release .
(Mentor – Steven A. Jones)
Title: Behavior of Platelets in Dynamic and Static Conditions
Introduction: Stroke and Heart attack are caused by arterial thrombi, which in turn depend on
platelet activation, adhesion and aggregation. Platelets secrete agents that act as platelet
activators, and they also secrete platelet inhibitors. We propose that the motion of blood and
associated mass transport phenomena affect the relative importance of activators and inhibitors.
The initial objectives of this set of experiments are to determine whether platelet adhesion is
enhanced or decreased when the fluid in contact with a glass slide is agitated.
Methods: Citrated bovine whole blood was centrifuged at 320 rcf for 35 minutes to extract the
platelet-rich plasma. Layer-by-layer self assembly was used to coat the glass slides, which were
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then exposed to the plasma in a Petri
dish. For each experiment, one slide
was static and the other was agitated by
a vortexer to which a metal plate had
been attached (Figure 10). A student Ttest was used to determine the statistical
differences between the adhesion for the
agitated and unagitated condition. The
slides were then stained with acridine
orange, rinsed, and then imaged by
fluorescent microscopy. The images
were analyzed with MATLAB program
to give us the percent surface area
Figure 10: Vortexer setup used to agitate the
coated by platelets. The null hypothesis
is that there is no difference and both
glass slide.
the stationary and the agitated samples
will give the same mean. The alternative hypothesis is that the agitated sample will have a higher
average surface coverage. A p-value of 0.05 was considered significant.
Results: Figure 10 shows a typical set of data for the static and dynamic conditions when the
slide was coated with one bilayer. Although this figure suggests a larger amount of adhesion for
the static case, other slides showed the opposite result, so the final results were not statistically
significant at the 0.05 level.
Figure 10: Percentage of surface coverage on a glass slide with one bilayer of fibrinogen under
static and dynamic conditions.
Additional experiments were performed with 3 and 5 bilayers. The adhesion results are
summarized in Figure 11. As the number of bilayers increased, the adhesion in the dynamic case
tended to be greater than the adhesion in the static case. However, none of the cases indicated
statistical significance at the 0.05 level.
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Conclusion: Although the results were not
statistically significant, they suggest that
significance can be shown with a larger
data set. Furthermore, the results indicate
that the adhesion will depend on the
number of bilayers in the layer-by-layer
process. Whether or not significance is
shown, the next step will involve the
addition of L-arginine to the platelet-rich
plasma to stimulate the production of
platelet-derived nitric oxide. Because nitric
oxide is small and has a high diffusion
coefficient, it is proposed that its transport
will be particularly important to the
inhibition of platelet adhesion.
Figure 11: Percentage of surface coverage for
static and dynamic conditions for different
numbers of bilayers.
(Mentor – Long Que)
Title: Optical biosensing with a polymer-based micromachined Fabry-Perot interferometer
Introduction: Numerous optical devices, such as gratings, mirrors, lenses, and interferometers
have been miniaturized and modified to sense on the micro level. The micromachined FabryPerot interferometer (μFPI), an especially versatile optical sensor, can be used for gas sensing,
chemical sensing, and optical modulation. The interferometer’s center cavity contains the gases
and chemicals and a spectrometer detects the effect of the chemical or gas on the light. The μFPI
includes a crosslinked electro-optic polymer in its cavity, which allows for high time bandwidth
modulation.
A Fabry-Perot interferometer is usually used to control and measure wavelengths of light
through shining a beam between two highly reflecting parallel plates. The beam goes through
multiple reflections where some of the beam gets transmitted and some of the beams get
reflected. If all of the reflected beams are in phase, then constructive interference takes place
and transmission peaks occur. The narrower the transmission peaks, the higher the reflectivity
and the easier it becomes to detect slight phase shifts between different chemicals through the
different indexes of refraction. In this project, the ultimate goal is a high-performing refractive
index sensor that can be used on the microscopic level.
Traditionally, -FPIs have been fabricated from silicon, polysilicon, silicon nitride, or silicon
oxide, which are all semiconductive materials based from the element silicon. Past μFPIs have
also operated on laser sources. However, such interferometers are expensive and cannot allow
for one-use disposals, despite risks for cross-contamination upon reuse. Therefore, a new μFPI
design has been proposed, which contains a polymer base using polydimethylsiloxane and is
operated by a white-light source. Polydimethylsiloxane (PDMS for short) is a silicon-based
organic polymer, a common plastic material. Unlike the traditional fabrication approach, which
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requires several mask-leveling processes and a laser source, this new design will make
fabrication less expensive, and require only a white light source.
A primary goal of this project is to monitor real-time bonding of antibodies and antigens and
detect the phase shifts before and after the bonding due to differences in the refractive indexes.
The long-term objective of the project is to utilize this ability in field of drug discovery and
screening.
Methods: The light source undergoes multiple reflections inside a cavity formed by a PDMS
layer and a gold-coated plate. Antibodies (BSA) are immobilized onto the gold surface. Then a
the liquid containing antigens or protein is injected into the Fabry-Perot interferometer cavity
with a KD Scientific syringe pump and allowed to interact and bond with the antibodies on the
gold surface. After bonding, the liquid is flushed from the cavity to remove the excess
unbounded antigens. The reflected signals, represented as intensity as a function of time, are
recorded at each stage of this process.
Results: Differences in sample content are indicated by phase shifts in the intensity signal from
the interferometer. Although there is some noise in these signals, the do show such phase shifts
between the samples corresponding to the bare cavity, the antigen and the antigen+antibody
combination (Figure 12).
Figure 12: Intensity output of the interferometer for the three conditions tested.
Conclusions: The interferometer provided signals that could distinguish between different cases.
However the signals are still noisy, and it was often difficult to remove unwanted bubbles from
the cavity. The fringe shapes also depended strongly on the pressure at the input tube. It will be
necessary to solve this problem before the device will be practical.
(Mentor – Eric Guilbeau)
Title: Immobilization of Glucose Oxidase for Temperature-Based Analysis Using Layer-byLayer Assembly
Introduction: Thermopiles are highly sensitive instruments for the detection of temperature, and
they can be used, in particular, to detect changes in temperature with high sensitivity. Therefore,
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the detection of endothermic or ectothermic reactions can be used as a basis for chemical
analysis in a wide variety of sensors. In such sensors, reactants or catalysist can be immobilized
on microchannels that pass over the thermopile, and the reactions that occur as the analyte flows
through the region are indicated by a change in the thermopile voltage output. One type of
sensor that is particularly important is the glucose sensor. In this project, we use layer-by-layer
self assembly to immobilize glucose on a thermopile system.
Methods: Glucose oxidase oxidizes glucose to gluconic acid, while releasing 80 kJ/mole. A
(negatively charged) glass substrate was used, and was first coated with a positive layer of PEI.
Two bilayers of alternating PSS and PEI were then added, and a final set of alternating Glucose
oxidase and PEI layers formed the surface (Figure 13). Atomic force microscopy was used to
analyze the layered surfaces and determine layer thickness.
Figure 13: Layering pattern for glucose oxidase immobilization.
Results: Figure 14 shows the atomic force microscopy images for the clean slide, the PEI/PSS
precursor layer and the completed assembly with glucose oxidase. The glucose oxidase has
more small-scale features than the PEI/PSS precursor layer. It also indicates that there is some
grouping of the layering, as suggested by the peaks in the image. The thickness of the PEI/PSS
layer was 10 to 15 nm, and the thickness of the glucose oxidase layer was 35 to 45 nm.
Figure 14: Atomic force microscopy images for the clean slide, the PEI/PSS precursor layer and
the completed assembly with glucose oxidase.
Conclusion: Layer-by-layer assembly is an effective method to immobilize glucose oxidase onto
the substrate.
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