1. 2016 Meeting of the Biophysical Society
Motivation
DIY Equipment and Biochemical Experiments in Undergraduate Curriculum
Kayla Washenberger, Benjamin L. Stottrupa
a) Department of Physics, Augsburg College, Minneapolis, MN b) Department of Chemistry, Augsburg College, Minneapolis, MN
Student Results
Many undergraduate physics majors will go directly to
industry after graduation. Incorporating laboratory research
into senior level courses facilitates the teaching of
interdisciplinary skills such as designing experiments,
careful measurement, and troubleshooting experimental
difficulties. In addition to these skills, hacking and making
are also valuable for physics majors seeking employment
directly after graduation. Two experiments were utilized to
engage students in building these skills. In the first,
traditional biochemical studies of phase behavior in lipid
membranes was done with fluorescent probes.
Fluorescence spectroscopy assays were used to measure
main chain phase transitions in lipid bilayers and
transbilayer flip-flop rates for phospholipid/cholesterol
liposomes. Results from both assay experiments were
compiled across teams of students. Each student was also
responsible for producing a technical written lab report
focusing on the experimental methods and results. In a
second experience students attempted to build
components of a Brewster Angle microscope using 3D
printed parts and a simple cell phone camera. This project
introduced students to CAD software and 3D printing. We
frame these experiences within membrane biophysical
studies.
Procedural Steps
The wavelengths at 440 and 490
have the highest peaks indicating
the excitation intensities.
A home brew spectrometer was assembled using
SpectraSuite software, temperature control,
thermometer, voltage input, and water pump for
water circulation. A laser pointer from eBay
provided an early light source for our experiments.
DIY Equipment
Activity: Students spent 2 weeks designing and printing parts for a home made Brewster
Angle Microscope. This included filter holders, an iPhone 5 camera mount, as well as
some connecting apparatus.
As Cholesterol increased in the DMPC vesicles, so did
the Generalized Polarization respectively. Students
were responsible for 0% Cholesterol and one of the
three added Cholesterol amounts, 10%, 20%, or 30%.
H2O
H2O
H2O
Vesicle
Biological Relevance:
Cholesterol is a major modifier to
the phospholipid bilayer structure
and is seen everywhere within
animal cell membranes. Important
questions still remain regarding the
influence of cholesterol on
dynamic properties pertaining to
gel and liquid-crystalline phases.
For this reason we use studies of
cholesterol/phospholipid systems
to place experiments into a
broader context.
Lipids: Lipids are amphiphilic molecules that contain a hydrophilic head
and a hydrophobic tail. This property determines the structures that lipids
form such as a micelle, monolayers, bilayers or multilamellar vesicles. A
vesicle is a spherical lipid bilayer as seen in the cartoon below. Vesicles
are often used to model properties of a cell membrane and as potential
drug delivery systems. Two assays were used to test the characteristics
of vesicles. First, the phase transition within the bilayer membrane using
the fluorescent probe, Laurdan, can be determined using a Laurdan
Assay. Under biologically relevant conditions the lipids making up a
vesicle are free to diffuse both laterally and between the leaflets of the
membrane. The second assay, which measures the tranbilayer diffusion
rates is a sodium dithionite assay.
Spectrometer
Apparatus
GeneralizedPolarization
Temperature (C)
Laurdan Emission at 27°C
Wavelength (nanometers)
Intensity(arb.units)
Lab Organization: Students had 100 minutes of
supervised lab time each week. The Laurdan
experiment was set up so that students would extrude an
already prepared multilamellar vesical solution. Each
week a group of lab students would test one solution.
Each group of students had the opportunity to test two
binary mixtures. To calibrate and determine the
uncertainty across lab groups all groups of students
tested 100 mol % DMPC.
Laurdan Assay
Laurdan Overview: The objective for the Laurdan Assay is to detect
the phase transition within the bilayer membrane using the fluorescent
probe, Laurdan. Bilayer membranes are sensitive to the polarity of their
environment. Apolar solvents leave the emission spectra to be bluer. A
large spectra shift is found due to dipolar relaxation processes occurring
in the liquid-crystalline phase. Liposomes produce a spectra transition
as the lipids within the membrane shift from a liquid-crystalline (ordered
to a gel (disordered phase, and they are calculated using the
Generalized Polarization (GP). Wavelengths at 440 and 490 are where
the excitation intensities are.
Course Format
This four credit course meets 70 minutes on MWF for a
traditional lecture format. Then students are asked to
work outside of class for 2 to 3 hours in the lab each
week with 100 mintues of supervised time. The text
used is Biological Physics, by Nelson.
Labs: Students were able to attend 1 of 2 lab
sessions. Each lab session had about 9 students in
it. Students worked in groups of three or pairs. A lab
activity lasted 2 weeks total and there were 6
activities for students across the course. The majority
of these activities focused on studies of lipid systems.
Sodium Dithionite Assay
NBD-PE is a fluorescently tagged-lipid used
to monitor fluorescence intensity. Adding
NBD tagged lipids during the formation of
vesicles will results in both leaflets contain
fluorescent probe.
Sodium dithionite quenches the fluorescence
of only the outer leaflet by reducing the
electron withdrawing nitro group to an
electron donating amine.
A column is used to separate lipids from
sodium dithionite based on size exclusion.
After passing through the columns the
vesicles are fluorescently labeled only on the
inside. Since sodium dithionite is not in
contact with the lipids, fluorescence will not
be quenched until sodium dithionite is added
again, quenching only the outer leaflet.
amine group
nitro group
At various times a sample of asymmetric
lipids was placed in the spectrometer to
find the fluorescence intensity. Sodium
dithionite was again added to quench
lipids that flipped to the outer leaflet while
being incubated
The ratio of lipids that have flipped to the
outer leaflet was determined by
This ratio was plotted versus time and fit
with the equation:
using a Matlab program to extract rate
constants (k1 and k2) for the inward flip
and outward flop rates.
)1(
)(
)(
21
1
2/1
21 xkk
e
kk
k
t 
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

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
initial
finalinital
I
II
R
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Conclusions
10%
22%
33%
40%
Transbilayer diffusion for vesicles made
with dihydrocholesterol. Increasing the
concentration of dihydrocholesterol results
in a decreased rate of flip-flop.
Time (min)
Ratiooflipidsinthe
OuterLeaflet(%)
Comparison to previous experimental results
obtained by undergraduate researcher.
A B
C
D
Various Maker Items Printed for Lab:
A) Work Drive Assembly B) Mounts
for Pumps C) Holder for microfluidic
cell D) Barrier mounts for Langmuir
Trough
Logistically this lab was a significant challenge. The student TA needed to be present
for all successful experimental runs. Pitfalls included: 1) a significant fraction of the
class had never run a column. 2) The time required for experiments meant that there
was no opportunity for error. 3) Variability across student groups made experiments
too difficult to compare.
iPhone Holder Filter holders & Objective Mount
Temperature (C)
GeneralizedPolarization
0% Cholesterol with DMPC
Over the course, every student spent a
week running the Laurdan assay with
cholesterol and without. The curves are
generally in the same area.
Students were able to successfully run the Laurdan Assay
and collectively record useful data for writing lab reports.
The Sodium Dithionite Assay was difficult within the short
time allotted for the lab. Most data observed did not reach
maximum quenching on the outer leaflet. Incorporating
bilayer systems into the biophysics lab has brought
diversity and a learning opportunity for the biophysics
course and the Augsburg Community. Other STEM
departments are going to begin including vesicle relations
within their faculty research.
Monolayer
Acknowledgements: This work was supported by Augsburg College’s office of Undergraduate Research and Graduate opportunities, The Sundquist Scholars program at Augsburg College, NSF DMR 1207544 (National Science Foundation). Professor
Bankers-Fulbright, Dr. Ravi Tavakley, Oscar Martinez, Eleni Beyene, Cain Valtierrez and the rest of the Augsburg Biophysics Research team.