Building a Bilayer Overtone Analysis (BOA) Instrument

Emma Rogers

NIST Center for Neutron Research

Neutron-Condensed Matter Science Group

SURF Colloquium August 2019
Physiological Homeostasis

TolTech VH Dissector Copyright © 2019 Touch of Life Technologies Inc. All rights 

reserved.

Almén MS, Nordström KJ, Fredriksson R, Schiöth HB (August 2009). "Mapping the 

human membrane proteome: a majority of the human membrane proteins can be 

classified according to function and evolutionary origin". BMC Biology. 7: 50. 

doi:10.1186/1741-7007-7-50. PMC 2739160. PMID 19678920.

Organization:

● Internal environment ≠ External 

environment

Compartmentalization:

● Cells → Tissues → Organs → Organ 

systems

● No global variables!

Unsteady equilibrium:

● Is complete stability a good thing?

● Newton’s Third Law of Motion

Maintaining unsteady equilibrium 

between constantly fluctuating internal 

and external environments

Kidney
Lipid Bilayer

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. 

New York: W. H. Freeman; 2000. Section 3.4, Membrane Proteins. 

Available from: https://www.ncbi.nlm.nih.gov/books/NBK21570/

Structure: BI-LAYER (two leaflets)

● Separated by some distance

● Hydrophobic (non-polar) and hydrophilic 

(polar) organization

● Separation of charge on two surfaces 

across a distance?

Transport: proteins and ions!

● Diffusion vs. transport

● Many classes of 

membrane proteins

● Structure, function, 

membrane interaction

● Signaling, communication, 

etc.

Transmembrane

Peripheral
Lipid Bilayer: Functional Model

Biophysical translation:

- +

Basic principle:

● Asymmetry of the membrane results in an 

intrinsic transmembrane potential

○ Charges (ions) build up on each side of the 

membrane

● Membrane behaves like a squishy capacitor

○ Interactions with ions and proteins result in 

changes in the transmembrane pressure

○ Bilayer shrinks and expands! And we can 

model that!

+

-
Lipid Bilayer: Functional Model

Katz D.M. Physics for Scientists and Engineers: Foundations and 

Connections. Cengage Learning; Boston, MA, USA: 2016

Biophysical 

translation:

Capacitor equations:

- +

+

-

ii2 =
What is a BOA? 

Component pieces:

● Lock-in amplifier- SR860

● Control software and GUI- for DAQ!

● Physical cell, teflon partition, 

aluminum chamber

● Silver chloride electrodes

● Electromagnetic shielding, vibration 

isolation stage

Electric 

shielding

Biophysical tool: semi-bio, semi-physical!
Electrical Signal & Measurement

Tubulin on biomimetic mitochondrial membranes, David P. Hoogerheide, 

Sergei Y. Noskov, Daniel Jacobs, Lucie Bergdoll, Vitalii Silin, David L. 

Worcester, Jeff Abramson, Hirsh Nanda, Tatiana K.Rostovtseva, Sergey M. 

Bezrukov, Proceedings of the National Academy of Sciences May 2017, 

114 (18) E3622-E3631; DOI:10.1073/pnas.1619806114

SR860 Lockin amplifier:

● f0 ≈ 2kHz, measure current at 2f0 

● Measure capacitance (first 

harmonic), VVac = 75mV

● Scan VVdc from -50 - 50 mV over 8s

○ Wait at 0V for transients

○ Measure second harmonic 

amplitude (slope) and phase 

(offset)

50mV

-50mV

X – in-phase component

Y – out-of-phase component

DC - voltage scan

ΘΘ – degree offset
Experimental Methods

Redondo-Morata, L.; Giannotti, M.I.; Sanz, F. Structural impact of cations on 

lipid bilayer models: Nanomechanical properties by AFM-force 

spectroscopy. Molecular Membrane Biology 2014, 31, 17.

Lipids: DOPC, DOPS, DOPC/PS

● ~75ug lipid suspended on top of 1M KCl 7.0pH 

buffer solution (~1mL total cell volume)

● 1% hexadecane- to make things “sticky” at ~13um

● ~30-60min equilibration period

○ Foil shielding, stirring, voltage scan

● Divalent ion series: 100uM-100mM in 5-25uL 

increments

○ 2x concentration KCl buffer of equal volume 

added to opposite side as control

○ Contents weighed for more accurate 

concentration

Zwitterionic (net neutral) Anionic (charged)

cis

trans
Experimental Results: Asymmetric membrane

Offset voltage (mV) Standard deviation

-62.148 0.050

Asymmetric membrane: DOPC (cis), DOPS (trans)

Charge density (cis):

−62mm ≈ 1ee−/0.66nn 2

-

Trans

+

Cis

DOPC

(Net neutral)

DOPS

(Charged)

- - - - -

-

-

-

DOPS vs DOPC: high salt
Experimental Results

DOPS: high salt

[CaCl2] (mM)

Capacitance (pF)

DOPS: high salt

Slope (pA/mV)

Time (s)

Trial 1: 

Trial 2: 

Trial 3: 

Charge density (cis):

−62mm ≈ 1ee−/0.66nn 2

Voltage Offset (mV)

Asymmetric membrane: DOPC (cis), DOPS (trans)

Offset voltage (mV) Standard deviation

-62.148 0.050
Experimental Results

Trial 1: 

Trial 2: 

Trial 3: 

Time (s)

Slope (pA/mV) Capacitance (pF)

DOPC: high salt

[CaCl2] (mM)

Trial 1: 

Trial 2: 

Trial 3: 

Charge density (cis): −62mm ≈ 1ee−/0.66nn

2

Voltage Offset (mV)

Asymmetric membrane: DOPC (cis), DOPS (trans)

Offset voltage (mV) Standard deviation -62.148 0.050
Experimental Conclusions

DOPC (uncharged): 

DOPS (charged): 

Charge density (cis):

−62mm ≈ 1ee−/0.66nn 2

DOPC vs DOPS: high salt

DOPS: high salt

Asymmetric membrane: DOPC (cis), DOPS (trans)

Offset voltage (mV) Standard deviation

-62.148 0.050
Discussion & Future Experiments

Pros of this set-up:

● We can ‘see’ everything!

Cons of this set-up:

● We can see everything

Future experiments:

● Phospholipases and mutant forms

○ Study integral and peripheral membrane proteins

● Try more concentrated solutions of divalent ions

● Optimize membrane formation at a lower salt concentration

○ Study less concentrated solutions of divalent ions

Lingering questions:

● What are the markers of a stable membrane at high salt?

○ At low salt we can look at the slope

● What are the effects of hexadecane?

○ At the torus and bilayer interior

Complementary technique:

Integrate with other biophysical tools to get the complete picture1
ACKNOWLEDGEMENTS

Dr. David Hoogerheide

• Mentor, NCNR Project Leader

Dr. Joseph Dura & Dr. Julie Borchers

• NCNR SURF Directors

Dr. Brandi Toliver

• SURF Program Director

Lisa Fronczek

• EL SURF Director, Office of Advanced 

Manufacturing

Fellow NCNR SURFers!

• Shark Tank, Submarine, and Fish Bowl
Emma Rogers

NIST Center for Neutron Research

Neutron-Condensed Matter Science Group

SURF Colloquium August 2019