Membrane technologies for
channel protein-based sensing
Schmidt Group
UCLA Department of Bioengineering
schmidtlab.seas.ucla.edu
Channel proteins are natural sensors
• Channel proteins are typically 5-15nm in
size and inhabit lipid bilayer membranes
• There is a water-filled channel which
runs down the center of the protein
• Channel proteins can exhibit charge or
size selectivity due to the presence of
charged or steric constrictions within the
channels
• Natural sensing: Applied voltage or
binding of ligands to the channel can
induce conformational changes which
gate its conductance
Probing channel proteins
experimentally
• The conductance state of the channel
can be probed electrically by measuring
ionic currents flowing through the
channel in response to an applied
voltage
– The lipid bilayer membrane is electrically
insulating, with resistance typically
greater than 10GW (conductance <
100pS) for membranes hundreds of mm in
size
– The conductance of a typical channel
protein is 5-1000pS, which gives rise to a
.5-100pA ionic current in response to a
100 mV applied potential
– Binding foreign material to the channel
interior can significantly block current
High-throughput drug screening of
channel proteins
Source: Meyer et al. Assay and drug development technologies. 2:507-514 (2004)
Sensing using engineered proteins
His4
•
•
The Bayley group has engineered a
large number of mutants of the
bacterial pore a-hemolysin to contain
different binding sites within the
channel
Example: cation binding sites using
His4 bind to a number of different
cations, each distinguishable through
examination of the magnitude and
temporal signature of the ionic current
it blocks
•
Stochastic Sensing
– The occurrence and duration of each
binding event is random, but
statistically show the concentration of
analyte in solution, as well as its
affinity for the binding site
– Measurements occur on time scales
on the order of minutes or less
Zn2+
Co2+
Both
Bayley and Cremer, Nature 413, 226 (2001)
Fast single molecule
nanopore DNA sequencing
•
Initial work by Kasianowicz (PNAS
1996) looked the current through
aHL modulated by the passage of
polymers of RNA and DNA through
it
•
Since the membrane is highly
insulating and the rest of the
solution highly conductive, there is
a huge electric field in the pore
which drives the charged polymer
through very rapidly
– All 100 of these bases traverse the
pore in <2ms, about 10-20us/base
(Akeson Biophys J 1999)
– We need to measure pA currents in
high bandwidths
CA
Obstacles toward the technological
exploitation of channel proteins
• We can direct the self-assembly of lipids to
create membranes with a planar or spherical
geometry
• Although vesicles are generally more robust
than planar membranes, the planar geometry
ensures that we have access to both sides of the
membrane for full control of the electrical and
chemical environment of the protein
• The primary hurdle in the creation of practical
devices using channel proteins is the short life
and fragility of planar membranes
Freestanding planar lipid bilayer
membrane fabrication
Figure from Mayer, M., et al., Biophys. J. 85(4):2684-2695. (2003).
“Painted” membranes
(Black Lipid Membranes)
Figures from White in Ion Channel Reconstitution
“Painted” membranes (Mueller-Rudin)
(Black Lipid Membranes)
Membranes are short-lived, ~12 hours
“Solvent-free” membranes
(Montal-Mueller method)
Figures from White in Ion Channel Reconstitution
Langmuir films of lipid form at the air-water interface and form a
membrane when the water level is raised beyond a hole that has
had a suitable pretreatment with a lipid/organic solution
Not really solvent-free. Membranes are short-lived, ~12 hours
Addressing these shortcomings
• Freestanding planar membranes are metastable and have intrinsic lifetime limits
• Fixes:
– (Get rid of the membrane and protein channel?)
– Substitution of lipid with biomimetic polymers
– Supported membranes
• Membranes in contact with solid surfaces
• Membranes in contact with porous (gel) surfaces
– Automated microfluidic formation
Lipid substitutes
• Amphiphilic polymers
– E.g. pluronics
– There is a lot of interest in manipulating
amphiphilic polymers to self-assemble into
a range of macromolecular structures for
drug delivery and other applications
– Di-block copolymers (Bates, Discher)
– Di-block copolypeptides (Deming)
– Tri-block copolymers (Meier)
• A number of experiments creating
biomimetic membranes (9nm thick!) formed
from these polymers containing protein
• The hydrophilic PMOXA groups also have a
methacrylate group on the end, enabling
them to be crosslinked
– Increases vesicle lifetime and robustness
Nardin et al., Langmuir 16 1035 (2000)
Discher, Science 284, 1143 (1999)
Channel proteins can be functionally
incorporated into polymer vesicles
•
Meier incorporated a number of
channel and pore-forming proteins
(OmpF, LamB, Alamethicin, etc.) and
demonstrated that these proteins
retain their ability to form channels as
well as their native properties
– Lambda phage docking with LamB
incorporated into polymer vesicles
– OmpF gating in the presence of a
Donnan potential
•
Creation of asymmetric ABC triblock
copolymers with controlled A and C
blocks can control the orientation of
inserted protein (Stoenescu,
Macromol. Biosci. 2004, 4, 930)
Graff PNAS 99, 5065 (2002)
Planar polymer membranes
•
All of the work above was done with protein
incorporation into polymer vesicle solutions
and the results measured with bulk
fluorescence or spectroscopy
– Although we can see that the protein can insert
and function in the membranes, we still don’t
know if the membrane environment is having
some effect on the protein
– Measurement at the single molecule level
sheds some light on this
•
•
Electrical transport measurements of OmpF
and maltoporin inserted into planar polymer
membranes show protein activity at the few
molecule level (~27 trimeric pores)
Following protein insertion, membrane
showed conductance decrease upon
polymerization (B), then further decreases
upon the addition of sugar (arrows)
Nardin Langmuir 2000, 16, 7708
Polymer membrane lifetime and single
molecule transport measurements
• Using a shorter version (5-6 nm) of Meier’s PMOXAPDMS-PMOXA polymer (9-31-9, previous 15-68-15) we
created freestanding membranes on conventional Teflon
substrates as well as micromachined orifices in Si to
measure membrane lifetime
– Average lifetime of polymer membranes is >50% greater than
that of lipid
• Commonly exceeds 24 hours
• Obtained a 4 day polymer membrane on a 150um Si hole
– Resistance typically exceeds 100GW, and is over 30x that of lipid
membranes on average.
• Also probed the effects of the polymer environment on
protein insertion and function
Single molecule measurements of
α-hemolysin in
DPhPC
polymer
Conductance: 0.79 nS
Conductance: 0.72 nS
Summary of our single molecule
measurements
• Other proteins incorporated
and measured at the single
molecule level (for thin
polymer- for thicker polymer,
OmpG inserted, but not
aHL!):
– OmpG (80 mV applied)
– MscL (16 mmHg applied)
– Alamethicin
Conductance of alamethicin in Polymer 40mV 2008_008
7
5
4
3
2
1
Time (s)
2.05
1.95
1.85
1.76
1.66
1.56
1.46
1.37
1.27
1.17
1.07
0.98
0.88
0.78
0.68
0.59
0.49
0.39
0.29
0.2
-1
0.1
0
0
Conductance (nS)
6
Stabilizing membranes with a solid surface:
Tethered lipid bilayer membranes
• Can create these structures in two ways
1) Must covalently attach lipid to solid surface (silane or thiol SAMs)
2) Non-specifically absorb lipid onto surface through vesicle fusion
• These membranes generally show outstanding
robustness and can withstand dehydration and
rehydration, although it is unknown whether small
defects develop (e.g., ~nS in conductance)
• Any protein incorporated into the tethered membranes
must be spaced from the surface to avoid any
deleterious interactions with it
Sensors using
tethered BLMs on
gold
•
We cannot perform any DC
measurements because the
bottom surface, if conductive,
is usually gold and therefore
can only function capacitively
•
First experiment of this kind
was Cornell et al. Nature 387
580 (1997)
– Used gramicidin
• Dimeric ion channel, whose
conductance would be
disrupted when one half of it
would be pulled away to bind
to an analyte
– Looked at complex
conductance as a function of
time as analytes were
introduced
Tethered BLMs
on gold
• Using impedance spectra
for capacitively probed
membranes
– Complicated to interpret
– Need to model
capacitance of electrode,
double layer, and
membrane as well as the
resistance of the
membrane, incorporated
ion channels, and the
surrounding solution
– Look at real and
imaginary components of
impedance as a function
of frequency
Advances in tBLMs
• If the resistance of the tBLM is sufficiently large, there can be a large
RC time constant for the ions in the double layer (between the
membrane and the electrode) to deplete
• When this happens, pseudo-DC (.01 Hz or slower) measurements of
ion channels in the membrane are possible
• As of yet, none of these resistances are high enough to show single
channels, but patterning the surface to limit the membrane area can
cut down on membrane resistance and there is a path to single
channel current measurements
– This would be a significant advance as these membranes are typically
stable, long-lived and the substrates are easily integrated into a device
configuration
– Duran group reported these results at recent ACS meeting this week
Porous membrane supports using
gels
• By surrounding a BLM with an
agarose gel on one or both sides,
mechanical or other interactions
with the gel may alleviate various
membrane failure modes
• Early attempts at gel supported
membranes used standard
techniques to paint membranes on
a Teflon partition and then bring
gels in contact with membrane on
either side
• Gel allows mechanical support
while allowing ions and other
analytes to diffuse to and from the
membrane
Gel supported membranes
• Ide and Yanagida formed bilayer membranes
on agarose gels using applied pressure, but
instead used the relaxation of a compressed
material to apply negative pressure to the
bottom of the membrane, causing the
membrane to immediately thin out
– Membrane formed in < 10s
• Measured a number of proteins at the single
channel level
Ide and Ichikawa, Biosensors and
Bioelectronics 21 (2005) 672
In situ gel-encapsulated
membranes
• In recent work, we have
created Mueller-Rudin
DPhPC lipid membranes in
the presence of a hydrogel
precursor solution
• Polymerization of the gel
solution encapsulates the
membrane within it, forming a
mold of the membrane in
almost continuous contact
with it
PEG-DMA (1 kDa)
O
O
O
n
O
hν
photoinitiator
O
O
O
O
O
O
O
O
n
O
O
O
n
O
O
O
O
O
O
n
O
O
n
O
n
O
O
O
O
n
In situ gel-encapsulated
membranes
• Initial observations
– Gel polymerization also accelerated
membrane thinning and resulted in a
stable solvent annulus at the
membrane periphery
– Encapsulated membranes have longer
lifetimes, and enabled measurements
of single channels for days
Jeon, Malmstadt, Schmidt, JACS, 128, 42 (2006)
In situ gel-encapsulated
membranes
• Mechanical perturbation- shaking/hitting
the air table
16
In situ gel-encapsulated
membranes
• Mechanical perturbation- poking the gel
15
Mechanical perturbation
• Facilitating membrane formation by
manipulating the gel
17
Susceptibility of membrane to
pressure (1)
• Experiments
– 500 um hole, 200x microscope
– 7.5% (w/v) PEG-DMA hydrogels with 1% Irgacure, 400W (5 min. polymerization)
exp1
oil
exp2
gel
water
gel
water
control
oil
exp3
gel
gel
Susceptibility of membrane to
pressure (2)
• Experiment 1
– 500 um hole, 200x microscope
– 1ml added at once  membrane fails
water
3
control
Susceptibility of membrane to
pressure (3)
• Cont’d
– 1ml added at once and then removed  membrane recovers
water
4
control
Susceptibility of membrane to
pressure (4)
• Cont’d
– 50 ul added at each point
water
control
membrane area (mm2)
Membrane failed at
higher pressure
2.00
1.50
1.00
0.50
0.00
0
0.1
0.2
0.3
0.4
volume added (ml)
0.5
Susceptibility of membrane to
pressure (5)
• Experiment 1
– 500 um hole, 200x microscope
– 7.5% (w/v) PEG-DMA hydrogels with 1% Irgacure, 400W (5 min. polymerization)
exp1
oil
120 ul
gel
120 ul
120 ul
Susceptibility of membrane to
pressure (6)
• Experiment 2
– 500 um hole, 200x microscope
– 7.5% (w/v) PEG-DMA hydrogels with 1% Irgacure, 400W (5 min. polymerization)
membrane area (mm2)
exp2
water
0.250
gel
0.200
0.150
0.100
0.050
0.000
0
0.5
1
1.5
2
volume added (ml)
2.5
Susceptibility of membrane to
pressure (7)
• Experiment 3
– 500 um hole, 200x microscope
– 7.5% (w/v) PEG-DMA hydrogels with 1% Irgacure, 400W (5 min. polymerization)
120 ul
oil
exp3
120 ul
gel
120 ul
120 ul
120 ul
gel
The gel traps solvent within the
membrane (1)
•
1 sec time lapse  30 frames x 13 sec  390 sec
exp
gel
control
5
Real time
1sec time lapse
6
The gel traps solvent within the
membrane (2)
7
exp
gel
gel
Robustness to applied voltage
• Experiments
– 500 um hole, 200x microscope
– 1 sec step function (with 5 mV increments)
exp1
exp2
+
gel+
+
+
-
exp3
+
+
+
+
control
+
+
+
+
-
- gel
-
+
gel+
+
+
gel
-
Robustness to applied voltage(2)
7
6
control
5
nA
4
3
+
+
+
+
2
1
0
0
0.1
0.2
0.3
0.4
0.5
V
Bigger annulus, broke at 245mV
8
-
Smaller annulus, broke at 215mV
9
Robustness to applied voltage(3)
exp1
0 ~ 500 mV (with 5mV increments, 1sec each)
10
+
gel+
+
+
-
Robustness to applied voltage(4)
exp1
+
gel +
+
+
0 ~ 500 mV (with 5mV increments)
11
-
Poking the gel after electro-compression
12
Robustness to applied voltage(5)
0 ~ 500 mV (with 5mV increments), broke at 215mV
13
exp2
+
+
+
+
- gel
-
Robustness to applied voltage(6)
0 ~ 500 mV (with 5mV increments), broke at 375mV
14
exp3
+
gel+
+
+
gel
-
Possible slowing of DNA translocation
by the encapsulating gel
• 150 base pair singlestranded DNA was added
atop the hydrogel.
• The hydrogel appears to
significantly slow the DNA
diffusion through the mesh
to the nanopore.
• Blockades as slow as ~1
ms/base were detected.
Planar lipid bilayer fabrication by solvent
extraction in a microfluidic channel
Design criteria for an automated lipid bilayer
fabrication device
• Simple: no need for operator intervention or human
monitoring
• Fast: new membranes can be formed in a matter of
minutes
• High-quality membranes: gigaohm seals for ion channel
research and applications
• Ability to measure single-molecules
PDMS solvent “incompatibility”
Log(PDMS swelling ratio)
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
Cross-linked
poly(dimethylsiloxane)
(PDMS) elastomer
he
n-
an
pt
e
n
pe
n
ta
e
n
be
n
ze
e
c
o
or
l
h
rm
fo
e
no
a
th
l
w
at
t
er
er
uo
Fl
After Lee et al., Anal. Chem.
75(23):6544-6554 (2003).
rin
Membrane formation by solvent
extraction: Principle of operation
Aqueous
phase
Lipid
solution
Aqueous
phase
Device design
Membrane isolation valves
Aqueous
inlet
Outlet
Fluidic channels
Ag/AgCl
Pneumatically
electrodes
actuated valve
(100 µm width)
channels
(200 µm width)
Lipid Peristaltic pumps
solution
inlet
Experimental apparatus
Applied voltage in
V
dV
I=C
dt
Amplifier
Measured current out
I
+
+
+
+
+
+
+
-
CCD
C 
 0 A
d
Fluid compositions
Aqueous phase
Organic phase
•
•
•
•
1 M KCl
5 mM Hepes
pH 7.0
•
•
Solvent composed of 1:1 ndecane: squalene
Lipid: 0.025% (w/v)
diphytanoylphosphatidylcholine
(DPhPC)
50 ppm perfluorooctane
O
O
N
+
O P O
O
O
O
O
Lipid solution droplet formation
Lipid solution
stream
100 µm
Solvent extraction
Lipid solution droplet
100 µm
5x replay speed
Membrane capacitance during
solvent extraction
16
10
5
0
14
Input voltage
Output current
12
Capacitance (pF)
Voltage (mV)/Current (pA)
15
10
dV
I=C
dt
8
6
C 
4
-5
2
-10
 0 A
d
0
0
0 50
100
2
Time (ms)
150 4
200
6 250
Time (seconds)
8
10
12
Measured current (pA)
Observed membrane resistances of 50100 GΩ
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1.6
-1.8
-2
-100
-80
-60
-40
-20
0
20
40
60
Applied voltage (m V)
This membrane has a resistance of 91 GΩ
80
100
Insertion of a-hemolysin into a
microfluidic membrane
3
2.5
2
1.5
1
0.5
0
0
5000
10000
15000
Time (ms)
2000
Count
Conductance (nS)
3.5
0
-0.20.0 0.2 0.4 0.60.8 1.0 1.2 1.4 1.6 1.82.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
Conductance (nS)
Design criteria for an automated lipid
bilayer fabrication device
• Simple: no need for operator intervention or human
monitoring
– Valves are computer controlled
• Fast: new membranes can be formed in a matter of
minutes
– True, but lifetime is limited; 15 minutes for full integrated device,
45 minutes for PDMS solvent extraction only
• High-quality membranes: gigaohm seals for single
molecule ion channel research and applications
– Unique geometry results in minimal background capacitance,
resulting in very low noise measurements
Future work: Hydrogel encapsulation
Optimize Organic phase
•
•
Solvent composed of 1:1 ndecane: squalene
Lipid: 0.025% (w/v)
diphytanoylphosphatidylcholine
(DPhPC)
50 ppm perfluorooctane
•
•
•
Lipid concentration
Solvent choice and concentrations
Fluorocarbon
•
Mask channel and initiate
photopolymerization.
Future work: Ion channel assay
platform
Acknowledgements
• Schmidt Group
–
–
–
–
–
Tae-Joon Jeon
Noah Malmstadt
Jason Poulos
Robert Purnell
Denise Wong
• Funding provided by DARPA and ACS-PRF