Technical Background

Tethered Membrane Technology

A tethered bilayer lipid membrane (t-BLM) is a lipid bilayer that is chemically anchored to a gold electrode providing a stable model system for the study of membrane biophysics.

Lipid Gold Chemistry

The formation of a t-BLM occurs in four steps as shown in Figure 1.

SDx provides lipid coated gold electrodes (steps 1 and 2). The researcher forms a bilayer with the addition of second layer lipids (steps 3 & 4).

To coat the gold electrodes lipids are chemically bound to the gold substrate using a benzyl disulphide (Figure 2a) which is more stable against oxidation than a simple thiol and also provides optimal spacing of the ethylene glycol linkers (Figure 2b) to eliminate conduction limitation of ionic species within the reservoir region between the BLM and the gold electrode.

Figure 1. Formation of a t-BLM.

step 1: gold immersed in ethanol-lipid solution step 2: lipids reacts with gold and tethers to surface. step 3: addition of non-tethered lipids in typically ethanol. step 4: rinse with PBS to form t-BLM.

Figure 2. Tethered lipids

(a) tethered phytanyl bis-tetra-ethyleneglycol

(b) hydroxyterminated-bis-tetra-ethyleneglycol

A range of slide coatings with different tether densities is available. They are described by Tm(x) where x= the ratio expressed as the % of the tethered fraction that penetrates the bilayer and forms the inner layer leaflet of the t-BLM (see figure 2(a) above), relative to the tethered fraction that terminates in an OH group at the surface of the inner leaflet of the bilayer (see figure 2(b) above), The advantage of a range of % tethers within the inner leaflet of the t-BLM is that it permits experiments to be designed where ion channels or polypeptides can be optimally incorporated into the bilayer.

The average spacing of the t-BLM tether (figure 2(a) above) can be used as an approximate guide to the molecular weight of moieties to be accommodated within the untethered fluid lipid fraction of the t-BLM. The basis of this estimate is a simple volume calculation based on the molecular weight of the membrane associated component and an assumed thickness of 4nm for the t-BLM. All slide coatings ranging from 0 < x < 100 will form bilayers however a trade-off exists between the stability and ionic leakage of the t-BLM and the volume of fluid lipid bilayer available to incorporate additional membrane associated species such as an ion channel.

When designing an experiment we recommend using the highest tether density possible for a particular molecular weight species being incorporated into the t-BLM. The molecular weights are calculated according to this simple geometric model. Of course this molecular weight may only be a fraction of the molecular weight of a whole ion channel or other membrane associated compound under study.

Forming a Bilayer

In preparation for use the gold electrodes supplied are rinsed with ethanol, then dried. A further ethanol solution containing mobile outer layer lipids (figure 3) is added and the slide rinsed with PBS spontaneously forming a t-BLM.

Figure 3. Mobile layer lipid species.

(a) diphytanyletherphosphatidylcholine

(b) Glycerodiphytanylether

The mole ratio of the two components is 70:30 for compounds 3(a):3(b). The reason for the mixture is to achieve an average area per molecule at the hydrocarbon-aqueous interface that is commensurate with the average area per molecule at the centre of the bilayer. This ratio may require adjustment for different included species within the membrane. This ratio is best determined empirically for any particular included species.

Measuring Conductance

The SDx Impedance Reader in its most basic operation reports the conductance across a tethered bilayer membrane. With this conductance measure the properties of the membrane itself or the change in conductance in response to the insertion of ion channels can be studied without a requirement for an in-depth understanding of Impedance Spectroscopy.

Tethered Membranes because of the limited ions available in the reservoir use AC current rather than the standard DC current used in patch clamp equipment. The SDx membranes have a area that is an order of size greater patch clamp and therefore has a seal of a mega Ohm scale rather than the giga Ohm scale of patch clamp. Although single channel measurements are not available the tethered membrane principle provides robust with sensitivity and flexibility of technique.

For more sophisticated electrophysiology studies the SDx Reader offers additional capability. Both phase and impedance are calculated at each frequency providing a range of metrics to determine the electrical properties of the membrane. These include the admittance at the frequency for minimum phase (YminP: provides membrane conductance), and the admittance at 1kHz (Y1kHz from which the membrane thickness may be determined).

The equivalent electrical circuit of a tethered membrane is shown in Figure 4.

Ions in the saline solution create two capacitors: C1 (~0.5µF/cm2), due to the insulating barrier of the membrane; and C2 (~3.5µF/cm2,) due to ions crowding at the gold surface. A 10 to 50mV swept frequency a.c. excitation in the range 1 to 1000HZ is applied between the gold electrode and a large area gold return electrode on the facing surface of the flow cell cartridge. Current passing through C1 and C2 is measured across the reference resistor R. The voltage across R due to the current flow is amplified in amplifier A and the output supplied via a USB port to a computer for processing and analysis.

Figure 4. Electrically Equivalent Circuit

Software packages are provided which further permit a calculation of the kinetics of admittance changes of the membrane when channels are introduced or when ligands are added which block the channel.

Inserting Ion Channel Proteins and Peptides

a. Ionophore (channel or carrier) incorporation

The incorporation of Ionophores, into a membrane can occur by many mechanisms the major approaches being:

Spontaneous insertion for Ionophores such as peptides like Valinomycin or protein ion channels such as the Chloride Intracellular Ion Channel (CLIC), or the bacterial ion channel ?-Haemolysin into a preformed t-BLM.
Detergent assisted insertion for Ionophores such as the mechanosensitive channel (MscL). PBS rinsing of coated electrodes exposed to detergent dispersions of MscL forms t-BLMs containing MscL.
Proteoliposomal fusion for Ionophores such as the voltage dependent anion channel (VDAC) where the t-BLM forms from liposomes containing the VDAC ion channel.
Fusion of Proteoliposomes containing an ion channel with a preformed t-BLM.

In each case empirical optimization of the insertion conditions is advised. The % tethering of the t-BLM may be adjusted to optimize the balance between the stability and seal of the membrane against the the amount of material that may be inserted. In the case of liposomal fusion to form a t-BLM, the fusion process requires >Tm(50) coated slides to stimulate the fusion process from aqueous liposomal dispersion. This may be reduced to lower Tm(x) values by using detergent solutions to increase the fusion rate of the liposomes.

References

Tethered Membranes

Title: Making lipid membranes even tougher
Author(s): Prashar J, Sharp P, Scarffe M, et al.
Source: JOURNAL OF MATERIALS RESEARCH Volume: 22 Issue: 8 Pages: 2189-2194 Published: AUG 2007

Title: Tethered bilayer membranes containing ionic reservoirs: Selectivity and conductance
Author(s): Krishna G, Schulte J, Cornell BA, et al.
Source: LANGMUIR Volume: 19 Issue: 6 Pages: 2294-2305 Published: MAR 18 2003

Title: Tethered bilayer membranes containing ionic reservoirs: The interfacial capacitance
Author(s): Krishna G, Schulte J, Cornell BA, et al.
Source: LANGMUIR Volume: 17 Issue: 16 Pages: 4858-4866 Published: AUG 7 2001

Title: Tethered-bilayer lipid membranes as a support for membrane-active peptides
Author(s): Cornell BA, Krishna G, Osman PD, et al.
Source: BIOCHEMICAL SOCIETY TRANSACTIONS Volume: 29 Pages: 613-617 Part: Part 4 Published: AUG 2001

Title: Tethered lipid bilayer membranes: Formation and ionic reservoir characterization
Author(s): Raguse B, Braach-Maksvytis V, Cornell BA, et al.
Source: LANGMUIR Volume: 14 Issue: 3 Pages: 648-659 Published: FEB 3 1998

Small peptides

Title: A tethered bilayer sensor containing alamethicin channels and its detection of amiloride based inhibitors
Author(s): Yin P, Burns CJ, Osman PDJ, et al.
Source: BIOSENSORS & BIOELECTRONICS Volume: 18 Issue: 4 Pages: 389-397 Published: APR 2003

Biosensor

Title: A biosensor that uses ion-channel switches
Author(s): Cornell BA, BraachMaksvytis VLB, King LG, et al.
Source: NATURE Volume: 387 Issue: 6633 Pages: 580-583 Published: JUN 5 1997

Title: The ion channel switch biosensor
Author(s): Woodhouse G, King L, Wieczorek L, et al.
Source: JOURNAL OF MOLECULAR RECOGNITION Volume: 12 Issue: 5 Pages: 328-334 Published: SEP-OCT 1999

Title: Gramicidin ion channel-based biosensors: Construction, Stochastic dynamical models, and statistical detection algorithms
Author(s): Krishnamurthy V, Luk KY, Cornell B, et al.
Source: IEEE SENSORS JOURNAL Volume: 7 Issue: 9-10 Pages: 1281-1288 Published: SEP-OCT 2007

Title: Kinetics of the competitive response of receptors immobilised to ion-channels which have been incorporated into a tethered bilayer
Author(s): Woodhouse GE, King LG, Wieczorek L, et al.
Source: FARADAY DISCUSSIONS Volume: 111 Issue: 111 Pages: 247-258 Published: 1998

Title: Rapid detection of influenza A virus in clinical samples using an ion channel switch biosensor
Author(s): Oh SY, Cornell B, Smith D, et al.
Source: BIOSENSORS & BIOELECTRONICS Volume: 23 Issue: 7 Pages: 1161-1165 Published: FEB 28 2008

Title: Ion Channel Biosensors Part I Construction Operation and Clinical Studies
Author(s): Krishnamurthy V, Monfared SM, Cornell B
Source: IEEE Transactions on Nanotechnology May 2010 Volume 9 No 3: Pages: 303-313 Published: MAY 2010

Title: Ion Channel Biosensors Part II Dynamic Modelling, Analysis and Statistical Signal Processing
Author(s): Krishnamurthy V, Monfared SM, Cornell B
Source: IEEE Transactions on Nanotechnology May 2010 Volume 9 No 3: Pages: 313-322
Published: MAY 2010

Links

Tethered Membrane Seminar by Dr Bruce Cornell 2010
http://www.youtube.com/watch?v=PofLZSJ71nU

Schematic of the Principle of the Ion Channel Switch
http://www.youtube.com/watch?v=6Ti83oO2ml4

SDX Research