Lipid Bilayers at Gel/Gel Interface for Ion Channel Recordings

We have developed a practical method to produce durable artificial lipid bilayers using a hydrogel-hydrogel interface for ion channel measurements. Bilayers were formed by forcing a hydrogel-bead into contact with the hydrogel layer (hydrogel plate) in a lipid solution. The immediate formation of a bilayer was observed (< 1 s). This allows channel recordings to be repeated more easily and quickly as compared to conventional methods. Currents of various types of channel such as gramicidin, hemolysin and BK-channel have been recorded. Our channel property results mirrored those of other techniques and were reproducible. Hydrogel solutions containing gramicidin were extremely stable and could be used months after preparation for bilayer experiments. [DOI: 10.1380/ejssnt.2008.130]


I. INTRODUCTION
Biosensors have been used to characterize the physiological and pharmacological properties of various membrane channels.Recently, advancements in artificial bilayer techniques have allowed for even more elaborate studies of these channels.However, the fragility makes the application of artificial bilayers difficult.A great deal of skill and practice is necessary in order to acquire practical data from these channel recordings meaning that although the information acquired is extremely useful, the labour is laborious and frustrating.Thus several proposals have been made for the construction of durable bilayers [1][2][3][4][5].We have developed a practical method to produce such durable bilayers for channel measurements.This method allows experiments to be repeated more easily and quickly than other methods.We have previously reported the fundamental technique by constructing an agarose gel/aqueous solution interface bilayer [6,7].Here, we report the next stage by describing the construction of a hydrogel-hydrogel interface bilayer combined with measuring the corresponding ion channel currents.

A. Hyrogel bead formation
Hydrogel-beads were made by heating hydrogel (0.5-2 % agarose VII, 1 % κ-carragenan or Sepharose 4B, 150 mM KCl, 10 mM Hepes, pH 7.2) in an oven, mixing the dissolved hydrogel with a lipid solution (20-50 mg/mL asolectin or DOPC in decane or hexadecane) and finally stirring the solution by a vortex mixer.The suspension Figures 1(B) and (C) show hydrogel beads made at a tip of a fine tube.Figure 2 shows how to generate a hydrogel bead at polyethylene or glass tube.Hydrogel was dissolved by heating and then inserted into the tube.Pressure applied at one end formed a tiny bead at the tip of the tube (Fig. 2(A)).The bead was placed onto the interface by inserting the tube into the lipid layer.However, sometimes these beads were too large for experimental purposes.To create even smaller ones, a tube filled with dissolved gel was brought into contact with individual hydrogel-beads resulting in even smaller beads forming at the tip (Fig. 2(B)).

C. Bilayer formation
In Fig. 1(A), a layer of hydrogel was placed onto a culture dish followed by a layer of lipid solution (20-50 mg/mL phospholipids in n-decane or hexadecane) placed on top.The mixture was left for several minutes when the lipid solution containing hydrogel-beads was added.An Ag-AgCl electrode controlled by a micromanipulator was used to push a bead through the lipid layer to the hydrogel-lipid interface.By forcing a hydrogel-bead into contact with the hydrogel layer (hydrogel plate), the immediate formation of a bilayer was observed.This was indicated by the formation of a bulky annulus that formed around the bilayer (Fig. 1(C)).Bilayers occurred almost instantly (< 1 s).The condition shown in Fig. 1(A) with the bead coming into contact with the hydrogel plate can be confirmed by measuring the membrane capacitance.

D. Electrical measurements
The Ag-AgCl electrode inserted into the hydrogel was attached to a patch clamp amplifier (CEZ2400, Fig. 2) in order to measure the channel current potential.Virtual ground was defined at the hydrogel layer.The signal was filtered at 2 kHz, sampled at 10 kHz, digitized and stored onto a PC.

III. RESULTS AND DISCUSSIONS
In Fig. 1A, hydrogel was poured into a culture dish to form a layer several millimeters thick.Over this hydrogel layer, a lipid solution (20-50 mg/mL phospholipids in n-decane, hexadecane or squalene) was poured and left to sit for several minutes allowing for the two solutions to separate with the lipid layer on top.Next, we heated a lipid solution containing hydrogel-beads and allowed it to cool in order to become a gel.This gel was added to the top of the lipid layer.In order to form a bilayer, the beads were slowly pushed downward to contact the hydrogel layer (hydrogel plate) by an Ag-AgCl electrode using a micromanipulator.However, the electrode prevented the formation of the bilayer from being directly observed.Therefore, for this purpose, the electrode was substituted with a glass pipette or polyethylene pipette filled with the same gel (Fig. 1(B) and (C)).The pressure of the tip on the hydrogel layer pushed away any excess lipid solution to form an almost instant bilayer.Visually, a bilayer was identified by the presence of a bulky annulus (Fig. 1(C)), the result of pushing the hydrogel-beads onto the hydrogel layer.The diameter of the bilayer was positively dependent on the pressure applied by the tube.
The choice of solvent is significant.If hexadecane or squalene is used, the gel beads must be incubated in the lipid solution for 15 minutes before coming into contact with the hydrogel layer at the bottom of the mixture.Otherwise, the bilayer will gradually become unstable.This is not a concern when using decane.Nevertheless, bilayers could be made for all three solvent.The thickness separating the two layers also depends on the solvent.Decane results in the greatest separation meaning that these bilayers are the most stable.
Figure 3 shows the results of a gramicidin channel incorporated into our bilayers.Gramicidin is an ideal choice because it can be introduced randomly into the hydrogel layer and yet will still form channels in the bilayer at the hydrogel-hydrogel-bead interface.The electric properties of the gramicidin channels formed in our artificial bilayers resemble those from self standing bilayer experiments indicating the channels function like those developed using other methods.The electrode was moved up ( ) and down ( ) as indicated by the arrow heads.Lifting the electrode caused the current level to reach zero, coinciding with the disappearance of the bilayer.Lowering the electrode resulted in current step increases indicating the formation of a new bilayer and incorporation of the channels.The gel plate contained 0.2 µg/mL gramicidin D. The membrane potential was held at −100 mV.Furthermore, various types of gel beads (Sepharose 4B, carrageenan, water-drops) could be used without affecting the preparation of the bilayers as shown in Fig. 4(A)-(C).
An additional, unexpected result was seen in the gramihttp://www.sssj.org/ejssnt(J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) cidin experiments.With immaculate care, one could generate a viable channel without pressing a hydrogel-bead onto the hydrogel layer.Rather, directly pressing the tip of the Ag-AgCl electrode to the layer sometimes proved sufficient (Fig. 4(D)).We surmise that water molecules adhered to the tip due to polar interactions creating the aqueous environment necessary for bilayer formation.However, this result was not consistently achieved and although it potentially makes the process of bilayer formation easier, it needs to be further developed before being recommended.
We were also able to successfully make bilayers that incorporated α-hemolysin [8] or lysenin [9]. Figure 5(A) shows the electrical current from one of our artificial bilayers incorporating α-hemolysin.Single channel currents showed no gating characteristics (Fig. 5(B)).In Fig. 5(C), the channel was blocked using βCD.Similar measurements were also attained in bilayers that incorporated lysenin, although sphngomyelin had to be included in these cases (Fig. 6).
To this point, our examples have not involved channels from living tissue, which are of greater interest.However, we have also been able to incorporate these channels into our bilayers without compromising function.Suspensions from bovine tracheal vesicles were mixed into the lipid layer from Fig. 1.As before, hydrogel-beads were pressed onto the hydrogel layer resulting in bilayers, which were accompanied by simultaneous current signals (Fig. 7).Removing the pressure on the bead eliminated the current signal indicating that the current was a consequence of the channel embedded within the bilayer.These results were reproduced with regularity, strongly suggesting that the bilayer occurred between the hydrogel-bead and hydrogel layer.Because these are currently only preliminary studies, the ideal conditions have not been established.Nevertheless, because our artificial bilayer technique is easier http://www.sssj.org/ejssnt(J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) e-Journal of Surface Science and Nanotechnology to implement than the self standing bilayer method, our method will likely prove to be preferable for channel analysis.

IV. CONCLUSIONS
Overall, we have successfully developed a method to produce artificial bilayers that is easier and faster than traditional ones.Our channel property results mirrored those of other techniques and were reproducible.Furthermore, hydrogel solutions containing gramicidin were extremely stable and could be used months after preparation for bilayer experiments.In future work, we will report on the progress of this artificial bilayer technique including our goal to automate the procedure by applying laboratory chip technology that will allow for a more rapid, voluminous method [10].

FIG. 1 :
FIG. 1: (A)The method to form a bilayer at the gel-gel interface.Pushing a gel bead on a gel plate with an electrode removes excess lipid solution resulting in a thin bilayer between the gels.(B) A gel bead made at the tip of a polyethylene pipette for optical observation of the bilayer formation.(C) A bilayer formed at the agarose gel-agarose gel interface.The boundary between the bilayer and bulky annulus (arrowhead) is clearly seen.

FIG. 3 :
FIG. 3: Channel current of gramicidin D recorded from the bilayer at the gel-gel interface.

FIG. 5 :
FIG. 5: (A) An α-hemolysin channel current recorded from the bilayer at the gel-gel interface.The gel bead contained 10 µg/mL α-hemolysin.The membrane potential was held at 100 mV.(B) The single channel current recorded from our bilayer.(C) The channel was inhibited by adding 10 µM βcyclodextrin to the gel plate.

FIG. 6 :
FIG.6: Lysenin current from a bilayer at an agarose-agarose interface.The bilayer was formed from lipid solution containing 20 mg/mL asolectin and 5 mg/mL sphingomyelin in n-decane.The gel-bead solution contained 7.5 pM lysenin.