Formation of tBLM by using mixed DPhyPC + CHAPS
micelles(2)
This is the report on how to form tBLM by using mixed DPhyPC + CHAPS micelles and the
kinetic is studied by surface plasmon resonance and impedance spectroscopy
The SPR kinetic curve is obtained by getting the minima of SPR dip as a function of time.
While the impedance spectra is measured and fitted to the equivalent circuit in figure 1 to get
the capacitance and resistance of resulting tBLM. The capacitance and resistance values
reported here are without normalization. To get the normalized values, capacitance has to be
multiplied by 3 while resistance has to be divided by 3.
Figure 1: Equivalent circuit for fitting of impedance spectra
The buffer used in all cases is 100mM NaCl + 10mM HEPES at pH7
This report is divided into 2 major parts in which the 1st set of experiment utilizes ethanol as
catalyst to break the micelles apart and the 2nd set of experiment which does not use ethanol
as catalyst.
(1) 1st set of experiment(with ethanol as catalyst):
- SAM: FC16:betaME = 1:1
- Method: 40microliter of ethanol is injected into the cell and later 400microliter of
1mg/ml DPhyPC + 6mM CHAPS micellar solution is injected. The volume
of ethanol to volume of micellar solution is 1:10. The existence of ethanol
actually break the micelles apart and the solution becomes more and more
opaque. tBLM is largely formed in this stage as well as seen by SPR. The final
product of this solution is actually opaque solution with precipitation(there
might be other morphologies existed in the solution as well such as vesicles,
bicelles and micelles but at this point it is impossible to tell what are they).
When the SPR signal is almost stable the solution is diluted by 10-folds to
0.1mg/ml DPhyPC + 0.6mM CHAPS. When the SPR signal is stable again the
solution is diluted by 10-folds to 0.01mg/ml DPhyPC + 0.06mM CHAPS.
Finally the solution in the cell is exchanged for fresh buffer.
- Result: The corresponding SPR kinetic curve is shown in figure(2) while the
impedance parameters of tBLM at various stages of experiment are shown in
table(1)
Kinetic of tBLM Formation
ethanol
750
0.1mg/ml DPhyPC + 0.6mM CHAPS + ethanol
SPR minimum/pixel
700
0.01mg/ml DPhyPC + 0.06mM CHAPS + ethanol
650
(a)
rinse
600
(b)
(c)
1mg/ml DPhyPC + 6mM CHAPS + ethanol
550
0
1000
2000
3000
time/second
4000
5000
Figure 2: The kinetic of tBLM formation by using ethanol as catalyst on FC16:betaME = 1:1
SAM. The corresponding impedance parameters at point (a), (b) and (c) are shown in table(1)
a
b
c
CPE of tBLM
(x 10e-7 Farad)
3.06  0.03
2.72  0.02
2.72  0.01
αof CPE
0.961  0.002
0.977  0.001
0.990  0.001
Resistance
(x mega ohms)
0.92  0.02
2.63  0.12
3.39  0.14
Table 1 : The impedance parameters of tBLM at various stages of the experiment as shown in
figure(2). The fitting is from 1 to 30kHz
6000
(2) 2nd set of experiment(without ethanol):
Result from 2 samples will be reported in which 1 of the tBLM is formed on
FC16:betaME = 1:1 SAM while another tBLM is formed on FC16:betaME = 35:65 SAM
The 1st sample:
- SAM : FC16:betaME = 1:1
- Methods: 2.5mg/ml DPhyPC + 10mM CHAPS micellar solution is injected into the
cell. Later the micellar solution is diluted to 1mg/ml DPhyPC + 4mM
CHAPS. At this lipid:detergent ratio the micelles break apart without
ethanol as catalyst. The solution become more and more opaque and
precipitation is formed. When the SPR signal is almost stable the solution is
diluted by 10-folds to 0.1mg/ml DPhyPC + 0.4mM CHAPS. Again when
the SPR signal is stable the solution is diluted by 10-folds to 0.01mg/ml
DPhyPC + 0.04mM CHAPS. Finally the solution in the cell is exchanged
for fresh buffer.
- Result: The corresponding SPR kinetic curve is shown in figure(3) while the
impedance parameters of tBLM at various stages of the experiment are
shown in table(2)
0.1mg/ml DPhyPC + 0.4mM CHAPS
800
(a) 0.01mg/ml DPhyPC + 0.04mM CHAPS
SPR minimum/pixel
750
(b)
700
rinse
(c)
650
600
1mg/ml DPhyPC + 4mM CHAPS
Kinetic of tBLM Formation
550
500
1000
2.5mg/ml DPhyPC + 10mM CHAPS
2000
3000
4000
time/second
5000
6000
Figure 3: The kinetic of tBLM formation without ethanol on FC16:betaME = 1:1
SAM. The corresponding impedance parameters at point (a), (b) and (c) are shown in table(2)
7000
αof CPE
CPE of tBLM
(x 10e-7 Farad)
2.74  0.05
2.53  0.03
2.49  0.03
a
b
c
0.951  0.004
0.973  0.003
0.979  0.002
Resistance
(x mega ohms)
8.2  0.6
15.6  1.3
22.5  2.1
Table 2 : The impedance parameters of tBLM at various stages of the experiment as shown in
figure(3). The fitting is from 0.1 to 30kHz
The 2nd sample:
- SAM : FC16:betaME = 35:65
- Methods: Same as the 1st sample
- Result: The corresponding SPR kinetic curve is shown in figure(4) while the
impedance parameters of tBLM at various stages of the experiment are shown
in table(3)
700
(a)
Kinetic of tBLM Formation
650
SPR minimum/pixel
0.1mg/ml DPhyPC + 0.4mM CHAPS
(b)
600
0.01mg/ml DPhyPC + 0.04mM CHAPS
rinse
550
1mg/ml DPhyPC + 4mM CHAPS
(c)
500
1000
2.5mg/ml DPhyPC + 10mM CHAPS
2000
3000
4000
5000
6000
tme/second
Figure 4: The kinetic of tBLM formation without ethanol on FC16:betaME = 35:65
SAM. The corresponding impedance parameters at point (a), (b) and (c) are shown in table(3)
7000
a
b
c
CPE of tBLM
(x 10e-7 Farad)
3.94  0.03
3.65  0.02
3.60  0.02
αof CPE
0.958  0.001
0.965  0.001
0.979  0.002
Resistance
(x kilo ohms)
434  10
909  27
891  21
Table 3 : The impedance parameters of tBLM at various stages of the experiment as shown in
figure(3). The fitting is from 2 to 30kHz
Discussions:
(1) So we see that in figure(2) to (4) that when the micelles are broken apart(with and without
ethanol) the SPR signal rises. Strictly speaking, the rise of the SPR signal could be due to
the deposition of lipid(or detergent) material to the SAM surface and due to the ‘phase
transition’ of the micelles to precipitation in the bulk solution. At this point it is hard to
say how does this ‘phase transition’ changes the refractive index of bulk solution and how
much does it contribute to the rise of SPR signal. But since the volume fraction of
lipid+detergent molecules in the bulk solution is small(<1%), I will say most of the rise of
the SPR signal is due to the deposition of lipid material to SAM to form tBLM
(2) So when the solution in the cell is diluted 10-folds for the 1st time to 0.1mg/ml DPhyPC,
again we see a small rise in the SPR signal. The rise of the signal is most probably due to
the exchange of CHAPS in tBLM with DPhyPC in the bulk which leads to a tighter
packed tBLM
(3) The last question to be answered is how does the lipid molecules transfer to the surface
from the bulk. Is it deposited to the surface as monomer/micelles/bicelles/or closed-shell
vesicles? In other controlled experiments(data not shown) in which SAM was incubated
with the already opaque solution(with precipitation) of 1mg/ml DPhyPC + 4mM CHAPS
(I suspect there might be some closed-shell vesicles in these solution), tBLMs are formed
as check by impedance spectroscopy but the time required to form tBLM is much longer
than the time reported in this report as checked by SPR. This might indicate that the
process to form tBLM as reported here is not mediated by closed-shell vesicles.
Conclusions:
It is been shown that the tBLM will be formed by using mixed DPhyPC + CHAPS micelles
and its kinetic could be monitored by SPR