Tsuneo Urisu, Md. Mashiur Rahman, Hidetaka Uno, Ryugo Tero, PhD, Yoichi Nonogaki, “Formation of high resistance supported lipid bilayer on the surface of Si substrate with micro electrodes” Nanomedicine 1 (2005) 317-322.

1. Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317 – 322
www.nanomedjournal.com
Experimental
Formation of high-resistance supported lipid bilayer on the surface
of a silicon substrate with microelectrodes
Tsuneo Urisu, PhD,4 Md. Mashiur Rahman, Hidetaka Uno,
Ryugo Tero, PhD, Yoichi Nonogaki, PhD
Department of Vacuum UV Photoscience, Institute for Molecular Science, The Graduate University for Advanced Studies,
Myodaiji, Okazaki, Japan
Received 28 August 2005; accepted 10 October 2005
Abstract We have developed two basic technologies for fabrication of supported planar lipid bilayer
membrane ion channel biosensors: a defect-free lipid bilayer formation on the substrate surface with
electrode pores and a patterning technique for the hydrophobic self-assembled-monolayer to form
the guard ring that reduces the lipid bilayer edge-leak current. The importance of the supported-
membrane structure to achieve low noise and high-speed performance is suggested on the basis of
the observed relation between the single-ion-channel current noise and the pore size.
D 2005 Published by Elsevier Inc.
Key words: Supported membrane; Lipid bilayer; Membrane protein; Gramicidin; Self-assembled monolayer; Ion channel;
Biosensor
Signal transmission and processing in the living body many applications [1]. The ion channel and/or receptor-
takes place via life body molecules, specifically neuronal reconstructed lipid bilayer as a key component of the
transmitter molecules, as the signal carrier. It is a unique detector of neuronal transmitter molecules is useful not
communication system comparable to electrical and optical only as a biosensor but also in an in vitro study of cell
communication, which use electrons and photons, respec- membrane biological functions. In reports of single-ion-
tively, as the signal carriers. Thus it can be called channel biosensors, membrane protein reconstructed lipid
bmolecular communication.Q Neurotransmitter molecules bilayers are suspended in a small pore on the substrate
discharged from the presynaptic membrane are received by made by Si, SiO2, glass, or other materials [2-13].
ion channels on the surface of postsynaptic membranes, However, technological problems still exist with stability
and the electrical signal (ie, the depolarization of the of the single-ion-channel biosensor. Development of the
membrane) is generated by the channel current flowing supported-membrane sensor is considered to be one way to
though ion channel pores. The development of molecular solve these problems. However, single-ion-channel record-
communication devices such as a detector and a transmitter ing has not yet been successful in supported-membrane
of neurotransmitter molecules has the potential to facilitate devices. The challenges are believed to consist in the
important medical applications such as diagnostics, treat- fabrication of a defect-free supported planar lipid bilayer
ment of diseases, and screening in drug development. (SPLB) on the substrate surface and the reduction in edge-
Combination with the Si LSI technology allows a leak current through the thin water layer under the lipid
significant scale-down to nanosized devices suitable for bilayer [14].
insertion into the body, or efficient integrations useful in Here we have developed two basic process technolo-
gies necessary to fabricate the ion channel supported-
membrane biosensors: formation of a defect-free lipid
bilayer on the Si surface with microelectrode pores, and
No conflict of interest was reported by the authors of this paper.
4 Corresponding author. Institute for Molecular Science, Myodaiji, the deposition and patterning of the hydrophobic self-
Okazaki, 444-8585 Japan. assembled monolayer (SAM) as a guard ring to reduce the
E-mail address: urisu@ims.ac.jp (T. Urisu). edge-leak current.
1549-9634/$ – see front matter D 2005 Published by Elsevier Inc.
doi:10.1016/j.nano.2005.10.002

2. 318 T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322
Materials
Palmitoyl-2-oleoyl-sn-3-[phosphor-l-serin] (POPS), 1,2-
diphytanoyl-sn-glycero-3-phosphocholine (DfPC), and
fluorescence-labeled lipid diacyl phosphoethanolamine-N-
lissamine rhodamine B sulfonyl (Rb) were purchased from
Avanti Polar Lipids Co. (Alabaster, AL) Dipalmitoylphos-
phatidylcholine (DPPC) was provided by Nippon Fine
Chemical Co. (Osaka, Japan) HF, H2O2, H2SO4, HCl, and
HNO3 solutions, as well as CaCl2 and KCl, were
analytical grade and purchased from Sigma-Aldrich
(St. Louis, MO). Octadecyltrichlorosilane (OTS) and
toluene were also purchased from Sigma-Aldrich. All of
the chemicals and solvents were used without further
purification. The purities of Co and SiO2 sputter targets
and Ag wires (0.5 mm diameter) were 99.99%. Spin-on
glass (SOG) was purchased from Rasa Industries Co.
(Tokyo, Japan) Si(100) wafers (p type, B doped, 0.018 V
cm, and 525 Am in thickness) were purchased from
Miyoshi Co. Water (Kanagawa, Japan) with a typical
resistivity of greater than 18 MV cm was produced using a
Milli-Q purification system (Millipore Co., Billerica, MA)
Formation of defect-free SPLB
Formation of the SPLB with sufficiently high resistivity
(greater than gigaohms) is the necessary condition for
single-ion-channel recordings. To make a defect-free Fig 1. Schematics of the fabrication process of the supported-membrane
SPLB, It is crucial that the surface roughness be substrate with AgCl/Ag microelectrodes and the SPLB.
minimized. In this work we have successfully made the
pore with about 1 Am diameter for the microelectrode,
keeping the Si substrate surface extremely flat (Ra b 1 (0.05 torr) and O2 (0.002 torr) as the etching gas. The SR
nm), by using femtosecond laser ablation patterning and etching provides a vertical side wall and completely stops at
synchrotron radiation (SR) etching. the CoSi 2 surface [15]. The Co contact mask was
successfully removed without damaging the substrate by
immersion into 0.1 M HNO3 aqueous. Ag (50 nm thickness)
Methods and results
was deposited on CoSi2 electrode surfaces by electroplating.
Fig. 1 shows the fabrication process of the well structures Then, AgCl/Ag was also formed by electroplating.
with microelectrodes. Co (10 nm thick) and Ag (100 nm Unilamellar giant vesicles [16] of DPPC/POPS/Rb (ratio
thick) thin films were sputter-deposited on the mirror- 89.5:10:0.5) were prepared as follows. A chloroform
polished and reverse-side rough Si(100) surfaces after solution of a lipid mixture (10 mg/mL) was dried under
conventional wet cleaning. After that, a SiO2 thin film N2 flow using a rotary evaporator for about 30 minutes and
consisting of SOG (400 nm thickness) and sputtered SiO2 subsequently vacuum-dried for 10 hours to completely
(200 nm thickness) were deposited and the sample was remove the solvent; a buffer solution (10 mM KCl) was
annealed at 5408C for 10 minutes. By this process the Co added to the lipid thin film obtained and gently agitated. The
layer was changed to CoSi2 and the Co/Si interface became lipid concentration of the suspension obtained was 0.1 mg/
ohmic. The sample was then annealed by SR irradiation to mL. All the processes were carried out at room temperature
remove gas from the SOG [15]. A Co layer as an etching (RT). After incubation at 488C for 10 hours, dialysis was
contact mask was deposited on the SOG surface by then carried out for the suspension of giant vesicles using a
sputtering, and electrode hole mask patterns were made 5-Am filter for 1 hour in the buffer solution (10 mM KCl,
using a femtosecond laser (E = 1560 nm, average power = pH 6.6) at RT. For the deposition of lipid bilayer
250 mW, frequency = 258 kHz, pulse width = 900 femto- membranes, the substrate was incubated for 1 hour at
seconds, and irradiation time = 4 milliseconds). The 508C under a buffer solution formed by mixing 200 AL of
diameter of an electrode hole was about 1 Am. SR etching the vesicle suspension and 50 AL of a 50 mM solution of
of the SiO2 layer for making the well on the electrode was CaCl2. Then the sample was washed five times at RT with
carried out at beam line 4A2 of the SR facility (UVSOR) at the buffer solution. Atomic force microscopy (AFM)
the Institute for Molecular Science, using a mixture of SF6 observations were carried out using a SPI3800 scanning

3. T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322 319
Fig 2. A, The surface morphology measured by AFM around the electrode holes of the substrate after AgCl electroplating. B, Cross-sectional profile.
probe microscopy system (Seiko Instrument Inc.) in the
dynamic force mode (tapping mode) using a Si cantilever.
The spring constant of the cantilever for measuring the
surface roughness of the substrate in air was 43 N/m, and
1.5 N/m for the in situ characterization of the lipid bilayer.
Fig. 2, A shows the AFM topography of the substrate
surface around the electrode wells made by the process
shown in Fig. 1. The cross-sectional profile in line with X-Y
is also shown in Fig. 2, B. These data show that the surface
around the electrode well is very flat (Ra ~0.8 nm). To
obtain such a flat surface, it was important to control the
irradiation power of the femtosecond laser such that only the
Co film was removed while causing negligible damage to
the SiO2 layer beneath. If the SiO2 layer was also sputtered
by the laser, particles (composition unknown) of 100 to
200 nm diameter, which were difficult to remove using the
usual etching solutions such as HF, HCl, H2SO4, and HNO3, Fig 3. I-V characteristics of the substrate measured in 10 mM KCl solution
were deposited around the wells. The electric characteristics (A) before and (B) after SPLB formation, and (C) the equivalent circuit of
were determined using a patch clamp amplifier (CEZ-2400, the system.
Nihon-Koden, Tokyo, Japan) through the AgCl/Ag elec-
trode in conjunction with the eCell (Version 2.12) software. lipid thin film. Because the bilayer was formed using the
Line a in Fig. 3 shows the current-voltage (I-V) character- same protocol as that used in the formation of the single
istics of the substrate under the buffer solution before bilayer patches shown in Fig. 4, A, it is considered from the
vesicle fusion. From these data, the series resistance Rs in fluorescence microscopy image that the single bilayer was
the equivalent circuit shown in Fig. 3, C is given to be 10 F formed on the microelectrode area. From the I-V character-
3 MV. Mixing of negatively charged lipid POPS with istics of the system measured after the lipid bilayer
neutral lipid DPPC was essentially effective in forming formation (shown as line b in Fig. 3), the resistance of the
unilamellar giant vesicles without aggregation. Addition of lipid bilayer (Rm in Fig. 3, C) was estimated to be 1.2 GV.
Ca2+ was also necessary to induce the rupture of vesicles on The capacitance (Cm in Fig. 3, C) of the system measured
the SiO2 surface [17]. Fig. 4, A shows a fluorescence using a patch clamp amplifier was 10.7 pF. These values
microscopy image of a single SPLB formed on the were observed with extremely good reproducibility during
SiO2/Si(100) surface by the rupture of the giant vesicles. our experiments for more than 5 hours. Because the
The diameter of the bilayer was typically about 200 to resistance almost completely returned to the original value
300 Am, large enough to cover the electrode area (10 to 30 Am of 10 MV when the bilayer was broken by adding 5 AL of
diameter). The thickness of the bilayer, 4.5 nm, observed gramicidin solution (1 mg/mL), the high resistance observed
by AFM corresponds to the height of the single bilayer [18]. subsequent to formation of the bilayer was considered not to
The lipid bilayer covering the electrode well was formed be due to small vesicles clogging the electrode hole. Dark
by giant vesicle fusion. A fluorescence microscopy image spots at the electrode holes in Fig. 4, B are not due to the
(Fig. 4, B) after lipid bilayer formation on the microelec- nonexistence of the bilayer on the well. In the region of
trode area clearly shows the existence of the homogeneous 600-nm-thick SiO2, the fluorescence microscopy image is

4. 320 T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322
Fig 4. Fluorescence microscopy image of the lipid bilayer formed by the rupture of the giant vesicle on (A) the SiO2/CoSi2/Si surface and (B) the electrode area.
very bright because of the fluorescence interference contrast
effect [19]. On the other hand, the electrode hole area, in
which there is no back surface reflection, is relatively dark.
Because the observed capacitance of 10.7 pF is almost
explained by the calculated capacitance of 10 pF due to the
SiO2 thin film (600 nm in thickness and 0.5 mm in diameter,
determined from the upper electrode, were assumed), the
capacitance due to the lipid bilayer formed on the well is
considered to be almost equal to the value estimated from
the specific capacitance of the single bilayer, 0.5 to 0.8 AF
[4,20]. From these considerations, it is concluded that the
gigaohm seal by the single bilayer was formed on the
microelectrode area.
Patterning of OTS-SAM and vesicle fusion on the surface
Resistance of 1.2 GV is still insufficient to permit a
single-channel recording with a level of 1 pA channel
current. Much higher resistance is expected to be obtained
by reducing the edge-leak current. Working from the Fig 5. The AFM image after the patterning of OTS-SAM on SiO2/Si.
concept of using the hydrophobic SAM guard ring to
reduce the edge-leak current, we have developed a SiO2/Si surface is very flat (Ra = 0.22 nm). OTS-SAM was
patterning technique for SAM of OTS, of which the deposited by immersing the SiO2/Si(100) substrate in a 10
hydrophobic carbon chain length is close to that of the lipid. mM water-saturated toluene solution of OTS for 5 seconds at
The sample treatment and the OTS deposition were RT. The OTS/SiO2 sample was sonicated in toluene, acetone,
carried out according to our earlier work [21]. Briefly, a ethanol, and pure water to remove the excess OTS molecules
mirror-polished Si(100) wafer covered with the native oxide from the OTS/SiO2 surface.
was first sonicated in acetone, ethanol, and Milli-Q water A negative-resist pattern was formed on the OTS/SiO2
(N 18 MV cm; Millipore Co.) for 5 minutes each. Then surface by using conventional photolithographic technique.
the substrate was boiled in a solution of concentrated H2SO4 A 50-Am line-and-space conventional photomask pattern
and H2O2 (30%) (7:3 in volume ratio) at 1208C for 5 minutes was used. The sample was then exposed to UV light in air
to remove the organic contaminants and immersed in a HF for 30 minutes to remove the OTS-SAM from the open
solution (2.5%) for 2 minutes to remove the surface oxide area, where the distance between the sample and the lamp
layer. After this cleaning, the SiO2 film of 120 nm in was also 3 cm. Finally, the photoresist was removed using
thickness was deposited on the Si(100) surface by sputtering. a negative-resist remover (NS, Tokyo Ohka Kogyo Co.,
The sample was then exposed to UV light (UVL20US-60, Nakagawa, Japan) followed by rinsing with distilled water
Sen Lights Co.) for 10 minutes. The distance between the and drying by blowing N2. The OTS line-and-space pattern
sample and the lamp was 3 cm. AFM images show that the obtained and measured by AFM is shown in Fig. 5.

5. T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322 321
Fig 6. A, Fluorescence microscopy image of SPLB formed on the patterned OTS-SAM. B, Intensity distribution on the A-B line in A.
Lipid bilayers were deposited on the patterned OTS-
SAM area by the rupture of giant unilamellar vesicles.
When the substrate was immersed in an aqueous solution of
lipid vesicles, the vesicles adhered to the surface, broke up,
and spread to form a bilayer on hydrophilic surfaces and a
monolayer on hydrophobic surfaces [21]. Fig. 6, A shows a
fluorescence microscopy image of the OTS-SAM–patterned
SiO2 surface after immersing in the giant vesicle suspen-
sion. Fig. 6, B shows the intensity distribution on the A-B
line in Fig. 6, A. Earlier study had shown that after the
vesicle fusion, a bilayer forms on hydrophilic SiO2 surfaces
and a monolayer forms on OTS-SAM hydrophobic surfaces
[21]. In the present case, a monolayer was formed on the
hydrophobic OTS-SAM area (i region in Fig. 6) and a
bilayer was formed on the hydrophilic SiO2 area (ii region
in Fig. 6), so the fluorescence intensity from the lipid layer
containing Rb is different between these areas. In the iii
region it is very dark, because neither a bilayer nor a
monolayer has formed on the SiO2 surface.
The fluorescence microscopy image of Fig. 6 confirms Fig 7. Total root mean square current noise as a function of the pore
that the bilayer and the monolayer are successfully 5
diameter. The bandwidths of the patch clamp amplifier were (O) 5 kHz, (5 )
10 kHz, and (D) 20 kHz.
deposited on the bare SiO2 and the OTS/SiO2 regions,
respectively. A substantial body of evidence suggests that a
thin water layer of approximately 1 to 2 nm in thickness is considered that the OTS-SAM surface and the lipid
trapped between the substrate surface and the head-groups monolayer undersurface adhere completely to each other
layer in the lower leaflet of the bilayer [22]. This water layer in the i region because of the strong hydrophobic interaction
causes the edge-leak current of the SPLB system. The of both surfaces. This indicates that OTS-SAM patterns can
calculated length of the OTS molecule is 2.75 nm [23]. On be used sufficiently as a guard ring to reduce the leak
the other hand, the lengths of the DPPC acyl group and head current from the SPLB edge.
group are 2.0 nm and 1.5 nm, respectively. The length
mismatch seems to exist between the OTS and DPPC
Discussion
molecules. However, in our previous experiments with
DPPC monolayer deposition by the Langmuir-Blodgett We have investigated the relation between single-ion-
method on the SiO2 surface with OTS-SAM islands, the channel current noise and pore size using the conventional
height of the DPPC monolayer area was observed to agree arrangement of the black membrane formed at the pore of
with that of the OTS-SAM island area [24]. Therefore, in the Teflon chip partitioning the two chambers. A lipid
the lipid layer–OTS-SAM structure shown in Fig. 6, it is bilayer was formed at the pore of the chip by contacting

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