Arrangement of ferritin molecules on a gold disk array fabricated on highly ordered anodic porous alumina substrate

Article abstract of DOI:10.1246/cl.2004.812

An ordered array of ferritin, in which the arrangement of ferritin coincided with the arrangement of Au disks, was prepared on Au nanodisk arrays fabricated on a highly ordered anodic porous alumina substrate. Selective adsorption of ferritin onto the Au disk by controlling the modification time and ferritin concentration in the modification solution was confirmed by scanning electron microscopy.

Full text of DOI:10.1246/cl.2004.812

812
Chemistry Letters Vol.33, No.7 (2004)
Arrangement of Ferritin Molecules on a Gold Disk Array Fabricated on Highly Ordered
Anodic Porous Alumina Substrate
Hideki Masuda,ꢀy;yy Hiromi Hogi,y Kazuyuki Nishio,y;yy and Futoshi Matsumotoyy
yDepartment of Applied Chemistry, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397
yyKanagawa Academy of Science and Technology, 5-4-30 Nishi-Hashimoto, Sagamihara 229-1131
(Received February 9, 2004; CL-040148)
An ordered array of ferritin, in which the arrangement of fer-
(a)
(d)
ritin coincided with the arrangement of Au disks, was prepared
on Au nanodisk arrays fabricated on a highly ordered anodic po-
rous alumina substrate. Selective adsorption of ferritin onto the
Au disk by controlling the modification time and ferritin concen-
tration in the modification solution was confirmed by scanning
electron microscopy.
The arrangement of functional biological molecules such as
proteins, DNA, and antibodies into biomolecular arrays has at-
tracted considerable interest because of the potential application
of such arrays to useful tools for estimating specific signals in
living cells and molecular electronics.1–3 To date, many studies
on the arrangement of biological molecules have been limited to
submicron-level patterning within two dimensions using con-
ventional photoresist lithography, microcontact printing, and a
self-assembled monolayer.4–6 In order for the biological arrays
to become widely used, arrays with smaller disk interval and di-
ameter, on which biological molecules are patterned on the
nanometer level, need to be developed. In addition, in order to
confirm the arrangement of biological molecules on the nanome-
ter level on 2-D substrates and detect accurately and rapidly sig-
nals from each pattern, the patterns of biological molecules
should be highly ordered with nanometer-sized intervals. In this
study, we show for the first time the highly ordered arrangement
of biological molecules on the nanometer scale using Au nano-
disk arrays (Figure 1) fabricated on a highly ordered anodic po-
rous alumina substrate, which has a nanometer-scale porous
structure with hexagonally arranged close-packed cells over a
large area,7,8 by filling the pores with Au. The selective adsorp-
tion of biological molecules onto the Au disk surface, that is, the
specific interaction between biological molecules and the Au
disk surface, leads to the simple formation of a highly ordered
biomolecular array with a high density. The fabrication of an ar-
ray of ferritin molecules, which has a ferric oxide core and a
spherical shape and whose molecular size is 13 nm,9,10 as a mod-
el for nanometer-sized biological molecule array, was performed
on the Au nanodisk array with a disk period (center-to-center
distance between the disks) of 63 nm and a disk diameter of
46 nm.11
(b)
(c)
Figure 1. Schematic of highly ordered ferritin molecules
obtained using a highly ordered Au disk array fabricated with
anodic porous alumina. (a) porous alumina, (b) aluminum, (c)
Au, and (d) biological molecule.
a 5% (by weight) phosphoric acid solution for 10 min at 30 ꢁC to
widen the pore diameter. Then, Au was deposited in the pores of
the porous alumina by AC electrolysis at 11 V until the deposited
Au overflowed the pores using a Pt plate as a counter electrode in
an aqueous solution containing 2.5 mM HAuCl4, which was ad-
justed to pH 1.2 with H2SO4.12 The over-flowed Au was then
etched using Ar ion milling to planarize the surface. Ferritin
from an equine spleen (90% purity) was obtained from Sigma
and used without further purification. Ferritin patterns on the
Au disk array were observed using a scanning electron micro-
scope (SEM, JSM-6700F, JEOL).
Figure 2 shows typical SEM images of the ordered Au disk
array fabricated using highly ordered anodic porous alumina
with a pore diameter of 46 nm and a pore period of 63 nm. The
Au disks are observed as bright spots having uniform diameters,
(a)
(b)
A Au disk array with a disk period of 63 nm and a disk di-
ameter of 46 nm was prepared on an anodic porous alumina sub-
strate having the same structure. Aluminum (99.99% purity) was
anodized using the Al plate as a counter electrode for 4 h in
0.3 M sulfuric acid solution under a constant voltage of 25 V
at 16 ꢁC with stirring, after electrochemical polishing using a
mixture of perchloric acid and ethanol under a constant current
of 100 mAꢂcmꢃ2 for 4 min at 0 ꢁC. The substrate was dipped into
100 nm
50 nm
Figure 2. Typical SEM images of an ordered Au disk array
with a disk diameter of 46 nm and disk period of 63 nm. (a) lower
magnification and (b) higher magnification.
Copyright Ó 2004 The Chemical Society of Japan

Chemistry Letters Vol.33, No.7 (2004)
813
and can be seen to be arranged uniformly over the sample except
for a few holes in which the Au metal was not fully deposited.
The average diameter of the Au disks as estimated from the
SEM observation was 46.2 nm. The high-resolution image (b)
of the Au disk surface confirms that it is clean and uniform.
On the other hand, in the image of the Au disk array on which
the ferritin molecules adsorbed (Figure 3), the ferritin molecules
are observed as brighter spots because of the ferric oxide
core.9,10 The figure shows that many ferritin molecules are ad-
sorbed selectively on the Au disks to form a clearly defined array
of ferritin with a Au disk arrangement, while very few ferritin
molecules are adsorbed onto the surrounding alumina. The se-
lective adsorption of ferritin onto the Au disk is thus confirmed
to occur. From the SEM image of Figure 3, three to five bright
spots, due to the ferric core of the ferritin molecules, can be ob-
served on each Au disk. The SEM images in Figure 4 show the
dependence of the amount of adsorbed ferritin on the concentra-
tion of ferritin in the modification solution. The extent of adsorp-
tion depends strongly on the concentration of ferritin. At a ferri-
tin concentration of 0.125 mgꢂmLꢃ1, a few ferritin molecules
adsorb onto the Au disk. The Au disk onto which ferritin mole-
cules do not adsorb can also be observed. On the other hand, at a
ferritin concentration of more than 0.5 mgꢂmLꢃ1, ferritin adsorbs
not only onto the Au disk but also onto the surrounding alumina.
A number of ferritin molecules are strongly attracted by ferritin
adsorbed onto the Au disk surface with van der Waals attraction
between molecules. Ferritin layers larger than the Au disks are
formed on the Au disk array. It is difficult to form a ferritin array
with the size coincided with one of the Au disk at a ferritin con-
centration of more than 0.5 mgꢂmLꢃ1. The trend in the surface
coverage with modification time is similar. At a modification
time of more than 30 min, ferritin molecules start to adsorb onto
the alumina. A modification time of 30 min and a ferritin concen-
tration of 0.25 mgꢂmLꢃ1 are therefore considered most suitable
for achieving a well-separated arrangement of ferritin. More-
over, the amount of adsorbed ferritin on the Au disks was sensi-
tive to the change in pH of the modification solutions. Selective
adsorption of ferritin onto the Au disk could not be observed in
the acidic solution with a pH of less than 6.5 and an alkaline so-
(a)
(b)
100 nm
100 nm
Figure 4. Typical SEM images of ferritin molecules adsorbed
onto the Au disk array with a disk diameter of 46 nm and disk
period of 63 nm. Modification with ferritin was performed by
immersion in (a) 0.125 and (b) 0.5 mgꢂmLꢃ1 ferritin phosphate
buffer solutions (pH 8.0) for 30 min.
lution with a pH of more than 9. The adsorption behavior of fer-
ritin onto the Au disk surface may be attributed to the surface
charge of the ferritin molecule and the porous alumina substrate.
Therefore, the control of the concentration of ferritin, immersion
time in the modification solution, and solution pH are important
in the fabrication of a biomolecular array of regular size and ar-
rangement.
In conclusion, an ordered array of ferritin molecule using a
Au nanodisk array, which has a disk period and diameter of
nanometer sizes, was fabricated using a highly ordered anodic
porous alumina substrate. Ferritin was confirmed by scanning
electron microscopy to adsorb selectively onto the Au disk by
controlling the modification time and ferritin concentration in
the modification solution. The process used here has the advant-
age that the size and shape of the Au disk array is highly control-
lable, and thus it allows for the preparation of biomolecular ar-
rays having various shapes, sizes, and spot spacing. Further
reductions in disk area and spacing could lead to high-density,
highly ordered isolated biomolecular arrays for the analysis of
signal transduction and molecular electronics.
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Figure 3. Typical SEM image of ferritin molecules adsorbed
onto the Au disk array with a disk diameter of 46 nm and disk
period of 63 nm. Modification with ferritin was performed by
immersion in 0.25 mgꢂmLꢃ1 ferritin phosphate buffer solutions
(pH 8.0) for 30 min.
Published on the web (Advance View) June 7, 2004; DOI 10.1246/cl.2004.812