Selective functionalization of In2O3 nanowire mat devices for biosensing applications

Article abstract of DOI:10.1021/ja0503478

A strategy to covalently attach biological molecules to the electrochemically active surface of indium oxide nanowire (In₂O₃ NW) mat devices is presented. A self-assembled monolayer (SAM) of 4-(1,4-dihydroxybenzene)butyl phosphonic a

Full text of DOI:10.1021/ja0503478

Published on Web 04/26/2005
Selective Functionalization of In2O3 Nanowire Mat Devices for Biosensing
Applications
Marco Curreli,† Chao Li,‡ Yinghua Sun,‡ Bo Lei,‡ Martin A. Gundersen,‡ Mark E. Thompson,*,† and
Chongwu Zhou*,†,‡
Departments of Chemistry and Electrical Engineering-Electrophysics, UniVersity of Southern California,
Los Angeles, California 90089
Received January 19, 2005; E-mail: met@usc.edu (M.E.T.): chongwuz@usc.edu (C.Z.)
Nanomaterials, such as carbon nanotubes (CNTs)1 and nanowires
(NWs),2 have been employed in nanoscale biosensing devices.3 The
detection of DNA and proteins is an important goal, where the
diagnosis and treatment of genetic diseases and the identification
of infectious agents are of central importance.4 Nanoscale biosensing
devices give very low detection limits for biological molecules and
biologically important ions, largely due to their high surface-to-
volume ratio, such that even low levels of analyte binding markedly
affects the properties of the nanomaterial, for example, its conduc-
tivity. Selectivity to a given analyte has been accomplished by
anchoring an analyte-specific recognition group to the surface of
the CNT or NW.
Figure 1. (a) Schematic representation of an In2O3 NW mat device (only
one NW is shown for clarity). The NW is functionalized with a SAM of
HQ-PA and is placed between two gold electrodes protected by a SAM of
dodecane-1-thiol. (b) (i) The monolayer of HQ-PA, deposited on the In2O3
NW or ITO, can be reversibly oxidized to Q-PA in an electrochemical cell.
(ii) Addition of the probe, thiol-terminated DNA (HS-DNA) to Q-PA. (iii)
Attachment of complementary DNA strand (dye-DNA′) to the probe DNA.
Indium oxide (In2O3) NWs have been used to fabricate both
chemical sensors and biosensors.5 They can work as valid alterna-
tives to CNTs or Si NWs. Unlike Si NWs, In2O3 NWs do not have
a native oxide coating between the active semiconductor and the
bound analyte. Bare In2O3 NWs, when previously employed as gas
sensors, showed ultrahigh sensitivity down to parts per billion level,
fast response, and fast recovery time.5a To detect biomolecules the
In2O3 NW, surfaces must be coated with analyte binding groups.
To this end, we have extended the surface chemistry reported by
Mrksich and co-workers6 toward selectively functionalizing In2O3
NWs with DNA strands, as confirmed using voltammetric and
fluorescence studies. This procedure can be readily generalized to
selectively immobilize biological molecules onto various metal
oxide (e.g., SnO2 and ZnO) NW surfaces. A related process for Si
NW functionalization has been reported,7 in which the linking
molecules are anchored to silicon NW surfaces using UV radical
photoactivation, followed by deprotection of the hydroquinone
residue using BBr3. Our approach achieves the In2O3 NW surface
functionalization via a simple and mild self-assembling process,
utilizing 4-(1,4-dihydroxybenzene)butyl phosphonic acid (HQ-PA).
This eliminates the use of UV photoactivation and BBr3, a very
corrosive Lewis acid we have found to attack many metal oxide
NWs.
We have generated a self-assembled monolayer (SAM) of HQ-
PA on the In2O3 NW surface (Figure 1a). HQ-PA is an electro-
chemically active molecule containing a hydroquinone group that
undergoes reversible oxidation/reduction at low potentials.6b Oxi-
dized HQ-PA (Q-PA) reacts with a range of functional groups,
which can be easily incorporated into biomolecules and other
materials, such as thiols, azides, cyclopentadienes, and primary
amines. Here, we have attached a thiol-terminated DNA, electro-
chemically addressing only the desired NWs (Figure 1b).
Derivatives of phosphonic acid are well-known to bind to indium
tin oxide (ITO),8 which has a surface composition very similar to
that of In2O3 NWs. Therefore, preliminary studies were carried out
Figure 2. (a) Fifteen consecutive CVs showing the reversible oxidation/
reduction of a SAM of HQ-PA on an ITO glass sheet. (Inset) Chrono-
amperometry shows the amount of charge necessary to oxidize a predefined
area of HQ-PA: an average of 56.8 µC. (b) A fluorescence image of ITO
surface that was oxidized to Q-PA, reacted with SH-DNA, and the DNA
paired to its complementary DNA strand labeled with a fluorescence dye.
(c) A fluorescence image of ITO with the HQ-PA monolayer went through
the same DNA attachment procedure as that of the ITO sheet in (b), showing
little or no DNA binding.
on ITO-coated glass. A monolayer of HQ-PA was deposited on
freshly cleaned ITO surfaces by submerging the substrate into a
0.1 mM aqueous solution of HQ-PA for 16 h. The surface coverage
and stability were characterized by electrochemical analysis. A
cyclic voltammetry trace (CV) of a derivatized film is shown in
Figure 2a. The film was scanned multiple times (shown for 15
consecutive scans) in the range from -450 to +600 mV. The
oxidation wave is centered at +330 mV, and the reduction wave is
centered at -200 mV (Figure 2a), which are in agreement with
† Department of Chemistry.
‡ Department of Electrical Engineering-Electrophysics.
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C O M M U N I C A T I O N S
chemical cell with both electrodes submerged in the electrolyte but
with potential applied across only select Au electrodes (upper
electrodes in Figure 3a). The potential of the cell was held at +450
mV for 5 s. In this way, the monolayer of HQ-PA coating on
specific NWs was oxidized to Q-PA. Then, the entire device was
submerged in the SH-DNA solution for 2 h, resulting in the
selective, covalent linkage of DNA to the NWs with Q-PA.
Fluorescence studies were used to confirm the selective func-
tionalization of the In2O3 NW array. Typical fluorescence images
from similar nanowire devices with Q-PA and HQ-PA, which have
been treated with the complementary DNA strand containing a
fluorescent dye label, are shown in Figure 3d and e, respectively.
In Figure 3d, the gold electrodes appear as dark lines, whereas the
NW mat with Q-PA, derivatized with DNA, appears as a bright
network. In contrast, the NWs with HQ-PA, which went through
the same DNA treatment, do not show any fluorescence, as seen
in Figure 3e. This demonstrates that there is no DNA binding to
the NWs with HQ-PA.
Figure 3. (a) A photograph of a NW mat sample contacted by two groups
of electrodes. Only the HQ-PA attached to the NWs between the upper
electrodes was converted to Q-PA. (b) An SEM image of the In2O3 NWs
before functionalization. The brighter stripes are gold electrodes covering
the NW mat. (c) The same sample imaged at higher magnification, where
the NW mat is clearly visible. (d) A fluorescence image of the NWs with
Q-PA taken after DNA attachment. The gold electrodes, passivated with
an alkanethiol, appear dark under the fluorescence microscope. (e) A
fluorescence image of the NWs with HQ-PA after DNA incubation. The
NWs appear dark, indicating no DNA attached to HQ-PA.
In summary, we have demonstrated selective functionalization
of an array of In2O3 NW-based devices by electrochemically
activating their surfaces and then immobilizing single-strand DNA.
This can be considered a key step for the future fabrication of large-
scale biosensor arrays or chips for inexpensive multiplexed detec-
tion. The methodology described here may also be adapted to
selectively functionalize individual NW devices.
previously published data.6,7 Also, chronoamperometric measure-
ments on a predefined area of the ITO sheet (0.57 cm2) reveal that
a charge of 57 µC is consumed for the complete oxidation of the
HQ-PA SAM, at +400 mV (Figure 2a inset). This gives a surface
coverage of 4.9 × 10-10 mol/cm2 or 33.7 Å2/molecule, indicating
that HQ-PA forms a densely packed monolayer.8,9 The potential
of the cell was then brought to +450 mV and held for 5 s to create
a monolayer of Q-PA (active state). A separate ITO sheet was held
at -350 mV to ensure the complete reduction of the monolayer of
HQ-PA (inactive state). Both ITO sheets were then submerged in
a solution of a 10 µM thiol-terminated DNA10a for 2 h (solvent )
PBS buffer containing a catalytic amount of triethanolamine, pH
7.40; Figure 1b, step ii). The ITO sheets were next incubated for
30 min in an aqueous solution containing the complementary
DNA10b tagged with a red fluorescence dye (Figure 1, step iii).
The ITO surface was rinsed with a PBS buffer (pH 7.00) containing
1 M NaCl to wash away any excess of complementary DNA.
Fluorescence microscopy images of the activated (Figure 2b, bright)
and inactivated (Figure 2c, dark) ITO sheets demonstrate the success
of this selective functionalization strategy. This demonstrates that
DNA binds only to the activated ITO sheet in a uniform manner.
In control experiments using a mismatched DNA-dye,10c the ITO
sheet appears dark, indicating no pairing (not shown).
Acknowledgment. The authors would like to thank Peter Qin
and Qi Cai for helping with DNA purification, and James Heath
for sharing reference 7 prior to publication. We acknowledge
support from the DARPA MolApps Program (SPAWAR SysCtr
San Diego, #N66001-04-1-8902).
Supporting Information Available: Experimental details for the
synthesis of HQ-PA, device fabrication, surface functionalization, and
fluorescence microscopy. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) (a) Kong, J.; Franklin, N. R.; Zhou, C.; Chapline, M. G.; Peng, S.; Cho,
K.; Dai, H. Science 2000, 287, 622-625. (b) Chen, R. J.; Bangsaruntip,
S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, Y.; Kim, W.; Utz,
P. J.; Dai, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4984-4989. (c)
Star, A.; Gabriel, J. C.; Bradley, K.; Gruner, G. Nano Lett. 2003, 3, 459-
463. (d) Besteman, K.; Lee, J.-O.; Wiertz, F. G. M.; Heering, H. A.;
Dekker, C. Nano Lett. 2003, 3, 727-730.
(2) (a) Patolsky, F.; Zheng, G.; Hayden, O.; Lakadamyali, M.; Zhuang, X.;
Lieber, C. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14017-14022.
(b) Cui, Y.; Wei, Q.; Park, H.; Lieber, C. M Science 2001, 293, 1289-
1292.
Having established the successful functionalization strategy on
the ITO surfaces, the same reaction sequence was applied to In2O3
NWs. A mat sample of In2O3 NWs (average diameter of 10 nm
and length of 3 µm) was grown on a SiO2/Si wafer, followed by
photolithography and metal (Ti/Au, 3 nm/50 nm) deposition to
pattern an electrode array. The resultant device is shown in Figure
3a. We chose to work with NW mat devices as they have numerous
advantages, such as great reliability, high sensitivity, and ease of
fabrication when compared to individual NW devices. Figure 3b,c
shows typical SEM images of the NW mat sample used in this
study. Multiple nanowires were found bridging the Au electrodes.
A SAM of HQ-PA was created on a freshly cleaned In2O3 NW
mat sample using the same procedure as that for the ITO sheets.
The In2O3 NW device was placed into the electrochemical cell and
completely reduced to HQ-PA. To prevent the thiol-terminated
DNA from attaching to the gold electrodes, the sample was treated
with dodecane-1-thiol after HQ-PA SAM formation. This resulted
in the formation of a SAM of a C12 alkyl chain on the Au electrodes
surface (Figure 1a). The device was then placed into an electro-
(3) (b) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct.
1996, 25, 55-78. (b) Seker, F.; Meeker, K.; Kuech, T. F.; Ellis, A. B.
Chem. ReV. 2000, 100, 2505-2536.
(4) Wang, J.; Liu, G.; Jan, M. J. Am. Chem. Soc. 2004, 126, 3010-3011.
(5) (a) Zhang, D.; Liu, Z.; Li, C.; Tang, T.; Han, S.; Liu, X.; Lei, B.; Zhou,
C. Nano Lett. 2004, 4, 1919-1924. (b) Li, C.; Lei, B.; Zhang, D.; Liu,
X.; Han, S.; Tang, T.; Rouhanizadeh, M.; Hsiai, T.; Zhou, C. Appl. Phys.
Lett. 2003, 83, 4014-4016.
(6) (a) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 4286-
4187. (b) Chan, E. W. L.; Yousaf, M. N.; Mrksich, M. J. Phys. Chem. A
2000, 104, 9315-9320. (c) Yeo, W. S.; Yousaf, M. N.; Mrksich, M. J.
Am. Chem. Soc. 2003, 125, 14994-14995. (d) Yousaf, M. N.; Houseman,
B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992-5996.
(e) Sampson, N. S.; Mrksich, M.; Bertozzi, C. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 12870-12871.
(7) Bunimovich, Y. L.; Ge, G.; Beverly, K. C.; Ries, R. S.; Hood, L.; Heath,
J. R. Langmuir 2004, 20, 10630-10638.
(8) Gardner, T. J.; Frisbie, C. D.; Wrighton, M. S. J. Am. Chem. Soc. 1995,
117, 6927-6933.
(9) Bravo, B. G.; Mebrahtu, T.; Soriaga, M. P.; Zapien, D. C.; Hubbard, A.
T.; Stickney, J. L. Langmuir 1987, 3, 595-597.
(10) Oligonucleotides base sequence. (a) Thiol-terminated DNA: (5′-HS-GCT
TTG AGG TGC GTG TTT GT-3′); (b) complementary DNA with dye:
(5′-ALEX546-ACA AAC ACG CAC CTC AAA GC-3′); (c) mismatched
DNA with dye: (5′- ALEX546-ACA AAC ACT TTC CTC AAA GC-3′).
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