Polypyrrole nanotubes conjugated with human olfactory receptors: High-Performance transducers for FET-Type bioelectronic noses

Full text of DOI:10.1002/anie.200805171

Angewandte
Chemie
DOI: 10.1002/anie.200805171
Bioelectronic Noses
Polypyrrole Nanotubes Conjugated with Human Olfactory Receptors:
High-Performance Transducers for FET-Type Bioelectronic Noses**
Hyeonseok Yoon, Sang Hun Lee, Oh Seok Kwon, Hyun Seok Song, Eun Hae Oh,
Tai Hyun Park,* and Jyongsik Jang*
Significant advance in conducting polymers (CPs) has
prompted the development of devices such as organic light-
emitting diodes, solar cells, memories, and field-effect tran-
sistors (FETs). As alternatives to inorganic semiconductors or
metals, CPs provide great potential to produce low-cost,
large-area, lightweight, and flexible devices. Recently, CP
nanomaterials have received considerable attention because
of remarkable physical and chemical characteristics originat-
both transistor channel and sensitive element has been
limited, owing to difficulties in their synthesis and manipu-
lation. Herein, we describe the integration of human olfactory
receptors (hORs) and CP nanotubes into a FET platform
suitable for electronic control. Field-induced sensitivity was
observed, eventually leading to the recognition of a target
odorant at an unprecedentedly low concentration. To our
knowledge, this is the first example of FET-type bioelectronic
[
1]
[10]
ing from their small dimensions and high surface area. CP
nanomaterials also inherit advantages such as facile function-
noses based on hOR-conjugated CP nanotubes.
First of all, hOR 2AG1 (hOR2AG1) was expressed in
[2]
alization and biocompatibility from their bulk counterparts.
Escherichia coli (E. coli) as a fusion protein with a gluta-
[8]
Importantly, these features make CP nanomaterials highly
attractive for various future applications. Typically, there is an
increasing interest in the use of CP nanomaterials in
thione-S-transferase (GST) tag at its N terminus. The GST
tag was used as a fusion partner for efficient expression and
also for the Western blot analysis to confirm the expression of
olfactory receptor protein. The E. coli cells were sonicated to
obtain a membrane fraction. The hOR2AG1 was difficult to
solubilize with detergents. In contrast, most of the impurity
proteins were solubilized with Triton X-100 from the
insoluble fraction. To remove membrane-integrated proteins
other than hOR2AG1, the membrane fraction was treated
with 5% triton X-100 and then retrieved by centrifugation.
CP nanotubes were synthesized with the aid of cylindrical
[3,4]
analytical sciences.
Odor discrimination is a challenging research subject for
key applications in the fields of foods and beverages,
[5]
environmental monitoring, and disease diagnosis. The
challenges arise from the fact that only subtle differences in
the molecular structure of an odorant can lead to pronounced
modifications in odor quality. Human and animal noses can
perceive more than hundreds of thousands of odor mole-
[
6]
[11]
cules. Accordingly, particular attention has been paid to the
development of sensors that mimic the mammalian olfactory
system. A few significant studies regarding biotechnology-
based olfactory sensors, which are called bioelectronic noses,
have been reported, mainly based on quartz crystal micro-
micelle templates in an apolar solvent. Chemical copoly-
merization of pyrrole with pyrrole-3-carboxylic acid (P3CA)
on the cylindrical micelle surface yielded intrinsically func-
À1
tionalized CP nanotubes (four-probe conductivity: 10 –
0
À1
10 Scm ), namely carboxylated polypyrrole nanotubes
(CPNTs).
[
7,8]
balances.
However, their sensitivity and selectivity leave
room for improvement.
Although CPs have been extensively implemented in
different types of sensors, the use of CP nanomaterials as
Figure 1a schematically shows the FET sensor platform
[
12]
based on hOR-conjugated CP nanotubes. An interdigitated
microelectrode array (IDA) was patterned on a glass sub-
strate through a lithographic process. The IDA consisted of a
pair of gold electrode bands with 25 fingers each, in which the
bands served as source (S) and drain (D) electrodes,
respectively. The IDA was 2 mm wide, 1 mm long, and had
[
9]
[
*] S. H. Lee, H. S. Song, E. H. Oh, Prof. T. H. Park
School of Chemical and Biological Engineering
Institute of Bioengineering, Seoul National University
2
mm interfinger gaps. CPNTs were immobilized on the
599 Gwanangno, Gwanakgu, Seoul 151-742 (Korea)
electrode substrate through covalent linkages to maintain
stable electrical contact between the nanotubes and the
electrodes. The IDA substrate was treated with an amino-
silane (3-aminopropyltrimethoxysilane, APS), and the car-
boxy functional groups of the CPNTs were then coupled with
the surface amino group of the IDA substrate in a condensa-
tion reaction. Compared with conventional lithographic
methods, this immobilization approach offers critical advan-
tages, such as mild reaction conditions and a simple process
for the successful integration of CP nanomaterials into the
Fax: (+82)2-875-9348
E-mail: thpark@plaza.snu.ac.kr
Dr. H. Yoon, O. S. Kwon, Prof. J. Jang
School of Chemical and Biological Engineering
Seoul National University
599 Gwanangno, Gwanakgu, Seoul 151-742 (Korea)
Fax: (+82)2-888-1604
E-mail: jsjang@plaza.snu.ac.kr
Homepage: http://plaza.snu.ac.kr/~jsjang
[
**] This work was supported by a grant from the Center for Advanced
Materials Processing under the 21C Frontier Programs funded by
the Ministry of Knowledge Economy, Republic of Korea.
[13]
electrode substrate.
The hOR2AG1 contains terminal
amine groups on cysteine (Cys) residues. Therefore, the
covalent anchoring of hOR2AG1 on the nanotube surface
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200805171.
Angew. Chem. Int. Ed. 2009, 48, 2755 –2758
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
Figure 2. a) Current–voltage curves of CPNTs on the electrode sub-
strate before and after hOR immobilization in air (scan
À1
rate=10 mVs ). b) Schematic diagram of a liquid–ion gate FET using
Figure 1. The hOR2AG1-conjugated CPNT FET platform: a) Schematic
hOR2AG1-conjugated CPNTs. c) Cyclic ISD–E curve of the FET sensor
G
illustration. Only one nanotube is shown for clarity; covalent attach-
ments (1) and (2) were used to bind CPNTs on the electrode substrate
and to immobilize hORs to the nanotube, respectively. b) Typical FE-
SEM image of a hOR2AG1-conjugated CPNT.
À1
based on 1OR-CPNTs measured in phosphate buffer at Æ10 mVs
.
geous in making intimate contact with the nanotubes. To
could be achieved through a similar condensation reaction.
Figure 1b displays a typical field-emission scanning electron
microscopy (FE-SEM) image of hOR2AG1-conjugated
CPNTs deposited on the electrode substrate. It was clearly
observed that the nanotube surface was made considerably
more rugged by the attached hOR2AG1. Furthermore, there
was no hOR on the electrode substrate except on the
nanotubes, thus indicating that the hOR was selectively
deposited on the nanotubes through chemical coupling.
Compared with a noncovalent approach, covalent anchor-
ing provides better stability for analyte sensing in the liquid
phase and further offers the possibility to control the chemical
evaluate the FET characteristics, the gate potential (E ) was
G
applied between the reference electrode and the source
electrode through the buffer solution. The source–drain
current (ISD) was modulated with varying EG at a constant
VSD (50 mV), as shown in Figure 2c. The oxidation and
reduction potentials of the CPNTwere observed around + 0.9
and À1.0 V.
The principal function of hOR proteins is to bind specific
[6]
odorants and in turn to transduce a signal. Such hOR–
odorant interaction can affect the charge-carrier density of
conjugated CP nanotubes, and thus it may allow label-free
recognition of target odorants on the FET configuration. To
investigate the sensing capability of hOR2AG1-conjugated
[
14]
functionality of the nanotubes.
Under our experimental
conditions, controlled loading of hOR2AG1 on the nanotube
surface was achieved by adjusting its feeding amount in the
coupling reaction. CPNTs were functionalized with
hOR2AG1-to-CPNT weight ratios of 1:4 (1OR-CPNT), 1:2
CPNT FETs, the ISD was monitored in real time at VSD =
50 mV (E = 50 mV), a low operating voltage, upon addition
G
of odorants. It is known that hOR2AG1 specifically responds
[16]
to amyl butyrate, a common reagent for fruit flavor.
(
2OR-CPNT), and 1:1 (4OR-CPNT). Figure 2a exhibits
Figure 3a displays the response of 1OR-CPNT FETs to the
target odorant, amyl butyrate. The FET showed a concen-
tration-dependent increase in ISD when exposed to amyl
butyrate. The ISD increase was not attributed to electro-
chemical oxidation of amyl butyrate, because the applied EG
was well below its oxidation potential. It is believed that the
current increase is due to a change in the charge-transport
behavior of the CPNTs elicited by the hOR–odorant binding
event. Specifically, the cysteine residues of hORs adopt an
current–voltage (I–V) curves of pristine CPNTs and
hOR2AG1-conjugated CPNTs deposited on the electrode
substrate.
The dI/dV value of CPNTs gradually decreased with
[
2]
increasing amount of conjugated hOR. However, the ohmic
contact was retained after the coupling reaction and washing
process. This result means that the covalent immobilization of
CPNTs on the electrode substrate resulted in reliable
electrical contact. The recognition of odorants was carried
out in solution. A liquid–ion gate FET geometry was
constructed using a phosphate-buffered solution (10 mm,
À
uncharged (RSH) state or a negatively charged (RS ) state,
associated with the acid-to-base transition of the sulfhydryl
[6]
group. Importantly, the specific binding of odorants initiates
the structural rearrangement of hORs, finally leading to a
[12,13,15]
pH 7.4; Figure 2b).
The Ag/AgCl reference electrode
À
and platinum counterelectrode were immersed in a buffered
aqueous solution to provide gate control. Compared with
conventional back gating, the liquid–ion gating is advanta-
relative increase in negatively charged base state (RS ). The
increased negative charge density at the hOR2AG1–CPNT
interface can induce a p-type doping effect in the nanotubes,
2
756
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Angewandte
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[8]
magnitude lower than those of existing bioelectronic noses.
The sample with the highest degree of functionalization,
OR-CPNT, had the lowest detection limit (10 fm) as a result
4
of the enhanced hOR–odorant interaction. The sensitivity
was determined from the normalized ISD change recorded
after the addition of amyl butyrate. In all cases, the sensitivity
increased linearly with the odorant concentration on the log-
scale x axis. The sensitivity was in the order of 1OR-CPNT<
2
OR-CPNT< 4OR-CPNT over a wide concentration range.
In particular, at low concentrations (femtomole level), the
sensitivity of 4OR-CPNT was approximately twice that of
1
OR-CPNT or 2OR-CPNT. Therefore, it is expected that the
sensitivity of the CPNT FET sensor can be tuned by careful
control of the hOR loading.
In conclusion, we have demonstrated the feasibility of
FET-type bioelectronic noses using hOR-conjugated CP
nanotubes as the transistor channel and sensitive element.
Our chemical immobilization strategy enabled the facile
fabrication of the CPNT FET substrate with excellent
electrical properties as well as the quantitative control of
hOR functionalization. The FETs showed the possibility of
specific odorant detection and gave measurable signals down
to concentrations as low as tens of femtomoles. Considering
these facts, the hOR-conjugated CP nanotube FET system
offers a new direction for the highly sensitive and selective
recognition of odorants and could be expanded to allow the
multiplexed detection of various odorants after a judicious
optimization.
Figure 3. Real-time responses of 1OR-CPNT FET sensors measured at
VSD =50 mV. Normalized ISD changes upon addition of a) target odor-
ant (amyl butyrate, AB) and b) nontarget (butyl butyrate, BB; propyl
butyrate, PB; hexyl butyrate, HB) and target odorants.
which is probably responsible for the increase in ISD.
Figure 3b represents the selective response of the FET
toward a particular odorant of interest. Other odorants such
as propyl butyrate, butyl butyrate, and hexyl butyrate were
employed as nontargets to verify the specificity of
hOR2AG1–conjugated CPNTs. Upon addition of the non-
targets, no significant changes in ISD were observed, in
contrast with the case of amyl butyrate. Consequently, it
was evident that hOR2AG1-conjugated CPNTs had the
outstanding ability to translate and amplify hOR–odorant
interaction into a detectable signal as the transducer.
Experimental Section
First, hOR2AG1 was expressed in E. coli as a fusion protein. The GST
tag was used as a fusion partner. CPNTs were prepared by chemical
oxidation copolymerization of pyrrole and P3CA with the aid of
reverse-cylindrical micelles consisting of sodium bis(2-ethylhexyl)-
sulfosuccinate. An efficient condensing agent, 4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), was syn-
As mentioned above, the loading amount of hOR2AG1
on CPNTs was controlled during the covalent functionaliza-
tion process. The hOR2AG1 near the CPNT acted as an
[
17]
thesized according to the reported procedure. The related exper-
imental details are described in the Supporting Information.
effective gate, resulting in modulation of I . Accordingly, the
SD
An IDA was patterned on a glass substrate by a lithographic
process. To construct the FET platform, in the first stage the IDA
substrate was treated with 5 wt% aqueous APS for 6 h and then
exposed to a mixture of 1 wt% CPNT in ethanol (10 mL) and 1 wt%
DMT-MM in ethanol (10 mL) for 12 h. The resulting CPNT-immobi-
lized substrate was rinsed with distilled water. Subsequently, the
coupling reaction to attach hOR2AG1 to the CPNT surface was
carried out using a mixture of hOR2AG1 and 1 wt% aqueous DMT-
MM (10 mL) for 12 h. The feeding amounts of hOR2AG1 were 1:4,
loading amount of hOR2AG1 is considered to be an
important factor that affects the FET response. Figure 4
shows the dependence of the FET response on the amount of
conjugated hOR. The detection limits of the FETs were found
to be tens of femtomoles, approximately two orders of
2
4
:4, and 4:4 (w/w) relative to CPNT for 1OR-CPNT, 2OR-CPNT, and
OR-CPNT samples, respectively. Afterwards, the substrate was
rinsed with distilled water and dried with a stream of nitrogen gas.
All electrical measurements were conducted with a Keithley 2400
sourcemeter and a Wonatech WBCS 3000 potentiostat. A solution
chamber (50 mL volume) was designed and used for solution-based
measurements. The current change was normalized as DI/I = (IÀI )/
0
0
I , where I is the initial current and I is the measured real-time
0
0
current.
Figure 4. Calibration curves of the FET sensors. Sensitivity changes
were plotted on a log scale (x axis) as a function of amyl butyrate
concentration.
Received: October 22, 2008
Revised: January 22, 2009
Published online: March 9, 2009
Angew. Chem. Int. Ed. 2009, 48, 2755 –2758
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2757

Communications
Keywords: conducting polymers · field-effect transistors ·
nanotubes · olfactory receptors · sensors
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