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Biosensors and Bioelectronics 22 (2006) 318322

Short communication

Olfactory cell-based biosensor: A first step towards a


neurochip of bioelectronic nose
Qingjun Liu a,b , Hua Cai a , Ying Xu a ,
Yan Li a , Rong Li a , Ping Wang a,b,
a

Biosensor National Special Laboratory, Key Laboratory of Biomedical Engineering of Education Ministry,
Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China
b State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Shanghai 200050, PR China
Received 20 October 2005; received in revised form 23 December 2005; accepted 9 January 2006
Available online 29 March 2006

Abstract
Human olfactory system can distinguish thousands of odors. In order to realize the biomimetic design of electronic nose on the principle of
mammalian olfactory system, this article reports an olfactory cell-based biosensor as a real bionic technique for odorants detection. Effective cultures
of olfactory receptor neurons and olfactory bulb cells have been achieved on the semiconductor chip. Using light-addressable potentiometric sensor
(LAPS) as sensing chip to monitor extracellular potential of the neurons, the response under stimulations of the odorants or neurotransmitters,
such as acetic acid and glutamic acid, was tested. The results demonstrate that this kind of hybrid system of LAPS and olfactory neurons, which
is sensitive to odorous changes, has great potential and is promising to be used as a novel neurochip of bioelectronic nose for detecting odors.
2006 Elsevier B.V. All rights reserved.
Keywords: Bioelectronic nose; Cell-based biosensor; Neurochip; Olfactory cell; Light-addressable potentiometric sensor

1. Introduction
Since olfactory system plays an important role in recognizing environmental conditions, kinds of olfactory research have
been carried out due to its potential commercial applications.
Electronic nose, which mimics animals smell to detect odors
by its sensitive materials, is just one of these technologies. The
detection ability mainly depends on absorbability or catalysis of
those materials to special odors. Although great achievements
have been made, this method still has limitations in sensitivity
and specificity, compared with the biology binding of specific
odorants to the olfactory receptor neurons (Pearce, 1997).
Cell-based biosensors, which treat living cells as sensing
elements, can detect the functional information of biologically
active analytes. This novel biosensor technique, characterized
with high sensitivity, excellent selectivity and rapid response,
has been applied in many fields ranging from biomedicine to
environmental detection (Bousse, 1996; Wang et al., 2005).

Corresponding author. Tel.: +86 571 87952832; fax: +86 571 87951676.
E-mail address: cnpwang@mail.bme.zju.edu.cn (P. Wang).

0956-5663/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2006.01.016

Therefore, utilizing olfactory neurons as sensitive materials to


develop a bioelectronic nose is one of independent trends concerning the research and development of electronic nose, which
makes use of biomolecular function units to develop highly
sensitive sensors (Gopel, 2000). Some experiments, such as
insect antenna and human embryonic kidney-293 cells-based
biosensors, have been tried, and obtained high specificity and
sensitivity to drugs or odors (Gross et al., 1997; Schutz et al.,
2000; Hwi and Tai, 2005). However, the tissue or cells were not
olfactory neurons, and the parameters detected by those sensors
were also not the action potentials of the neurons.
The mechanism of signal detection and transduction in olfaction is an electrophysiological process, mainly taking place
among olfactory epithelium receptor cells and their corresponding mitral cells in olfactory bulb, and then the signals transferred
to the olfactory cortex (Laurent, 1999). Therefore, a satisfactory
bioelectronic nose should be a hybrid system of olfactory neurons and extracellular potential detection transducers.
Light-addressable potentiometric sensor (LAPS) is a commonly used semiconductor chip. Lots of experiments have been
done to investigate on LAPS as a possible cell-semiconductor
hybrid system to monitor the extracellular potentials of the cells

Q. Liu et al. / Biosensors and Bioelectronics 22 (2006) 318322

(Ismail et al., 2003; Stein et al., 2004). Our lab also has reported
the possibility of LAPS to monitor the potential of single exciting cell in a non-invasive way (Xu et al., 2005).
In the present study, we analyzed the interface between cells
and LAPS theoretically, and then based on the basic detection
theory of the extracellular potential, we cultivated olfactory neurons on surface of LAPS to monitor their extracellular potentials.
If the LAPS and olfactory neurons hybrid system is sensitive to
environmental changes, this bionic designed bioelectronic nose
study will be a first step to an olfactory neurochip, which has
potential to develop systems that monitor signals related closely
to animal odor sensation, and even to be used as electronic interfaces linking to the nose or brain directly.
2. Theories
LAPS is a surface potential detector. With light pointer illuminating on LAPS, the semiconductor absorbs energy and leads
to energy band transition, i.e. produces electron-hole pairs. If
LAPS is biased in depletion, the width of the depletion layer is a
function of the local value of the surface potential (Fig. 1a). Since
the surface of LAPS is laterally unstructured, cells can adhere
without any spatial restrictions. When the cell produces potential changes by the ionic currents of the Na+ and K+ (Fig. 1b),
which was equal to the change of bias voltage, and its photocurrent given corresponding fluctuation. Therefore, by focusing the
light pointer on the LAPS surface underlying a target cell, it is
possible to record changes of the extracellular potential by measuring the local surface potential at the illuminated region.
To understand how an action potential is generated, Hodgkin
and Huxley empirically modeled the ionic currents that flow
through the channels of excitable membranes. They combined

319

this model to predict the total transmembrane current (HH


theory):
dVM
(1)
+ Iionic
dt
where VM is the transmembrane potential, CM the membrane
capacitance per unit area, and Iionic is the total ionic currents
through the cellular membrane.
When neurons cultured on the oxidized silicon surface of
the LAPS (Fig. 1b), the simplified schematic circuit of the cellsemiconductor interface was shown in Fig. 1c. Based on the
theories of cellsilicon junctions and circuits (Fromherz, 2002),
we obtain the relationship as (2):

IM = CM

VJ
d(VM VJ )
= CM
+ Iionic
RJ
dt

(2)

where VJ is the transductive extracellular potential, VM the transmembrane potential, and RJ is the seal resistance. When the cell
produces VM changes, ionic and capacitive currents flow through
the membrane. The concomitant currents along the cleft give rise
to VJ between the cell and chip, which is equal to the change of
bias voltage of LAPS. This is the principle of the hybrid system
of the LAPS and the neurons cultured on it. The transductive
extracellular potential VJ represents general extracellular potential detected by LAPS.
3. Experiments
3.1. LAPS system
The LAPS chip and detecting setup were just similar to the
system we have reported (Xu et al., 2005). Here we describe it
briefly.

Fig. 1. The principle and the schematic diagram of the olfactory-LAPS system. (a) The scheme of cell-based biosensor using LAPS. (b) Simplified cell-semiconductor
interface. (c) Schematic circuit of the cell-LAPS hybrid system. (d) The scheme of experimental system.

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Q. Liu et al. / Biosensors and Bioelectronics 22 (2006) 318322

The LAPS consists of an electrolyte-insulator


[SiO2 ]semiconductor [Si] (EIS) structure. N-type silicon
wafers ( = 1.5 in.) with specific resistance of 1015  cm were
used as the LAPS chip. The upper side of the chip was insulated
with a layer of 30 nm SiO2 , thermally oxidized at 1000 C.
Bulk silicon was grinded to 100 m thick to increase the
sensitivity. A 1 m thick aluminum membrane was sputtered
on the backside of the wafer to create an ohmic contact.
Then, a 12 mm 5 mm 2 mm micro-chamber was formed by
polydimethylsiloxane on the chip to culture cells. A separated
platinum wire attached to the inside of the culture dish, serving
as a permanent ground reference. After attaching chip to the
carrier, a petri dish with a 5 mm diameter hole through the
bottom was sealed to the chip with biocompatible glue.
During experiments, LAPS chip with cultured olfactory neurons was mounted under a microscope objective in the set-up.
Then the modulated light was focused to less than 10 m by a
lens and highlighted on the desired cell. The light source was
a HeNe semiconductor laser (Coherent Co.), wavelength was
543.5 nm and power was about 5 mW. Photocurrent of the LAPS
shows corresponding fluctuation, and transmitted into peripheral equipments through working electrode. The electrodes of
potentiostat (EG&G Princeton Applied Research, M273A) were
used to detect the current. A 16 bit data acquired card and the
software of LABVIEW were employed to control the collection,
analysis and acquisition of the data. Culture medium or drug was
pumped alternatively by a peristaltic pump and passed through a
degasser, a selective valve, and flowed into micro-chamber. All
measurements were performed at 37 0.2 C. The experimental
system is shown in Fig. 1d.
3.2. Cells culture
To improve the biocompatibility of the silicon device,
we coated the surface of LAPS chip with poly-l-ornithine
and laminin mixture (100 g/ml poly-l-ornithine and 8 g/ml
laminin mixed by the rate of 1:1) prior to seeding cells, which
means depositing a layer that can promote the cells attaching to
the surface of chip (Ismail et al., 2003).
Olfactory receptor neurons and cells of olfactory bulb were
harvested from 5 to 7 days old rat pups. Cells were seeded on
the chip at densities of 1 105 cells/cm2 . After the second culture day, 10 g/ml 5-fluorouracil was added into the suspension
to inhibit the growth of fibroblasts and neuroglias. Cells were

maintained for one week in LAPS device, containing 10% fetal


calf serum, at 37 C under standard conditions of humidified
air with 5% CO2 . The cells were fed every 2 days with fresh
DMEM.
4. Results and discussion
4.1. Results obtained with cells culture
Olfactory receptor neurons are bipolar nerve cells. From their
apical pole the neurons extend dendrite to the epithelial surface,
where they expand cilia, which are specialized for odor detection. From basal pole of each olfactory receptor neuron projects a
single axon to the olfactory bulb, where the axon forms synapses
with neurons, such as mitral cells and granular cells, then relay
signals to the olfactory cortex. Fig. 2 shows olfactory neurons
growing on the surface of LAPS for 7 days, some neuronal networks even have been formed among the cells.
4.2. Extracellular recording of the mitral cells
Because glutamic acid (Glu) is one of the most important
neurotransmitters in the olfactory bulb, it was chosen as a stimulant for mitral cells to inspect the sensitivity of the neurochip.
The detailed process of the extracellular recording and samples
giving method refers to (Xu et al., 2005). Culture medium without drugs was pumped into micro-chamber, the chip detected
no valid signals yet, and this recording signals were taken as
baseline. When adding Glu (1 M) into micro-chamber, the
detected signals just liked the baseline, which implied that cells
had not been induced obviously potential changes. When we
took Glu with concentration of 25 M, we obtained the extracellular potential signal shape as Fig. 3a. There were several
obvious peaks in the middle of the curve. The amplitude of the
peaks was 1025 V. The peaks centralized just at the beginning of the stimulation of the Glu. All of these were similar to
extracellular potential signal of cortical cells to acetylcholine
(Ach) (Xu et al., 2005). The phenomena may be explained by
the desensitization produced by the agonist, Ach or Glu (Katz
and Thesleff, 1957; Heckmann and Dudel, 1997). The receptor
channels of the neurons opened and desensitized in response to
rapid applications of Glu or Ach. Then ion channels lost their
sensitivity. After we replaced the Glu with culture medium, the
signals could appear once again with the stimulation of the Glu.

Fig. 2. Neurons cultivated on the LAPS for 7 days. (a) Triangle mitral cell of olfactory bulb. (b) Bipolar olfactory receptor neuron of olfactory epithelium. (c)
Connected olfactory bulb neuronal network among mitral cells (triangle) and granular cells (bipolar). To display neuritis clearly, HE stain was used in (c).

Q. Liu et al. / Biosensors and Bioelectronics 22 (2006) 318322

Fig. 3. Extracellular recording of olfactory cells. (a) The response of mitral cell
under the effect of Glu. (b) Odor-elicited extracellular potential of the olfactory receptor cells before, during and after odor presentation. (c) The odorant
uniquely and consistently elicits strong 24 Hz frequency component extracellular potential.

However, when we increased concentration of Glu to 50 M,


we no longer got the potential signal. Probably, neurons were
killed by the excitable toxicity of the Glu, in this high concentration. At the same time, we utilized fast Fourier transform
(FFT) to analyze frequency components of each signals. A specific appearance of 24 Hz, was related to extracellular potential
signal of mitral cells under the effect of Glu.
4.3. Stimulation of the odor to the olfactory receptor cells
There are 20003000 distinct olfactory receptor neurons containing in the animal olfactory epithelium. Cultured rat olfactory
neurons are excitable and can respond to odors (Pixley and Pun,
1990). Although, there is a large family of odor receptors neuron
types, numbering approximately 1000, each receptor cell class
responds to many different odors. Thus any particular odor activates a substantial subset of these receptors (on the order of
hundreds of receptor types). The great variety, exquisite specificity, high sensitivity and fast response of olfactory receptor
neurons make them an ideal candidate for olfactory cell-based
biosensors.

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In order to primarily testify the feasibility of odorants detection, different concentrations (1, 25, 50 M) of acetic acid
(CH3 COOH, a organic acid, with a distinctive pungent odor)
are taken as stimulant to olfactory receptor neurons. We got the
typical peaks as those of mitral cells in Fig. 3b. It was sustained
in the whole course of the acetic acids stimulation to the receptor cells. The result proved the excitability and desensitization
of Glu to mitral cells more convincingly. With FFT analysis,
we also found that olfactory receptor neurons showed a specific
appearance of 24 Hz, occurred repeatedly to the stimulant. The
amplitude of the frequency was increased in a concentrationdependent manner, and disappeared along with the stop of the
odors stimulation 10 min later (Fig. 3c). Thereafter, we used
acetic acid as stimulation to mitral cells. Neither potential signals
nor frequency signals were found. The fact leads us to believe
that the frequency signal represented the binding of the odor to
the receptor neurons, and only the receptor neurons gained odor
sensitivity.
The olfactory processing can be achieved in the absence of
synaptic interactions between neurons, through phase locking to
a common underlying oscillatory potential (Brody and Hopfield,
2003). In the locust, odors puffed on an antenna cause the
synchronization of groups of antennal lobe projection neurons
(the functional analogs of vertebrate olfactory bulb mitral-tufted
cells), resulting in 2030 Hz local field potential oscillation in the
mushroom body (the functional analog of the piriform cortex)
(MacLeod and Laurent, 1996). Studies have found that such representations, for which the frequency characteristics are not odor
specific, are likely common to other olfactory systems, for example, amphibians, mammals and hamster (Laurent et al., 1996).
Our results suggest that in vitro olfactory neurons network of
rat also have the frequency characteristics with the stimulation
of acetic acid.
The patterns of interactions between pairs of neurons could
be studied by examining their cross-correlation function,
which reflect the mean firing rate of one result as a result
of the activity of another. If the cross-correlation between
neurons is recorded, it is not difficult to find whether there
were oscillators or not. Using the multi-light LAPS based on
digital compensation of frequency domain, the surface potential
at all illuminated regions can be measured simultaneously
by analyzing the resulting photocurrent (Zhang et al., 2001).
At present, we are designing multi-light systems to measure
potential changes of neurons simultaneously. This work is
necessary to develop bioelectronic nose, for each neuron in an
odor coding assembly responds with an odor-specific temporal
firing pattern consisting of periods of activity and silence
(MacLeod and Laurent, 1996). These correlations can suggest
whether olfactory neurons have influence on one another and
respond in synchrony to temporal pattern or not. Then, we
could monitor action potential to record the neural transmission
and the changes of olfactory neural network under the effect of
special external odors. And since the mechanism of olfactory
sensory neurons is a complex pattern of neuronal networks,
which makes the olfaction coding and decoding become very
difficult to study by the current electrophysiological recording
techniques, such as patch-clamp (Laurent, 1999). Observations

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Q. Liu et al. / Biosensors and Bioelectronics 22 (2006) 318322

of correlated firing also can provide more information of olfactory neurons connections and signal processing. This reveals
a new potential application of this novel olfactory cell-based
biosensor.
5. Conclusion
This article demonstrates an olfactory cell-based biosensor,
which is developed from our previous cell-based biosensor and
electronic nose research, to investigate the response of the olfactory neurons under stimulations of neurotransmitters and odorants. It has been proved by some primary experiments, olfactory
receptor neurons and olfactory bulb neurons cultivated on the
surface of chip are sensitive to environmental changes. All these
work suggests that the bionic designed hybrid system can be
used as a novel bioelectronic nose.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 30270387, 30570492), the
Project of State Key Laboratory of Transducer Technology of
China (Grant No. SKT0403), the Foundation for the Bureau of
Zhejiang Province of China (Grant No. 20040197).

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