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Short communication
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
(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
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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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).
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.
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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
322
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).
References
Bousse, L., 1996. Sens. Actuators B 34, 270275.
Brody, C.D., Hopfield, J.J., 2003. Neuron. 37, 843852.
Fromherz, P., 2002. Chem. Phys. Chem. 3, 276284.
Gopel, W., 2000. Sens. Actuators B 65, 7072.
Gross, G.W., Harsch, A., Rhoades, B.K., Gopel, W., 1997. Biosens. Bioelectron.
12, 373393.
Heckmann, M., Dudel, J., 1997. Biophys. J. 72, 21602169.
Hwi, J.K., Tai, H.P., 2005. Biosens. Bioelectron. 20, 13271332.
Ismail, A.B., Yoshinobu, T., Iwasaki, H., Sugihara, H., Yukimasa, T., Hirata, L.,
Iwata, H., 2003. Biosens. Bioelectron. 18, 15091514.
Katz, B., Thesleff, S., 1957. J. Physiol. 138, 6380.
Laurent, G., 1999. Science 286, 723728.
Laurent, G., Wehr, M., Davidowitz, H., 1996. J. Neurosci. 16, 28372847.
MacLeod, K., Laurent, G., 1996. Science 274, 976979.
Pearce, T.C., 1997. Biosystems 41, 6990.
Pixley, S.K., Pun, R.Y., 1990. Brain Res. 53, 125130.
Schutz, S., Schoning, M.J., Schroth, P., Malkoc, U., Weibecker, B., Kordos, P.,
Luth, H., Hummel, H.E., 2000. Sens. Actuators B 65, 291295.
Stein, B., George, M., Gaub, H.E., Parak, W.J., 2004. Sens. Actuators B 98,
299304.
Wang, P., Xu, G.X., Qin, L.F., Xu, Y., Li, Y., Li, R., 2005. Sens. Actuators B
108, 576584.
Xu, G.X., Ye, X.S., Qin, L.F., Xu, Y., Li, Y., Li, R., Wang, P., 2005. Biosens.
Bioelectron. 20, 17571763.
Zhang, Q.T., Wang, P., Wolfgang, J.P., George, M., Zhang, G.Y., 2001. Sens.
Actustors B 73, 152156.