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Biosensors and Bioelectronics 22 (2006) 318–322

Short communication

Olfactory cell-based biosensor: A first step towards aneurochip 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 2006Available online 29 March 2006

bstract

Human olfactory system can distinguish thousands of odors. In order to realize the biomimetic design of electronic nose on the principle ofammalian olfactory system, this article reports an olfactory cell-based biosensor as a real bionic technique for odorants detection. Effective cultures

f 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,uch as acetic acid and glutamic acid, was tested. The results demonstrate that this kind of hybrid system of LAPS and olfactory neurons, whichs 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.

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eywords: Bioelectronic nose; Cell-based biosensor; Neurochip; Olfactory cel

. Introduction

Since olfactory system plays an important role in recogniz-ng environmental conditions, kinds of olfactory research haveeen carried out due to its potential commercial applications.lectronic nose, which mimics animals’ smell to detect odorsy its sensitive materials, is just one of these technologies. Theetection ability mainly depends on absorbability or catalysis ofhose materials to special odors. Although great achievementsave been made, this method still has limitations in sensitivitynd specificity, compared with the biology binding of specificdorants to the olfactory receptor neurons (Pearce, 1997).

Cell-based biosensors, which treat living cells as sensinglements, can detect the functional information of biologicallyctive analytes. This novel biosensor technique, characterized

ith high sensitivity, excellent selectivity and rapid response,as been applied in many fields ranging from biomedicine tonvironmental 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).

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956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2006.01.016

ht-addressable potentiometric sensor

herefore, utilizing olfactory neurons as sensitive materials toevelop a bioelectronic nose is one of independent trends con-erning the research and development of electronic nose, whichakes use of biomolecular function units to develop highly

ensitive sensors (Gopel, 2000). Some experiments, such asnsect antenna and human embryonic kidney-293 cells-basediosensors, have been tried, and obtained high specificity andensitivity to drugs or odors (Gross et al., 1997; Schutz et al.,000; Hwi and Tai, 2005). However, the tissue or cells were notlfactory neurons, and the parameters detected by those sensorsere also not the action potentials of the neurons.The mechanism of signal detection and transduction in olfac-

ion is an electrophysiological process, mainly taking placemong olfactory epithelium receptor cells and their correspond-ng mitral cells in olfactory bulb, and then the signals transferredo the olfactory cortex (Laurent, 1999). Therefore, a satisfactoryioelectronic nose should be a hybrid system of olfactory neu-ons and extracellular potential detection transducers.

Light-addressable potentiometric sensor (LAPS) is a com-only used semiconductor chip. Lots of experiments have been

one to investigate on LAPS as a possible cell-semiconductorybrid system to monitor the extracellular potentials of the cells

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Ismail et al., 2003; Stein et al., 2004). Our lab also has reportedhe possibility of LAPS to monitor the potential of single excit-ng cell in a non-invasive way (Xu et al., 2005).

In the present study, we analyzed the interface between cellsnd LAPS theoretically, and then based on the basic detectionheory of the extracellular potential, we cultivated olfactory neu-ons on surface of LAPS to monitor their extracellular potentials.f the LAPS and olfactory neurons hybrid system is sensitive tonvironmental changes, this bionic designed bioelectronic nosetudy will be a first step to an olfactory neurochip, which hasotential to develop systems that monitor signals related closelyo animal odor sensation, and even to be used as electronic inter-aces linking to the nose or brain directly.

. Theories

LAPS is a surface potential detector. With light pointer illu-inating on LAPS, the semiconductor absorbs energy and leads

o energy band transition, i.e. produces electron-hole pairs. IfAPS is biased in depletion, the width of the depletion layer is a

unction of the local value of the surface potential (Fig. 1a). Sincehe surface of LAPS is laterally unstructured, cells can adhereithout any spatial restrictions. When the cell produces poten-

ial changes by the ionic currents of the Na+ and K+ (Fig. 1b),hich was equal to the change of bias voltage, and its photocur-

ent given corresponding fluctuation. Therefore, by focusing theight pointer on the LAPS surface underlying a target cell, it isossible to record changes of the extracellular potential by mea-

uring the local surface potential at the illuminated region.

To understand how an action potential is generated, Hodgkinnd Huxley empirically modeled the ionic currents that flowhrough the channels of excitable membranes. They combined

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ig. 1. The principle and the schematic diagram of the olfactory-LAPS system. (a) Thenterface. (c) Schematic circuit of the cell-LAPS hybrid system. (d) The scheme of ex

ectronics 22 (2006) 318–322 319

his model to predict the total transmembrane current (H–Hheory):

M = CMdVM

dt+ Iionic (1)

here VM is the transmembrane potential, CM the membraneapacitance per unit area, and Iionic is the total ionic currentshrough the cellular membrane.

When neurons cultured on the oxidized silicon surface ofhe LAPS (Fig. 1b), the simplified schematic circuit of the cell-emiconductor interface was shown in Fig. 1c. Based on theheories of cell–silicon junctions and circuits (Fromherz, 2002),e obtain the relationship as (2):

VJ

RJ= CM

d(VM − VJ)

dt+ Iionic (2)

here VJ is the transductive extracellular potential, VM the trans-embrane potential, and RJ is the seal resistance. When the cell

roduces VM changes, ionic and capacitive currents flow throughhe membrane. The concomitant currents along the cleft give riseo VJ between the cell and chip, which is equal to the change ofias voltage of LAPS. This is the principle of the hybrid systemf the LAPS and the neurons cultured on it. The transductivextracellular potential VJ represents general extracellular poten-ial detected by LAPS.

. Experiments

.1. LAPS system

The LAPS chip and detecting setup were just similar to theystem we have reported (Xu et al., 2005). Here we describe itriefly.

scheme of cell-based biosensor using LAPS. (b) Simplified cell-semiconductorperimental system.

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The LAPS consists of an electrolyte-insulatorSiO2]–semiconductor [Si] (EIS) structure. N-type siliconafers (ϕ = 1.5 in.) with specific resistance of 10–15 � cm weresed as the LAPS chip. The upper side of the chip was insulatedith a layer of 30 nm SiO2, thermally oxidized at 1000 ◦C.ulk silicon was grinded to 100 �m thick to increase the

ensitivity. A 1 �m thick aluminum membrane was sputteredn the backside of the wafer to create an ohmic contact.hen, a 12 mm × 5 mm × 2 mm micro-chamber was formed byolydimethylsiloxane on the chip to culture cells. A separatedlatinum wire attached to the inside of the culture dish, servings a permanent ground reference. After attaching chip to thearrier, a petri dish with a 5 mm diameter hole through theottom was sealed to the chip with biocompatible glue.

During experiments, LAPS chip with cultured olfactory neu-ons was mounted under a microscope objective in the set-up.hen the modulated light was focused to less than 10 �m by a

ens and highlighted on the desired cell. The light source wasHe–Ne semiconductor laser (Coherent Co.), wavelength was43.5 nm and power was about 5 mW. Photocurrent of the LAPShows corresponding fluctuation, and transmitted into periph-ral equipments through working electrode. The electrodes ofotentiostat (EG&G Princeton Applied Research, M273A) weresed to detect the current. A 16 bit data acquired card and theoftware of LABVIEW were employed to control the collection,nalysis and acquisition of the data. Culture medium or drug wasumped alternatively by a peristaltic pump and passed through aegasser, a selective valve, and flowed into micro-chamber. Alleasurements were performed at 37 ± 0.2 ◦C. The experimental

ystem is shown in Fig. 1d.

.2. Cells culture

To improve the biocompatibility of the silicon device,e coated the surface of LAPS chip with poly-l-ornithine

nd laminin mixture (100 �g/ml poly-l-ornithine and 8 �g/mlaminin mixed by the rate of 1:1) prior to seeding cells, which

eans depositing a layer that can promote the cells attaching tohe surface of chip (Ismail et al., 2003).

Olfactory receptor neurons and cells of olfactory bulb were

arvested from 5 to 7 days old rat pups. Cells were seeded onhe chip at densities of 1 × 105 cells/cm2. After the second cul-ure day, 10 �g/ml 5-fluorouracil was added into the suspensiono inhibit the growth of fibroblasts and neuroglias. Cells were

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ig. 2. Neurons cultivated on the LAPS for 7 days. (a) Triangle mitral cell of olfaonnected olfactory bulb neuronal network among mitral cells (triangle) and granula

ectronics 22 (2006) 318–322

aintained for one week in LAPS device, containing 10% fetalalf serum, at 37 ◦C under standard conditions of humidifiedir with 5% CO2. The cells were fed every 2 days with freshMEM.

. Results and discussion

.1. Results obtained with cells culture

Olfactory receptor neurons are bipolar nerve cells. From theirpical pole the neurons extend dendrite to the epithelial surface,here they expand cilia, which are specialized for odor detec-

ion. From basal pole of each olfactory receptor neuron projects aingle axon to the olfactory bulb, where the axon forms synapsesith neurons, such as mitral cells and granular cells, then relay

ignals to the olfactory cortex. Fig. 2 shows olfactory neuronsrowing on the surface of LAPS for 7 days, some neuronal net-orks even have been formed among the cells.

.2. Extracellular recording of the mitral cells

Because glutamic acid (Glu) is one of the most importanteurotransmitters in the olfactory bulb, it was chosen as a stim-lant for mitral cells to inspect the sensitivity of the neurochip.he detailed process of the extracellular recording and samplesiving method refers to (Xu et al., 2005). Culture medium with-ut drugs was pumped into micro-chamber, the chip detectedo valid signals yet, and this recording signals were taken asaseline. When adding Glu (1 �M) into micro-chamber, theetected signals just liked the baseline, which implied that cellsad not been induced obviously potential changes. When weook Glu with concentration of 25 �M, we obtained the extra-ellular potential signal shape as Fig. 3a. There were severalbvious peaks in the middle of the curve. The amplitude of theeaks was 10–25 �V. The peaks centralized just at the begin-ing of the stimulation of the Glu. All of these were similar toxtracellular potential signal of cortical cells to acetylcholineAch) (Xu et al., 2005). The phenomena may be explained byhe desensitization produced by the agonist, Ach or Glu (Katznd Thesleff, 1957; Heckmann and Dudel, 1997). The receptor

hannels of the neurons opened and desensitized in response toapid applications of Glu or Ach. Then ion channels lost theirensitivity. After we replaced the Glu with culture medium, theignals could appear once again with the stimulation of the Glu.

ctory bulb. (b) Bipolar olfactory receptor neuron of olfactory epithelium. (c)r cells (bipolar). To display neuritis clearly, HE stain was used in (c).

Q. Liu et al. / Biosensors and Bioel

Fig. 3. Extracellular recording of olfactory cells. (a) The response of mitral cellunder the effect of Glu. (b) Odor-elicited extracellular potential of the olfac-tul

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ory receptor cells before, during and after odor presentation. (c) The odorantniquely and consistently elicits strong 24 Hz frequency component extracellu-ar potential.

owever, when we increased concentration of Glu to 50 �M,e no longer got the potential signal. Probably, neurons wereilled by the excitable toxicity of the Glu, in this high concen-ration. At the same time, we utilized fast Fourier transformFFT) to analyze frequency components of each signals. A spe-ific appearance of 24 Hz, was related to extracellular potentialignal of mitral cells under the effect of Glu.

.3. Stimulation of the odor to the olfactory receptor cells

There are 2000–3000 distinct olfactory receptor neurons con-aining in the animal olfactory epithelium. Cultured rat olfactoryeurons are excitable and can respond to odors (Pixley and Pun,990). Although, there is a large family of odor receptors neuronypes, numbering approximately 1000, each receptor cell classesponds to many different odors. Thus any particular odor acti-ates a substantial subset of these receptors (on the order of

undreds of receptor types). The great variety, exquisite speci-city, high sensitivity and fast response of olfactory receptoreurons make them an ideal candidate for olfactory cell-basediosensors.

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ectronics 22 (2006) 318–322 321

In order to primarily testify the feasibility of odorants detec-ion, different concentrations (1, 25, 50 �M) of acetic acidCH3COOH, a organic acid, with a distinctive pungent odor)re taken as stimulant to olfactory receptor neurons. We got theypical peaks as those of mitral cells in Fig. 3b. It was sustainedn the whole course of the acetic acid’s stimulation to the recep-or cells. The result proved the excitability and desensitizationf Glu to mitral cells more convincingly. With FFT analysis,e also found that olfactory receptor neurons showed a specific

ppearance of 24 Hz, occurred repeatedly to the stimulant. Themplitude of the frequency was increased in a concentration-ependent manner, and disappeared along with the stop of thedors stimulation 10 min later (Fig. 3c). Thereafter, we usedcetic acid as stimulation to mitral cells. Neither potential signalsor frequency signals were found. The fact leads us to believehat the frequency signal represented the binding of the odor tohe receptor neurons, and only the receptor neurons gained odorensitivity.

The olfactory processing can be achieved in the absence ofynaptic interactions between neurons, through phase locking tocommon underlying oscillatory potential (Brody and Hopfield,003). In the locust, odors puffed on an antenna cause theynchronization of groups of antennal lobe projection neuronsthe functional analogs of vertebrate olfactory bulb mitral-tuftedells), resulting in 20–30 Hz local field potential oscillation in theushroom body (the functional analog of the piriform cortex)

MacLeod and Laurent, 1996). Studies have found that such rep-esentations, for which the frequency characteristics are not odorpecific, are likely common to other olfactory systems, for exam-le, amphibians, mammals and hamster (Laurent et al., 1996).ur results suggest that in vitro olfactory neurons network of

at also have the frequency characteristics with the stimulationf acetic acid.

The patterns of interactions between pairs of neurons coulde studied by examining their cross-correlation function,hich reflect the mean firing rate of one result as a resultf the activity of another. If the cross-correlation betweeneurons is recorded, it is not difficult to find whether thereere oscillators or not. Using the multi-light LAPS based onigital compensation of frequency domain, the surface potentialt all illuminated regions can be measured simultaneouslyy analyzing the resulting photocurrent (Zhang et al., 2001).t present, we are designing multi-light systems to measureotential changes of neurons simultaneously. This work isecessary to develop bioelectronic nose, for each neuron in andor coding assembly responds with an odor-specific temporalring pattern consisting of periods of activity and silenceMacLeod and Laurent, 1996). These correlations can suggesthether olfactory neurons have influence on one another and

espond in synchrony to temporal pattern or not. Then, weould monitor action potential to record the neural transmissionnd the changes of olfactory neural network under the effect ofpecial external odors. And since the mechanism of olfactory

ensory neurons is a complex pattern of neuronal networks,hich makes the olfaction coding and decoding become veryifficult to study by the current electrophysiological recordingechniques, such as patch-clamp (Laurent, 1999). Observations

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f correlated firing also can provide more information of olfac-ory neurons connections and signal processing. This reveals

new potential application of this novel olfactory cell-basediosensor.

. Conclusion

This article demonstrates an olfactory cell-based biosensor,hich is developed from our previous cell-based biosensor and

lectronic nose research, to investigate the response of the olfac-ory neurons under stimulations of neurotransmitters and odor-nts. It has been proved by some primary experiments, olfactoryeceptor neurons and olfactory bulb neurons cultivated on theurface of chip are sensitive to environmental changes. All theseork suggests that the bionic designed hybrid system can besed as a novel bioelectronic nose.

cknowledgements

This work was supported by the National Natural Science

oundation of China (Grant Nos. 30270387, 30570492), theroject of State Key Laboratory of Transducer Technology ofhina (Grant No. SKT0403), the Foundation for the Bureau ofhejiang Province of China (Grant No. 20040197).

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